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Age determination of body fluids by
mass spectrometry
Katarzyna Szykuła
Master Thesis
2015 – 2016
2
MSc Chemistry
Analytical Sciences
Master Thesis
Age determination of body fluids by mass spectrometry
by
Katarzyna Szykuła
June 2016
Supervisor: Second examiner:
Prof. dr. Garry Corthals Dr. Michelle Camenzuli
Analytical Chemistry Group
van’ t Hoff Institute for Molecular Sciences
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Acknowledgements
I would like to express my gratitude to professor Garry Corthals for his supervision and guidance
during this Master’s project. I have enjoyed my time carrying out this research and I would like to
thank everyone from the Analytical Chemistry group for their help and kindness. Additionally, I would
like to thank Dr. Michelle Camenzuli for reviewing this thesis.
Moreover, I would like to thank professor Maurice Aalders and his group for their help and the
opportunity to do experiments at Academic Medical Center.
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Abstract
In forensic science, biological traces have a significant importance for forensic investigators. They can
provide crucial information about the donor or about a crime itself, for example what happened on a
crime scene or when the criminal incident was committed. In order to estimate the latter one, aging
of body fluids found on a crime scene is studied. This research investigated aging process of four
body fluids: blood, saliva, semen and fingermarks. As all of them are protein-containing fluids, this
type of compounds was examined by using mass spectrometry. Samples of aforementioned body
fluids were analyzed at different age, and subsequently, the numbers of proteins identified as well as
their quantification were compared. For blood, saliva and semen, the number of unique proteins was
changing with the age of a sample. Additionally, some proteins showed variations in quantity when
sample was getting older. These findings can indicate that protein levels are changing over time in
biological traces, and these biomolecules can be potential biomarkers for age estimation of specific
body fluid found on a crime scene.
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Table of Contents
Acknowledgements ................................................................................................................................. 3
Abstract ................................................................................................................................................... 4
1. Introduction ......................................................................................................................................... 7
1.1 Forensic biological traces .............................................................................................................. 7
1.1.1 Blood ....................................................................................................................................... 7
1.1.2 Saliva ....................................................................................................................................... 8
1.1.3 Semen ..................................................................................................................................... 8
1.1.4 Fingermarks ............................................................................................................................ 8
1.2 Mass spectrometry ........................................................................................................................ 9
1.2.1 Proteomics .............................................................................................................................. 9
1.2.2 Quantitation in proteomics .................................................................................................. 11
1.2.3 Proteomics in forensic science ............................................................................................. 13
2. Materials and methods ..................................................................................................................... 15
2.1 Materials ...................................................................................................................................... 15
2.2 Methods ...................................................................................................................................... 15
2.2.1 Blood ..................................................................................................................................... 15
2.2.2 Saliva ..................................................................................................................................... 17
2.2.3 Semen ................................................................................................................................... 17
2.2.4 Fingermarks .......................................................................................................................... 17
3. Results and Discussion ...................................................................................................................... 19
3.1 Blood............................................................................................................................................ 19
3.1.1 Method optimization ............................................................................................................ 19
3.1.2 Examining aging of blood ..................................................................................................... 20
3.2 Saliva ............................................................................................................................................ 25
3.3 Semen .......................................................................................................................................... 30
3.4 Fingermarks ................................................................................................................................. 34
4. Conclusions and future research ....................................................................................................... 35
5. References ......................................................................................................................................... 36
Appendix 1. ............................................................................................................................................ 39
Appendix 2. ............................................................................................................................................ 44
Appendix 3. ............................................................................................................................................ 51
Appendix 4. ............................................................................................................................................ 57
Appendix 5. ............................................................................................................................................ 61
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Appendix 6. ............................................................................................................................................ 65
Appendix 7. ............................................................................................................................................ 70
Appendix 8. ............................................................................................................................................ 76
Appendix 9. ............................................................................................................................................ 82
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1. Introduction
orensic science is an enormously developing field nowadays. As an interdisciplinary study, it
utilizes the knowledge and technology from several fields, such as chemistry, physics, medicine
and mathematics to solve crimes. On a crime scene, investigators can find many forensically
important traces. Traces like: pieces of glass, hairs, fibers or paints are defined as physical traces. On
the other hand, body fluids, which can be commonly found on a crime scene as well, belong to the
group of biological traces. All traces found on a crime scene can play a crucial role during
investigations. They can provide information about the suspect/victim or what happened during the
criminal incident. However, this thesis will focus only on body fluids, therefore, only biological
evidence will be further described in details.
Examining biological evidence has a high importance for investigators as they can provide
intelligence about the donor (by DNA profiling) or about the crime of investigation. The latter one is
often studied by analyzing the bloodstain pattern, which can give the information about events that
occurred on the crime scene [1]. Moreover, by studying the aging of body fluids, it can be estimated
when a crime took place, which can narrow down the time intervals for detectives. To date, several
attempts have been performed to study body fluids aging by using mainly spectroscopic [2, 3] or
chromatographic methods [4, 5]. However, these studies did not provide with sufficient information
about the aging process. Therefore, the aim of this research was to use mass spectrometry
technique, suitable for trace analysis, to study the aging of body fluids.
1.1 Forensic biological traces
1.1.1 Blood
This body fluid is composed of blood cells (red cells, white cells and platelets) suspended in blood
plasma. The red color of blood is due to an iron-containing protein – hemoglobin, which its main
function is to transport the oxygen from lungs to tissues[6]. As blood contains proteins, lipids,
electrolytes, metabolites, hormones and more [6], this fluid is the most extensively studied, not only
in medical fields. In forensics, studying blood traces has various purposes. Besides obtaining DNA
profile, or studying blood stain pattern [1], also aging of this body fluid has been examined.
The idea to study blood aging is not new. Many attempts were made to investigate aging process by
studying different blood components with various methods, for example RNA with reverse
transcription polymerase chain reaction (RT-PCR) [7], erythrocytes with electron paramagnetic
resonance (EPR) [3] or globulins with immunoelectrophoresis [8]. More recent, three hemoglobin
derivatives: oxy-hemoglobin (oxy-Hb), methemoglobin (met-Hb) and hemichrome (HC) were studied
[2, 9, 10]. As the fractions of these derivatives are changing in time (fraction of oxy-Hb decreases and
met-Hb and HC increase), Bremmer et al.[2] applied reflectance spectroscopy to record spectra of
blood stains and calculated these fractions. They presented the potential of this method, as they
were able to discriminate age of blood stains with larger time intervals (e.g. 20 days). Edelman et al.
applied hyperspectral imaging to record the spectra of the described hemoglobin derivatives and
F
8
observed a decreased error compared to reflectance spectroscopy [10]. However, Bremmer et al. [9]
showed that conversion of oxy-Hb to met-Hb and HC is dependent on temperature and humidity.
Moreover, both methods were successful only when spectra were recorded against a white
background.
1.1.2 Saliva
This very essential body fluid has many functions, but the most important are: food solubilization,
lubrication, antibacterial, buffering and food digestion. Saliva is produced by salivary glands and it
contains water, electrolytes, proteins and other organic molecules [11]. Its chemical composition has
been studied widely, mostly for cancer [12], autoimmune diseases [13], hereditary diseases [14], or
viral diseases [15]. Saliva can also be used for drug analysis [16].
On a crime scene, this oral body fluid can be found for instance on food or cigarettes. In forensic
science, it is mainly studied for DNA profiling and aging studies have never been reported before.
Nevertheless, some study has shown that when saliva is in ex situ conditions [17], proteins can
degrade over time. This information is of importance and can indicate that saliva proteins have
potential to be used as a biomarker for age determination of saliva.
1.1.3 Semen
The main composition of semen is seminal fluid which may contain spermatozoa [18]. This fluid
contains high concentration of sugars (fructose and glucose) and proteins [19]. As study showed, in a
single individual seminal fluid, over 900 proteins can be identified [20] which can suggest that human
seminal fluid proteome can be used for biomarker studies, for example in cancer [21] or infertility
research[22]. In forensic science, this biological evidence can be found on a crime scene in a case of
sexual assaults. Semen has a high evidential importance as from this fluid donor DNA profile can be
obtained. For identification purposes, forensic investigators utilize the proteins’ natural fluorescence
from semen. However, there is yet no literature published to use this biomolecules in age
determination of seminal fluid.
1.1.4 Fingermarks
This type of traces is often found a crime scene. The chemical composition of fingermarks is diverse.
They contain elements (e.g. sodium, potassium, chloride), amino acids, proteins, fatty acids and
other lipids [23]. Forensic scientists study fingermarks to obtain as much information as possible
about the donors. For instance, from analysis of fingermarks information about the sex [24] or drug
abused [25] can be obtained. Similar to the other body fluids, the knowledge of the age of
fingermarks can be of significant importance for investigators. Wolstenholme et al. [26] attempted to
determine the age of fingermarks by investigating the degradation of olenaic acid. The other
research group, van Dam et al. [27], showed that the fluorescence signal is changing with the age of
the sample. However, the estimation error of aged samples was relatively high - only 55% of aged
male fingermarks estimated within the error of 1.9 days.
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1.2 Mass spectrometry
Mass spectrometry (MS) is a widely used method in analytical chemistry. Its very intense
development can be proved by five Nobel Prizes, starting from 1906 with J.J.Thomson for his work on
the electrical conductivity of gases. Subsequently, other prizes were for isotope discovery by mass
spectrograph, for cyclotron and for the development of the ion trap technique. The last Nobel Prize
in mass spectrometry, until now, was awarded to J. Bennet and K. Tanaka in 2002. The first one for
developing soft desorption ionization methods, and the second one for developing a new method for
mass spectrometric analyses of biological macromolecules; both extremely important in protein
analysis. Mass spectrometry, as a versatile technique, has found its application in various disciplines,
such as toxicology, forensic science, clinical or environmental chemistry. Its main advantage is high
sensitivity [28], which can be critical, especially, in forensic science for analyzing biological traces,
which often are found on the crime scene in scarce or mixed.
1.2.1 Proteomics
Proteomics is the field of science which studies the biological processes in organisms by protein
analysis. A proteome is the entire protein complement expressed by a genome. However, in contrast
to genes, the nature of proteins is not static but dynamic. Proteins can undergo modifications
(phosphorylation, oxidation), their abundance can change or they can form clusters. Those variations
are dependent on the physiological state of the organism. Therefore, the proteome reflects the state
of a cell or a tissue, and it is crucial to not only identify all the proteins but also to measure their
changes as well as pathways of change[29].
Protein analysis is a challenging task, as proteome is not only very complex because of all interactions
and modifications described before, but also, compared to genes, proteins cannot undergo
amplifications, which is the limiting factor when a sample amount is scarce. Hence, only appropriate
tools (i.e. high sensitivity) can be applied in proteomics, which makes mass spectrometry a suitable
method.
In proteomics, there are two ionization methods in widespread use: matrix-assisted laser
desorption/ionization (MALDI) and electrospray ionization (ESI). Both of them can generate ions from
large nonvolatile biomolecules. MALDI and ESI are knows as soft ionization methods, which means
that there is a minimal or no fragmentation of the analytes. The first one is mostly used for peptide
mass fingerprinting or tissue imaging. In MALDI, sample is mixed with a matrix, which molecules have
a strong absorption at the applied laser wavelength. The absorbed energy is transferred to the
analyte, which is desorbed and ionized together with matrix molecules. This solid-phase technique
mostly generates singly charged ions. Electrospray ionization is a liquid technique, which is easy to
couple to such separation methods as liquid chromatography or electrophoresis. Here, a high voltage
is applied, which generates highly charged droplets of the analyte. In contrast to MALDI, ESI
generates multiply charged ions [28].
The typical workflow in proteomics for LC-MS/MS analysis is presented on the Fig. 1. Briefly, proteins
in a sample undergo digestion into peptides, mostly by using enzyme digestion, trypsin for example.
Those peptides prior to MS/MS analysis are separated with reverse-phase liquid chromatography. In
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MS/MS analysis, the precursor ions as well as product ions are scanned, which provides crucial
information about the structure of a sample. After the analysis, by using appropriate software,
protein and peptide identification can be obtained.
Figure 1. Typical workflow in proteomics for LC-MS/MS analysis. Adapted from reference [30].
The instrument used in this project was TripleTOF mass spectrometer. The scheme below presents
the features in this instrument. Briefly, QJet and Q0 are used for ion focusing, Q1 selects the
precursor ion and Q2 is the collision cell, where the ions collide with neutral gas molecules
(nitrogen). That formed product ions leave the collision cell and are re-accelerated and focused by
ions optics into a parallel beam which constantly enters TOF analyzer. There, a pulsed electric field is
pushing ions orthogonally to their previous direction, and then they acquire their final energy [31].
The accelerated ions travel through the tube, where at the opposite site is an electrostatic ion mirror
which reverses the ions direction to the detector. Ions gain the same kinetic energy, however, their
velocities are different, due to the mass differences. And consequently, ions reach the detector at
different time.
MS1
MS2
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Figure 2. The scheme of triple TOF MS features. (a) The major platform features in details. (b) an image of this platform[32].
1.2.2 Quantitation in proteomics
Nowadays, only protein identification is no longer sufficient in proteomics studies. In order to gain
more knowledge about living organisms, tissues or cells, it is important to quantify the proteins. Their
abundances can change over a time or under specific conditions, such as disease for example. In
proteomics, there are two quantitative approaches, one which is based on labeling the sample and
the second one is label-free. Labeling techniques can be divided into three categories such as:
metabolic labeling, chemical labeling and addition of standard peptides. In those approaches, labeled
peptides have the same chemical properties as unlabeled ones, with the only difference in their
masses. In metabolic labeling, such as stable isotope labeling by amino acids in cell culture (SILAC) for
example, samples of “light” and “heavy” amino acids are mixed at early stage. In chemical labeling,
the label can be introduced at protein or peptide levels. One of commonly used chemical labeling is
the isotope-coded affinity tag (ICAT). This label is bound to sulfhydryl groups of cysteine residues and
is useful to study oxidation or reduction level of proteins. However, as cysteine belongs to low
abundant amino acids in proteins, it reduces the number of peptides that can be examined. The
other type of chemical labeling is isobaric mass tags. There are two tags commercially available:
tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)[33]. The
third technique of labeling approach in quantitative proteomics is by using synthetic isotope-labeled
peptides as a standard for absolute quantification (AQUA). These reference peptides allow to precise
measure the level of proteins and posttranslationally modified proteins. For AQUA, selected reaction
monitoring (SRM) analysis is performed [34].
In label-free approach, two quantitation techniques can be distinguished. First one is based on under
the curve (AUC) or signal intensity measurements, both based on precursor ion spectra. With these
methods chromatographic alignment is required. The second technique, spectral counting, is based
on counting the number of peptides assigned to a protein in MS/MS analysis. In spectral counting
methods, more abundant peptides are selected for fragmentation, and they produce a higher
abundance of MS/MS spectra. This process is proportional to protein amount in data-dependent
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acquisition. In order to assess protein abundance in the analyzed sample, protein abundance index
(PAI) is used. This index is defined as the number of identified peptides divided by the number of
observable generated peptides for every protein within a preferred mass range [35]. In order to
obtain absolute quantitation, PAI can be converted into exponentially modified emPAI, which is
equally to exponential form of PAI minus one, and this is proportional to protein content in analyzed
samples [36]. As in spectral counting, longer proteins generate more peptides than smaller ones, this
also can be used for quantification. In this case, normalized spectral abundance factor (NSAF) is used
which takes into account the length of the protein. In order to calculate NSAF, the number of spectral
counts for protein is divided by its length and this is defined as spectral abundance factor (SAF),
which has to be normalized by division by the sum of all SAFs for all proteins in a mixture [37].
Before applying a specific quantitation method, it is essential to consider which parameters are
crucial in the performed analysis. For example, it has to be determined what proteome coverage is
desired in the analyzed sample. This and other parameters for quantitative methods are overviewed
in the table below.
Table 1. Overview of quantitation methods applied in proteomics [33].
Method Proteome coverage Quantitative accuracy Quantitation type
Metabolic labeling
SILAC Medium Precise Relative
Chemical labeling
ICAT Poor Precise Relative
iTRAQ, TMT Medium Medium Relative
Standard peptide Poor Precise Relative/Absolute
Label-free
Ion intensities (PCP) Good Medium Relative
Spectrum count Good Poor Relative
Derived indices (APEX, emPAI)
Good Poor Relative/Absolute
Figure 3 shows that from all proteins in a sample, only a certain amount can be identified.
Furthermore, from this amount, even a smaller fraction can be successfully quantified. It emphasizes
how challenging protein analysis is and how important it is to perform appropriate sample
preparation with suitable enrichment and purification steps. Additionally, it also indicates how much
of the proteome is still unknown.
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Figure 3. Graphical representation of the amount of proteins that can be identified relative to all proteins in a sample [33].
1.2.3 Proteomics in forensic science
In forensic science it is very important to identify as many traces as possible which can be found on a
crime scene. The information of the trace’s origin can facilitate to reconstruct what has happened on
a crime scene. Especially, very crucial is the analysis of biological matrices as they can contain DNA,
which can have a huge value for investigators. However, as mostly those matrices are the mixtures of
several body fluids, such analysis is a complicated process. To identify such mixtures, an advanced
and sensitive method, like mass spectrometry, is needed. With this method, such body fluids as
blood, saliva, semen, vaginal fluids, urine and nasal secretions can be identified by a protein-specific
biomarker, which is unique for every body fluid [38, 39]. Moreover, analyzing biological traces with
MS is not destructive for DNA, which is an another advantage [40]. Hence, identification of the
source of a biological trace can be performed prior to DNA analysis.
The other approach of applying proteomics in forensic field is investigating organ-specific proteome.
This studies are especially useful in a case of ballistic reconstruction of the event [41] as the
information about which organ was damaged with the bullet can help to reconstruct the bullet’s
projectile.
Table 2. Organ-specific proteins [41].
Organ Protein
Heart
myosin binding protein C,cardiac-type glycogen phosphorylase, brain form troponin I, cardiac muscle myosin light chain 3 pyruvate dehydrogenase E1 component subunit beta, mitochondrial calpastatin NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 cytochrome c oxidase subunit 4 isoform 1, mitochondrial
Kidney
calbindin Na(+)/H(+) exchange regulatory cofactor NHE-RF3 low-density lipoprotein receptor-related protein 2 villin 1 retinyl ester hydrolase type 1 membrane metalloendopeptidase variant 2 phosphotriesteraserelated protein plastin-1
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Liver
hydroxymethylglutaryl- CoA synthase, mitochondrial carbamoyl-phosphate synthase [ammonia], mitochondrial phenylalanine-4- hydroxylase 3-oxo-5-beta-steroid 4-dehydrogenase catechol Omethyltransferase acetyl-CoA acetyltransferase cytochrome P450 2E1 dimethylaniline monooxygenase
Lung
plastin-2 cathelicidin-4 tubulin beta chain cysteine and glycine-rich protein 1 prostaglandin F synthase 2 myosin light polypeptide 6 calpain-2 catalytic subunit alpha-actinin-1
Muscle
bridging integrator 1 myosin-binding protein C, slow-type fructose-1,6- bisphosphatase isozyme 2 myosin-binding protein C, fast-type troponin C, skeletal muscle (fast type) myosin regulatory light chain 2, skeletal muscle isoform nebulin PDZ and LIM domain protein 3
As it can be seen, protein studies by mass spectrometry are common used in forensic science. It can
provide with very substantial information about the donor of a sample, or about the circumstances
that the crime happened. However, one of the very important information obtained from body fluids
has not been yet investigated using mass spectrometry – age determination of body fluids by protein
analysis. This thesis presents a novel approach of studying the aging process with LC-MS/MS method.
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2. Materials and methods
2.1 Materials
All samples were taken from healthy donors and kept in the dark at room temperature.
Chemicals:
Acetonitrile, methanol, water, formic acid (Biosolve)
Ammonium bicarbonate, DTT, iodoacetamide, Tris(2-carboxyethyl)phosphine hydrochloride, sodium
deoxycholate, Brilliant Blue G (Sigma Aldrich)
Sequencing Grade Modified Trypsin (Promega)
4-20% Tris-glycine precast gel, Tricine-Glycine SDS Running Buffer (Novex)
4-15% Tris-HCl precast gel (Biorad)
Apparatus:
Samples were collected on: Human ID Bloodstain Card; glass microfibers filter (Whatman) or into 2.0
ml Eppendorf vials.
Solid-phase extraction was performed with C18 solid-phase extraction cartridges, 4mm/1mL
(Empore).
LC system consist of nanoLC 425; separation column C18, 3µm, 120Å, 0.075x150mm, and trap
column C18, 3µm, 120Å, 350µmx0.5mm (Eksigent). Flow rate: 300nL/minute, injection volume 1-5
µL.
MS/MS analysis was performed with Triple TOF 5600+ (ABSciex) in positive mode. TOF m/z range:
400-1250, product ion m/z range 100-2000.
2.2 Methods
2.2.1 Blood
Dried blood spots were prepared by spotting 15µl of blood on Whatman filter paper. Samples
preparation for LC-MS/MS analysis was performed according to the protocol described by Chambers,
A.G., et al. [42]. Briefly, dried blood spots were excised from the filter paper and transferred to 1.5 ml
Eppendorf tubes. Fresh sample (15µl) was pipetted directly to the vial. In order to dissolve dried
blood spots and dilute fresh blood, 800µl of 25mM ammonium bicarbonate was added to the vials
and vortexed. Subsequently, 100 µl of 10% sodium deoxycholate and 10 µl of 0.5 M Tris(2-
carboxyethyl)phosphine hydrochloride were added and samples were incubated for an hour at 60°C.
Then, samples were alkylated for one hour in the dark (covered with aluminum foil) in room
temperature after addition of 52.63µl 200mM iodoacetamide. In order to consume remain
iodoacetamide, 55.4µl of 200mM dithiotreitol (DTT) was added and samples were incubated for 30
minutes at 37°C. Next, 10 µl of trypsin solution was added and protein digestion was performed
16
overnight at 37°C. Protein concentration in samples, before digestion, was estimated with Bradford
assay (Biorad). After digestion, sodium deoxycholate was precipitated by adding 40 µl of 2% formic
acid and the samples were centrifuged at 4000 g for 20 minutes. The supernatant was collected into
the new vials. Prior to LC-MS/MS analysis, samples were desalted by using solid-phase extraction.
Cartridges were first pre-washed twice with 50% ACN/water and 0.1% TFA. Afterwards, samples
were loaded and washed with 0.1% TFA. Analytes were eluted with 0.1% formic acid in 60% ACN.
The collected samples were, subsequently, evaporated to dryness and peptides were reconstituted
with water to obtain a final protein concentration of 0.2µg/µl.
In order to obtain the highest number of protein identification, three different LC methods were first
tested with fresh blood samples. For all methods, the same chemicals were used; mobile phase A:
0.1% formic acid, mobile phase B: 100% acetonitrile.
Table 3. Gradient profile for method 1 [43].
Time [min] Mobile phase A% Mobile phase B%
0 97.3 2.7
1.5 93.7 6.3
16 86.5 13.5
18 86.2 13.8
33 77.5 22.5
38 59.5 40.5
39 19 81
42.9 19 81
43 97.3 2.7
Table 4. Gradient profile for method 2.
Time [min] Mobile phase A% Mobile phase B%
0 100 0
10 95 5
40 40 60
45 20 80
60 100 0
Table 5. Gradient profile for method 3 [42].
Time [min] Mobile phase A% Mobile phase B%
0 95 5
90 55 45
92 10 90
94 10 90
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2.2.2 Saliva
Prior to LC-MS/MS analysis, proteins in saliva samples were first separated with SDS-PAGE using
precast 4-15% Tris-HCl gel. 10 µl of saliva was mixed with 10 µl of reducing sample buffer and
incubated for 5 minutes in 95°C. Then samples were applied to the gel lanes (20 µl per lane). Samples
were run at a voltage of 100V for 75 minutes. Afterwards, proteins were stained with Coomassie blue
and the gel was destained with distilled water.
Selected bands were excised from the gel and in-gel digestion was performed. Firstly, Coomassie
blue staining was removed from gel bands by incubation with 200µl of 0.04M NH4HCO3/50% ACN.
Next, proteins in gel bands were reduced with 100µl of 20mM DTT for 30 minutes in 37°C.
Afterwards, 100µl of 55mM iodoacetamide was added to every sample for alkylation in room
temperature for 20 minutes. Afterwards, proteins were digested with 10µl of 0.02µg/µl of trypsin
solution for 20 minutes on ice and subsequently 20µl of 40mM ammonium bicarbonate/10% ACN
was added and digestion was performed overnight in 37°C. Next day, digested proteins were
extracted from the gel bands with first 60µl ACN for 15 minutes in 37°C and then with 150µl of
50%ACN/5%HCOOH also for 15 minutes at the same temperature. Finally, peptides were dried and
dissolved in 10 µl of water prior to LC-MS/MS analysis and 94 minutes method was used, the same as
for blood samples.
2.2.3 Semen
Semen samples were analyzed similarly to saliva samples described in section 2.2.2. Firstly, proteins
were separated with SDS-PAGE (however, in this case, samples were first diluted 21 times with
phosphate-buffered saline) and later in-gel digestion was performed according to the same protocol.
LC-MS/MS analysis of semen peptides was performed with 20 minutes method (see Table 6).
Table 6. Gradient profile for semen samples.
Time [min] Mobile phase A% Mobile phase B%
0 95 5
15 70 30
16 5 95
18 5 95
20 95 5
2.2.4 Fingermarks
For fingermark samples collection, donors (males and females) were asked to put their finger onto a
glass surface, without previous washing their hands, in order to obtain “realistic” conditions.
Deposited fingermarks were later swabbed with cotton swabs. Swabs were subsequently transferred
into Eppendorf vials, 900 µl of 25mM ammonium bicarbonate was added and samples were
incubated at 37°C for 30 minutes. Afterwards, swabs were removed and samples were centrifuged
for 15 minutes at 3000g. The supernatants were collected and in-solution digestion was performed
18
according to the same protocol as for blood samples. For LC-MS/MS analysis, 66 minutes method
was used (see Table 7).
Table 7. Gradient profile for fingermark samples.
Time [min] Mobile phase A% Mobile phase B%
0 90 10
60 0 100
65 0 100
65.5 90 10
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3. Results and Discussion
Due to problems with the instrumentation, which had occurred during this research project, not all
samples were analyzed.
This chapter presents the results obtained from analysis of aging body fluids by mass spectrometry.
3.1 Blood
3.1.1 Method optimization
Prior to blood samples analysis, LC-MS/MS conditions were initially optimized with fresh blood
samples. Three different gradients were tested and the number of identified proteins and peptides
was compared. The graphs below present the obtained results.
Figure 4. Number of proteins identified in fresh blood with three different methods.
Figure 5. Number of peptides identified in fresh blood with three different methods.
0
50
100
150
200
Proteins identified
43 minutes method 60 minutes method 94 minutes method
0
200
400
600
800
1000
1200
Peptides identified
43 minutes method 60 minutes method 94 minutes method
20
From the foregoing graphs can be derived that the highest number of identification was obtained by
using 94 minutes method. With this gradient, 179 proteins and 953 peptides were identified. The 60
minutes method provided a significant lower number of proteins and peptide, 117 and 748
respectively. Finally, application of the shortest gradient resulted in 167 identified proteins and 748
identified peptides.
3.1.2 Examining aging of blood
Blood samples were analyzed at three different ages: fresh blood sample (liquid), 1 day old (stain)
and 7 days old blood (stain) with LC-MS/MS. Subsequently, the number of proteins and peptides
from every sample were compared (see Table 8). All measurements were performed in triplicates
and data was generated using human thorough protein database with Protein PilotTM software using
false discovery rate (FDR) 1%.
Table 8. Number of proteins and peptides identified in aged blood samples, in triplicates.
Sample Proteins #1
Proteins #2
Proteins #3
Peptides #1
Peptides #2
Peptides #3
Fresh blood
115 131 139 1280 1712 1646
1 day old 119 130 128 1552 1522 1512
7 days old 129 110 111 1458 1369 1058
Coefficient of variation (CV) in every sample for proteins and peptides, respectively, is as follow: fresh
sample 9.5%, 15%; one day old sample 4.7%, 1.4%; and seven days old sample 9.1%, 16.2%.
In the next step, data was generated using other software. First, Search Gui 2.7.1, and subsequently,
Peptide Shaker 1.8.1. Two combined search engines were used: Open Mass Spectrometry Search
Algorithm (OMSSA) and MS-GF+. The used database for identification was Target-Decoy version
Homo sapiens protein database + common contaminants. Quality control filtering was performed
with 1% FDR. Data from three technical replicates were pooled together.
The number of identified proteins from all three samples was plotted and presented in the Figure 4.
The complete lists of identified proteins in blood samples (using Search Gui and Peptide Shaker) can
be found in Appendices 1-3.
21
Figure 6. Comparison of proteins in all three blood samples.
78 proteins were detected in every blood sample. Nonetheless, the number of unique proteins in
every sample is different. These number decreased from 14 proteins in fresh blood to 12 protein in
one day old sample and finally to 4 proteins in 7 days old blood.
Protein quantification was performed by using normalized spectrum abundance factor (NSAF).
10 proteins were found with alterations in NSAF values. 9 proteins have decreasing NSAF value (Fig.
7-15) and for one it is increasing (Fig. 16).
Figure 7. NSAF values for alpha-1 acid glycoprotein-2.
0
0,02
0,04
0,06
0,08
0,1
fresh 1 day old 7 days old
NS
AF
Alpha-1 acid glycoprotein-2
22
Figure 8. NSAF values for alpha-2-macroglobulin.
Figure 9. NSAF values for ankyrin-1.
Figure 10. NSAF values for apolipoprotein B-100.
0
0,005
0,01
0,015
0,02
0,025
0,03
0,035
fresh 1 day old 7 days old
NS
AF
Alpha-2-macroglobulin
0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
fresh 1 day old 7 days old
NS
AF
Ankyrin-1
0
0,0005
0,001
0,0015
0,002
0,0025
0,003
0,0035
fresh 1 day old 7 days old
NS
AF
Apolipoprotein B-100
23
Figure 11. NSAF values for band 3 anion transport protein.
Figure 12. NSAF values for Ig gamma-4 chain region.
Figure 13. NSAF values for haptoglobin.
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
fresh 1 day old 7 days old
NS
AF
Band 3 anion transport protein
0
0,01
0,02
0,03
0,04
0,05
0,06
fresh 1 day old 7 days old
NS
AF
Ig gamma-4 chain C region
0
0,002
0,004
0,006
0,008
0,01
fresh 1 day old 7 days old
NS
AF
Haptoglobin
24
Figure 14. NSAF values for hemopexin.
Figure 15. NSAF values for putative uncharacterized protein.
Figure 16. NSAF values for putative bisphosphoglycerate mutase.
From all aforementioned proteins, two characterize with significant changes in NSAF values:
apolipoprotein B-100 and haptoglobin. The first one is responsible for lipids transporting and
proteins and phospholipids binding (See Table 9). The latter one is accountable for capturing and
0
0,01
0,02
0,03
0,04
0,05
0,06
fresh 1 day old 7 days old
NS
AF
Hemopexin
0
0,02
0,04
0,06
0,08
0,1
fresh 1 day old 7 days old
NS
AF
Putative uncharacterized protein
0,019
0,02
0,021
0,022
0,023
0,024
0,025
0,026
fresh 1 day old 7 days old
NS
AF
Bisphosphoglycerate mutase
25
combining free plasma hemoglobin and it allows for hepatic cyclin of heme iron which prevents the
damage of kidneys.
For two proteins, alpha-1 acid glycoprotein-2 and putative uncharacterized proteins, the NSAF value
is decreasing notably during first 24 hours, however, posteriorly, this decline is less noticeably.
Alpha-1 acid glycoprotein-2 is transporting protein, however, the function of putative
uncharacterized proteins in humans is still unknown. In other mammals, mouse for example, it has a
regulation function of proteins activity.
Only bisphosphoglycerate mutase protein demonstrates an increasing trend in NSAF values over
examined time period. It can be explained by the function of this protein, to regulate hemoglobin
oxygen affinity, which is stronger with the increasing age of the sample.
Table 9. Functions of proteins with changing NSAF values. Information took from http://www.uniprot.org/.
Protein Function
Alpha-1 acid glycoprotein-2 Transporting protein; binding hydrophobic ligands and synthetic drugs
Alpha-2-macroglobulin Inhibiting proteinases by unique 'trapping' mechanism
Ankyrin-1 Attaching integral membrane proteins to cytoskeletal elements
Apolipoprotein B-100 Lipid transporter activity, binding proteins and phospholipids,
Band 3 anion transport protein Transporting protein, mediator in electroneutral anion exchange across the cell membrane; structural protein
Bisphosphoglycerate mutase regulating hemoglobin oxygen affinity, exhibiting mutase and phosphatase activities
Ig gamma-4 chain C region Antigen binding
Haptoglobin Capturing and combining with free plasma hemoglobin (preventing kidney damage)
Hemopexin Binding heme and transporting it to the liver for breakdown and iron recovery
Putative uncharacterized protein Unknown function in homo sapiens
3.2 Saliva
Saliva samples at ages of one day old, one week old and three weeks old were analyzed.
Firstly, proteins were separated with SDS-PAGE (Fig. 17). The arrows indicate from which part of the
gel bands were excised. Per sample, three bands were excised and each band was analyzed
separately. Afterwards, results from three bands were pooled together.
26
Figure 17. SDS-PAGE of saliva samples.
Data were generated according to the second step with blood analysis, by using First Search Gui 2.7.1
and Peptide Shaker 1.8.1 software. The number of all identified proteins in all three samples is
presented in the Venn diagram below. The all lists of proteins identified in saliva samples can be
found in Appendices 4-6.
Figure 18. Comparison of proteins in all three saliva samples.
39 proteins were present in every saliva sample, nonetheless, the number of unique proteins
detected is increasing with the age of saliva samples, from 9 unique proteins in one day old sample to
22 in three weeks old.
27
Similarly to blood samples, saliva samples also underwent quantification with NSAF. For eight
proteins, an alteration of NSAF was observed (See Fig. 19-26).
Figure 19. NSAF values for keratin, type I cytoskeletal 13.
Figure 20. NSAF values for keratin, type I cytoskeletal 17.
Figure 21. NSAF values for keratin, type II cytoskeletal 2 epidermal.
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
one day one week 3 weeks
NS
AF
Keratin, type I cytoskeletal 13
0
0,005
0,01
0,015
0,02
0,025
one day one week 3 weeks
NS
AF
Keratin, type I cytoskeletal 17
0
0,005
0,01
0,015
0,02
0,025
one day one week 3 weeks
NS
AF
Keratin, type II cytoskeletal 2 epidermal
28
Figure 22. NSAF values for keratin, type II cytoskeletal 3.
Figure 23. NSAF values for keratin, type II cytoskeletal 6A.
Figure 24. NSAF values for lactotransferrin.
0
0,01
0,02
0,03
0,04
0,05
one day one week 3 weeks
NS
AF
Keratin, type II cytoskeletal 3
0
0,005
0,01
0,015
0,02
0,025
0,03
0,035
0,04
one day one week 3 weeks
NS
AF
Keratin, type II cytoskeletal 6A
0
0,002
0,004
0,006
0,008
0,01
0,012
one day one week 3 weeks
NS
AF
Lactotransferrin
29
Figure 25. NSAF values for leukocyte elastase inhibitor.
Figure 26. NSAF values for tubulin alpha-4A chain.
Interestingly, in this case, there are more proteins in which NSAF values were increasing with the age
of the sample. Moreover, most of proteins with variation in NSAF are keratin type proteins. The most
significant changes occur for keratin, type I cytoskeletal 13 with upward trend and for keratin, type II
3 with the downward trend. Furthermore, those two proteins have similar function: structural
molecule activity. The other two proteins with considerable alterations are leukocyte elastase
inhibitor and tubulin alpha-4A chain. The first one is responsible for the activity of neutrophil
proteases elastase, and the latter one for GTPase activity and GTP binding.
Table 10. Functions of proteins with changing NSAF values. Information took from http://www.uniprot.org/.
Protein Function
Keratin, type I cytoskeletal 13 Structural molecule activity; cellular response to retinoic acid; tongue morphogenesis
Keratin, type I cytoskeletal 17 Structural molecule activity
Keratin, type II cytoskeletal 2 epidermal Associated with keratinocyte activation; proliferation and keratinization
Keratin, type II cytoskeletal 3 Structural molecule activity; intermediate filament cytoskeleton organization
0
0,01
0,02
0,03
0,04
0,05
one day one week 3 weeks
NS
AF
Leukocyte elastase inhibitor
0
0,005
0,01
0,015
0,02
0,025
0,03
0,035
one day one week 3 weeks
NS
AF
Tubulin alpha-4A chain
30
Keratin, type II cytoskeletal 6A Involved in the activation of follicular keratinocytes after wounding
Lactotransferrin Iron ion binding
Leukocyte elastase inhibitor Regulating the activity of the neutrophil proteases elastase
Tubulin alpha-4A chain GTPase activity, GTP binding
3.3 Semen
Semen samples (fresh, one week old and two weeks old) were analyzed according to the same
protocol as used for saliva samples. Proteins were first separated with SDS-PAGE, then 7 bands per
lane were excised and analyzed separately. At the end, results were pooled together for every
sample.
Figure 27. SDS-PAGE of semen samples.
31
The Venn diagram below depicts the identified proteins in all semen samples. The lists of all
identified proteins in semen samples can be found in Appendices 7-9.
Figure 28. Comparison of proteins in all three semen samples.
In the case of this body fluid, 41 common proteins for all aging samples were found, however, the
number of the unique proteins is variable. In fresh samples 36 unique proteins were identified. In 7
days old samples this number increased to 37. Nevertheless, this number decreased to only 11
unique proteins in two weeks old samples.
Proteins in all semen samples were quantified with NSAF and 6 proteins showed variations in NSAF
values (Fig. 29-34).
Figure 29. NSAF values for clusterin.
0
0,005
0,01
0,015
0,02
0,025
fresh one week two weeks
NS
AF
Clusterin
32
Figure 30. NSAF values for dermcidin.
Figure 31. NSAF values for keratin, type I cytoskeletal 10.
Figure 32. NSAF values for keratin, type II cytoskeletal 6A.
0
0,05
0,1
0,15
0,2
fresh one week two weeks
NS
AF
Dermcidin
0
0,01
0,02
0,03
0,04
0,05
0,06
fresh one week two weeks
NS
AF
Keratin, type I cytoskeletal 10
0
0,002
0,004
0,006
0,008
0,01
0,012
fresh one week two weeks
NS
AF
Keratin, type II cytoskeletal 6A
33
Figure 33. NSAF values for semenogelin-2.
Figure 34. NSAF values for serum albumin.
Keratin proteins are also present in semen samples with the downward or upward trend for type I
and type II, respectively. However, there are two proteins which NSAF is changing significantly. First
one, dermcidin, with upward trend, has antimicrobial properties. The second one, with reducing
NSAF, is semenogelin-2, which is involved in gel matrix formation. This result can indicate why the
older semen is more liquid then the fresh one.
Table 11. Functions of proteins with changing NSAF values. Information took from http://www.uniprot.org/.
Protein Function
Clusterin Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones
Dermcidin
Displays antimicrobial activity; exhibits proteolytic activity
Keratin, type I cytoskeletal 10
Cellular response to calcium ion
Keratin, type II cytoskeletal 6A Involved in the activation of follicular keratinocytes after wounding
Semenogelin-2
Participates in the formation of a gel matrix
0
0,005
0,01
0,015
0,02
0,025
fresh one week two weeks
NS
AF
Semenogelin-2
0
0,005
0,01
0,015
0,02
0,025
0,03
0,035
fresh one week two weeks
NS
AF
Serum albumin
34
Serum albumin
Transporting
3.4 Fingermarks
7 female (2 donors) and 6 male (one donor) fingermarks samples were analyzed at different age:
fresh (only female), one day old, 10 days old and 17 days old. Results were generated with Protein
Pilot software. Table 12 presents proteins identified in each sample.
Table 12. Proteins identified in every fingermark sample.
Sample Proteins
Fresh female fingermark keratin, type I cytoskeletal (9, 10, 14, 16); keratin, type II cytoskeletal (1, 1b, 2 epidermal, 5, 6A, 78); dermcidin; keratinocyte proline-rich protein; filaggrin-2; skin-specific protein 32; loricrin; junction plakoglobin
One day old female fingermark keratin, type II cytoskeletal 1
One day old male fingermark keratin, type I cytoskeletal (9, 10, 13, 14, 15, 16, 17); keratin, type II cytoskeletal (1, 2 epidermal); charged multivesicular body protein 2a; serum albumin; phosphofurin acidic cluster sorting protein 1
10 days old female fingermark Keratin, type I cytoskeletal 9; keratin, type II cytoskeletal (1, 2 epidermal, 5, 6A, 73); apoptosis-enhancing nuclease;
10 days old male fingermark Keratin, type I cytoskeletal 9; keratin, type II cytoskeletal (1, 2 epidermal); sodium/potassium-transporting ATPase subunit alpha-4; dermcidin
17 days old female fingermark Keratin, type I cytoskeletal (9, 10, 14, 16, 17); keratin, type II cytoskeletal (1, 2 epidermal, 5); dermcidin
17 days old male fingermark Keratin, type I cytoskeletal 10; prolactin-inducible protein
All samples contain keratin proteins, which is a protein found in skin cells. Interestingly, some
samples contain proteins which were not found in other fingermarks. This can suggest that those
proteins originate from contamination, as the donors deposited their fingermarks directly on the
glass slides, without previous finger preparation.
The results from fingermark samples could not be quantified as the signal to noise ratio in the
spectra was too low.
35
4. Conclusions and future research
For the first time mass spectrometry method was applied to study the aging of blood, saliva, semen
and fingerprints. In this research, protein changes were investigated, which occurs when the body
fluid is aging. It was shown that for some proteins, their concentration is increasing with time, for
example biphosphoglycerate mutase in blood, or keratins, type II in saliva. There were also several
proteins with decreasing concentration, such as apolipoprotein B-100 or semengelin-2. In the present
study, it was observed that for saliva, more proteins increased in concentration in comparison to
blood and semen. With regard to fingermarks samples, in this case, different extraction method is
required to obtain enough protein concentration that can be measured. This work presented the
potential of mass spectrometry technique for age determination of four body fluids commonly found
on a crime scene, although more research is needed in this area.
For future research, it is recommended to broaden the range of analyzed samples. In the case of
investigating proper biomarkers, it is important to expand studies to several donors from both sexes
with a wider range of age. Additionally, in forensic science it is significant to investigate fluids on
different substrates. Also, environmental conditions are crucial, for example how proteins undergo
degradation at different temperatures and humidity levels. Furthermore, label-free quantification
methods used in this study, can also suggest that more accurate methods should be applied.
36
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39
The data presented in appendixes were generated by Search Gui 2.7.1 and Peptide Shaker 1.8.1.
Note that some proteins are identified more than one time per sample. emPAI - exponentially
modified protein abundance index; NSAF - normalized spectrum abundance factor; MW – molecular
weight.
Appendix 1. All validated proteins in fresh blood sample.
Accession
Protein Inference
Class
Protein name
Sequence Coverage
(%)
#Validated
Peptides
emPAI
NSAF
MW (kDa)
P01024 Unrelated Proteins
Complement C3
40.88995790739627
44 0.714125253
2913134 0.01152003971
2574301 187.0298753
1966258
F6KPG5 Single
Protein Albumin
(Fragment) 76.7521367
5213675 56
4.115178099293143
0.0685492934053836
66.48798147102801
P01023 Single
Protein Alpha-2-
macroglobulin 47.354138398914515
44 1.249054605
8357808 0.02915375633
5619455 163.1878914
6924135
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
17.501169864295743
26 0.249264143
7899512 0.00369787553
21113903 246.3157334
321971
A0A087WZE4
Related Proteins
Spectrin alpha chain,
erythrocytic 1
15.720164609053498
23 0.177572498
21932586 0.00339278233
15763428 280.9387841
720546
A0A087WTM7
Related and
Unrelated Proteins
Apolipoprotein B-100
6.883057090239411
18 0.091578487
87739664 0.00315395313
4692732 489.5273949
3234196
C8C504 Single
Protein Beta-globin
95.91836734693877
23 33.14548873
833603 1.08852418504
30756 15.98734254
8192922
P02730
Related and
Unrelated Proteins
Band 3 anion transport protein
24.259055982436884
15 0.674482438
0115348 0.05793226863
3375625 101.7274121
8512044
P02042 Related Proteins
Hemoglobin subunit delta
93.19727891156462
18 14.84893192
4611133 0.45956003636
3566 16.04528986
0997062
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
21 0.790655529
6923772 0.15593423608
704202 69.18136901
532891
A0A0K0K1H8
Related and
Unrelated Proteins
Epididymis secretory
sperm binding protein Li 71p
33.3810888252149
19 0.739828052
9303642 0.04192341630
458897 77.02963611
452142
P02787
Related and
Unrelated Proteins
Serotransferrin
32.5214899713467
19 0.739828052
9303642 0.02566325370
2962904 77.01362996
651181
P02787
Related and
Unrelated Proteins
Serotransferrin
32.95128939828081
19 0.739828052
9303642 0.02241122118
2637716 77.01362996
651181
P02671 Single
Protein Fibrinogen alpha chain
28.521939953810627
17 0.552427173
4347945 0.02193464511
4181275 94.91441497
589778
P04040 Related Proteins
Catalase 35.863377609108156
13 0.690714103
4735806 0.03718063723
218359 59.71875531
355211
P16157 Single
Protein Ankyrin-1
13.131313131313131
15 0.207722655
13446264 0.00599593074
032388 206.1369244
395263
P02675 Unrelated Proteins
Fibrinogen beta chain
50.30549898167006
16 0.887391822
1350972 0.04582730151
411469 55.89226078
115152
A0A0G2JL Related Complement 12.0141342 12 0.174268583 0.00282639279 187.5979404
40
54 Proteins C4-B 75618375 45092833 24914566 9222188
I1VZV6 Single
Protein Hemoglobin
alpha 1 92.9577464
7887323 15
13.251026703029977
2.749347740393739
15.269957551186833
P02647 Related Proteins
Apolipoprotein A-I
55.430711610486895
17 1.801356761
1988681 0.15414244973
89475 30.75893250
0112536
Q3LR79 Single
Protein Hemoglobin
beta 93.3333333
3333333 15
30.622776601683793
1.0646784733306542
11.474842789202496
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
40.19138755980861
13 1.198392648
8622894 0.13958117324
59462 46.67898813
8410034
Q9BWU5 Single
Protein
Mutant hemoglobin beta chain (Fragment)
85.71428571428571
13 14.19911082
9529339 1.16773237162
46961 11.49394786
3407726
D9YZU8 Single
Protein Hemoglobin,
gamma A 85.7142857
1428571 12
5.309573444801933
0.24368578322660492
16.11827504126487
C9JC84 Related Proteins
Fibrinogen gamma chain
33.839479392624725
10 0.687612475
7881477 0.05015322108
597598 52.30432646
809949
P00918
Related and
Unrelated Proteins
Carbonic anhydrase 2
48.07692307692308
9 1.154434690
0318838 0.08711270534
414638 29.22792218
6630536
Q4TZM4 Single
Protein
Hemoglobin beta chain (Fragment)
88.11881188118812
12 14.84893192
4611133 1.94561481731
5928 11.01563886
7792866
P02774 Unrelated Proteins
Vitamin D-binding protein
36.91983122362869
9 0.429461338
35684486 0.01950723327
3056056 52.92902967
385641
P02790 Single
Protein Hemopexin
36.58008658008658
11 0.778279410
0389228 0.05063651626
151628 51.64327297
890149
A0A075B6N9
Related Proteins
Ig mu chain C region
(Fragment)
31.346578366445915
11 0.947483039
9087562 0.03887085137
085134 49.40858023
156175
P30041 Unrelated Proteins
Peroxiredoxin-6
62.05357142857142
10 1.154434690
0318838 0.04061968537
4149665 25.01919029
056794
P06727 Single
Protein Apolipoprotei
n A-IV 15.151515151515152
5 0.242623671
9115049 0.01489071038
2513661 45.37146664
308862
P07195 Related Proteins
L-lactate dehydrogenas
e B chain
29.34131736526946
7 0.606506003
8537282 0.01930001682
6518603 36.61514296
895986
A0A087WU08
Related Proteins
Haptoglobin 39.8576512
455516 9
0.9952623149688795
0.035842008868324615
31.3878684863898
P04114 Related Proteins
Apolipoprotein B-100
2.4326101249178174
7 0.033029926
90742457 0.00166372245
1468622 515.2829109
17923
B7ZKJ8
Related and
Unrelated Proteins
ITIH4 protein 8.55614973
262032 5
0.13977251980662686
0.006619537275064268
103.81622401008842
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
3.0416471689284044
5 0.043728062944320634
0.0013099547156802832
246.3157334321971
P32119
Related and
Unrelated Proteins
Peroxiredoxin-2
49.494949494949495
10 1.993577294
7204895 0.16687101854
784764 21.87823782
783635
Q6MZU6
Related and
Unrelated Proteins
Putative uncharacteriz
ed protein DKFZp686C15
213
27.586206896551726
9 0.701254279
8525889 0.07749920340
380885 51.06619379
122771
A0A075B6N8
Related and
Ig gamma-3 chain C region
34.48275862068966
9 0.750827031
7357244 0.02486747170
9576934 41.29932388
898452
41
Unrelated Proteins
(Fragment)
O95408 Single
Protein Beta globin (Fragment)
71.9298245614035
8 99.0 1.61262328095
1787 6.262154763
39843
Q6J1Z9 Single
Protein
Hemoglobin alpha 1
(Fragment) 90.0 9 9.0
1.2764004083448635
9.585181362001366
Q53GY3
Related and
Unrelated Proteins
Angiotensinogen variant (Fragment)
25.56701030927835
8 0.692666615
037876 0.03076587301
587301 53.05051576
500968
E9PFZ2
Related and
Unrelated Proteins
Ceruloplasmin 8.87949260
0422834 5
0.1274137659327852
0.003986810551558753
108.75141618854433
P00491 Related Proteins
Purine nucleoside
phosphorylase
35.6401384083045
6 0.637893706
954064 0.02866205305
6516718 32.09715605
245075
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
25.10729613733906
8 0.567191080
3783076 0.10509241139
378137 51.12147771
750289
P30043 Unrelated Proteins
Flavin reductase (NADPH)
50.0 7 1.335721469
0901222 0.14358529819
694865 22.10542642
5215388
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
32.29461756373937
8 1.310129700
0831598 0.03864066547
120943 37.63063836
9583146
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
36.26062322946176
8 1.310129700
0831598 0.05537706714
484954 37.63063836
9583146
P01008 Related Proteins
Antithrombin-III
15.948275862068964
5 0.228247006
34692308 0.01217672413
7931035 52.56886431
029427
P07738 Unrelated Proteins
Bisphosphoglycerate mutase
24.324324324324326
4 0.321941148
46602906 0.02123552123
552123 29.98651036
8450627
P01042 Related Proteins
Kininogen-1 7.91925465
8385093 3
0.10529514112602167
0.009451795841209828
71.91215085210732
E9PFZ2
Related and
Unrelated Proteins
Ceruloplasmin 7.6109936575052854
5 0.127413765
9327852 0.00879296562
7498003 108.7514161
8854433
Q86YQ1 Single
Protein
Hemoglobin alpha-2
(Fragment)
87.91208791208791
6 4.623413251
903491 0.71365976578
52747 9.665182424
630526
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
31.443298969072163
7 1.448436746
8222272 0.18008751710
050394 21.38873696
621423
A0A0G2JPD4
Related and
Unrelated Proteins
Ig gamma-4 chain C region
(Fragment)
20.489296636085626
6 0.637893706
954064 0.05183673469
3877486 35.91786587
959916
A0A087WT59
Related Proteins
Transthyretin 38.3783783
7837838 5
1.2758459260747883
0.09604978354978352
20.132907042394372
P02787
Related and
Unrelated Proteins
Serotransferrin
9.025787965616045
6 0.191103133
28300688 0.03028455284
5528463 77.01362996
651181
A0A0C4D Related Haptoglobin 8.64553314 4 0.311133937 0.01055194805 38.42734787
42
GL8 Proteins 1210375 42156426 1948054 385203
G3V3A0
Related and
Unrelated Proteins
Alpha-1-antichymotry
psin
16.097560975609756
3 0.389495494
3731377 0.03170731707
317073 23.39110667
577842
P60174 Related Proteins
Triosephosphate isomerase
22.377622377622377
3 0.258925411
7941673 0.01923076923
076923 30.77169610
1193438
H0Y7Z1
Related and
Unrelated Proteins
Fibronectin (Fragment)
5.530371713508613
3 0.086787359
11863655 0.00180188504
89743114 120.6992441
0608232
Q86YQ4 Single
Protein
Alpha-1 globin
(Fragment)
90.9090909090909
5 3.216965034
2858223 0.77914141414
14315 9.476096235
395074
E5RFL2 Related Proteins
Carbonic anhydrase 1 (Fragment)
60.16949152542372
6 3.641588833
6127784 0.43644067796
61018 13.24825410
0518784
P02787
Related and
Unrelated Proteins
Serotransferrin
6.59025787965616
4 0.123654800
13875096 0.03523035230
352302 77.01362996
651181
P19652 Single
Protein Alpha-1-acid
glycoprotein 2 27.363184079601986
5 0.687612475
7881477 0.07718375693
835813 23.58763169
0019066
P02763 Related Proteins
Alpha-1-acid glycoprotein 1
33.33333333333333
6 0.930697728
8832501 0.06315513626
834378 23.49675931
597699
P13716 Related Proteins
Delta-aminolevulini
c acid dehydratase
22.12121212121212
5 0.417474162
9268053 0.02615384615
3846156 36.27141416
930379
P08603
Related and
Unrelated Proteins
Complement factor H
2.5995125913891144
2 0.037225095
40705721 0.00195694716
24266144 139.0046981
6836323
M0R009 Related Proteins
Alpha-1B-glycoprotein (Fragment)
10.130718954248366
2 0.185971012
33766966 0.00837520938
0234505 33.43379931
857088
P11171 Related Proteins
Protein 4.1 4.16666666
6666666 2
0.041223256048305235
0.003546099290780142
96.95728291972311
B2M1S7 Single
Protein
Beta-globin Showa
Yakushiji variant
(Fragment)
100.0 4 20.54434690
0318832 1.70899470899
47093 4.493394684
7851916
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
79.43925233644859
5 1.848035868
4358014 0.47472185135
7368 11.71166718
8377447
A0A087WV45
Related Proteins
Transthyretin 51.0791366
9064749 4
1.0309176209047357
0.13237410071942446
15.110588167739953
A0A087WU08
Related Proteins
Haptoglobin 14.9466192
1708185 4
0.35935639087852556
0.01361445571971888
31.3878684863898
E9PGN7 Related Proteins
Plasma protease C1
inhibitor
9.392265193370166
4 0.216483949
025851 0.00699440447
64188645 59.45455599
2296385
G3V595
Related and
Unrelated Proteins
Alpha-1-antichymotry
psin (Fragment)
15.081967213114755
3 0.258925411
7941673 0.01498127340
82397 34.60356994
9471684
Q13228 Related Proteins
Selenium-binding
protein 1
10.16949152542373
3 0.162030136
3862613 0.01059322033
8983049 52.35762502
132521
P02749 Related Beta-2- 19.4202898 4 0.311133937 0.01135265700 38.27265933
43
Proteins glycoprotein 1 55072465 42156426 4830919 588615
P04217 Related Proteins
Alpha-1B-glycoprotein
8.282828282828284
2 0.118872211
58742212 0.01318944844
1247004 54.21953944
303767
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
29.381443298969074
4 0.668100537
2000588 0.17207792207
792205 21.38873696
621423
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
15.979381443298967
4 0.668100537
2000588 0.17911255411
255414 21.38873696
621423
A0A0B4J231
Related and
Unrelated Proteins
Immunoglobulin lambda-
like polypeptide 5
32.093023255813954
4 0.623776739
1887217 0.06910112359
550558 23.13567037
3513683
C9JVG0 Related Proteins
Serotransferrin (Fragment)
26.865671641791046
4 1.030917620
9047357 0.13258426966
29213 14.68097224
2798342
E7EUT5 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e
25.76923076923077
4 0.425102670
3029981 0.05743243243
243244 27.85306080
5444024
A0A0A0MRQ5
Related and
Unrelated Proteins
Peroxiredoxin-1
29.896907216494846
3 0.873817422
8603839 0.04786450662
739325 10.66950712
5347321
C9JV77 Related Proteins
Alpha-2-HS-glycoprotein
21.467391304347828
4 0.389495494
3731377 0.03721374045
801526 39.38672460
1822
P02652 Related Proteins
Apolipoprotein A-II
19.0 3 0.778279410
0389228 0.065
11.16789735900159
H0YAS8 Related Proteins
Clusterin (Fragment)
28.57142857142857
2 0.258925411
7941673 0.05
16.166165820637204
E9PIT3 Related Proteins
Prothrombin 8.91938250
4288166 3
0.119902793272475
0.003569211669770329
65.36674524764112
P19827 Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H1
6.0373216245883645
3 0.082636733
87405435 0.00224791265
25369302 101.3256149
1857365
A0A024R6I7
Related and
Unrelated Proteins
Alpha-1-antitrypsin
10.76555023923445
3 0.199353946
2092342 0.10890151515
151522 46.67898813
8410034
P02652 Related Proteins
Apolipoprotein A-II
28.999999999999996
2 0.467799267
62206947 0.36666666666
666686 11.16789735
900159
P60709
Related and
Unrelated Proteins
Actin, cytoplasmic 1
17.333333333333336
3 0.211527658
62858836 0.022
41.709730139029176
P01009 Related Proteins
Alpha-1-antitrypsin
5.980861244019139
2 0.128837891
68468907 0.03219696969
69697 46.70701943
8538325
H0Y300 Related Proteins
Haptoglobin 8.82352941
1764707 3
0.16998910658906596
0.008426183844011143
49.07441753459848
E7EPV7 Related Proteins
Alpha-synuclein
31.30434782608696
2 0.359356390
87852556 0.06956521739
130435 11.76924943
3169955
E9PGN7 Related Proteins
Plasma protease C1
inhibitor
4.972375690607735
2 0.102943311
79161298 0.00419664268
5851319 59.45455599
2296385
A0A087WTE1
Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H2
4.338624338624339
3 0.073030940
52615622 0.00522252497
729337 106.5897031
5928195
P05109 Single Protein S100- 23.6559139 2 0.389495494 0.04838709677 10.82764922
44
Protein A8 78494624 3731377 419355 1794548
A2NYQ9 Single
Protein
Anti-folate binding protein
(Fragment)
31.666666666666664
3 0.778279410
0389228 0.01342592592
5925928 13.27443279
9882204
C9JV37 Related Proteins
Prothrombin (Fragment)
16.358024691358025
3 0.291549665
0148839 0.01378896882
4940049 35.90878157
243862
P55072 Unrelated Proteins
Transitional endoplasmic
reticulum ATPase
4.094292803970223
2 0.048113134154685655
0.004267425320056899
89.26571926128344
B7UCU6
Related and
Unrelated Proteins
Gamma-globin chain (Fragment)
19.047619047619047
2 0.519911082
9529336 0.64588744588
74546 11.57887621
4589096
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
6.866952789699571
2 0.118872211
58742212 0.03789954337
8995426 51.12147771
750289
A0A075B6P5
Related and
Unrelated Proteins
Protein IGKV2-28
(Fragment)
30.833333333333336
2 1.154434690
0318838 0.18604651162
790695 12.94845774
1318867
P11166 Related Proteins
Solute carrier family 2,
facilitated glucose
transporter member 1
4.67479674796748
2 0.136463666
38572482 0.02124183006
5359478 54.04853579
695947
A0A0C4DH32
Related and
Unrelated Proteins
Protein IGHV3-20
(Fragment)
25.64102564102564
2 0.668100537
2000588 0.01282051282
0512822 12.70929366
8088954
B4DJ51
Related and
Unrelated Proteins
Calmodulin 1 (Phosphorylas
e kinase, delta),
isoform CRA_a
20.13422818791946
2 0.425102670
3029981 0.02232142857
1428572 16.82683430
0721597
C9JF17 Related Proteins
Apolipoprotein D
(Fragment)
10.232558139534884
2 0.311133937
42156426 0.01162790697
6744186 24.14216018
7180192
A0A0C4DGV7
Related Proteins
Retinol-binding
protein 4
14.572864321608039
2 0.202264434
61741295 0.01005025125
6281407 22.85315160
2540063
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
15.979381443298967
4 0.668100537
2000588 0.17911255411
255414 21.38873696
621423
Appendix 2. All validated proteins in one day old blood sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
P01024 Unrelated Proteins
Complement C3
42.3331328923632
48 0.8001933328915447
0.014698652477377191
187.02987531966258
P01023 Single
Protein Alpha-2-
macroglobulin 47.21845318
8602446 44
1.2490546058357808
0.0229620407409577
163.18789146924135
A0A087WZE4
Related Proteins
Spectrin alpha chain,
erythrocytic 1
14.362139917695472
22 0.1692334586063975
0.004608800035677576
280.9387841720546
45
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
14.225549836218999
21 0.1969249348971422
5
0.004950891467032518
246.3157334321971
C8C504 Single
Protein Beta-globin
95.91836734693877
23 33.14548873833603
1.29374193690661
15.987342548192922
A0A087WTM7
Related and
Unrelated Proteins
Apolipoprotein B-100
3.4069981583793743
9 0.0447863359928655
2
0.0025306842333036477
489.52739493234196
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.05517801097802998
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
22 0.8410273525621605
0.04338165336213569
69.18136901532891
P02042 Related Proteins
Hemoglobin subunit delta
93.19727891156462
18 14.848931924611133
0.5795009151570016
16.045289860997062
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
21 0.7906555296923772
0.14441180197941006
69.18136901532891
P02647 Related Proteins
Apolipoprotein A-I
55.430711610486895
18 1.976351441631318
0.1752561778751475
30.758932500112536
A0A0K0K1H8
Related and
Unrelated Proteins
Epididymis secretory
sperm binding protein Li 71p
30.945558739255013
19 0.7398280529303642
0.045122985368780594
77.02963611452142
P02787
Related and
Unrelated Proteins
Serotransferrin
30.08595988538682
19 0.7398280529303642
0.028862822767154534
77.01362996651181
P02730
Related and
Unrelated Proteins
Band 3 anion transport protein
20.417124039517013
12 0.5104434757204741
0.04518471483416132
101.72741218512044
P02671 Single
Protein Fibrinogen alpha chain
23.556581986143186
16 0.5127782816797017
0.02231942486851851
94.91441497589778
Q3LR79 Single
Protein
Hemoglobin beta
(Fragment)
95.23809523809523
16 38.810717055349734
1.347167243856381
11.474842789202496
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
30.629139072847682
19 0.6940088022878714
0.03932995605270707
69.18136901532891
P02675 Unrelated Proteins
Fibrinogen beta chain
45.010183299389006
15 0.8139306939110624
0.0424301339685955
55.89226078115152
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
17 1.1877616239495525
0.11331533960494844
45.11835747484806
P16157 Single
Protein Ankyrin-1
8.452950558213717
11 0.1484425891565204
2
0.005237519370105122
206.1369244395263
Q9BWU5 Single
Protein
Mutant hemoglobin beta chain (Fragment)
87.61904761904762
14 17.73817422860384
1.3338945330661254
11.493947863407726
I1VZV6 Single
Protein Hemoglobin
alpha 1 85.21126760
56338 15
13.251026703029977
2.006994337243872
15.269957551186833
46
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
17 1.1877616239495525
0.05791823516680009
45.11835747484806
P04040 Related Proteins
Catalase 33.77609108
1593925 12
0.6237767391887217
0.04250658276431473
59.71875531355211
Q4TZM4 Single
Protein
Hemoglobin beta chain (Fragment)
90.0990099009901
13 18.952623149688797
2.304581351004367
11.015638867792866
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
29.545454545454547
17 1.1877616239495525
0.13331887535040376
45.11835747484806
D9YZU8 Single
Protein Hemoglobin,
gamma A 89.11564625
850339 15 9.0
0.25476829053070255
16.11827504126487
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
40.19138755980861
13 1.1983926488622894
0.10672025260093485
46.678988138410034
P00918
Related and
Unrelated Proteins
Carbonic anhydrase 2
60.38461538461538
11 1.5550970903525068
0.0862691166184616
29.227922186630536
C9JC84 Related Proteins
Fibrinogen gamma chain
32.97180043383948
10 0.6876124757881477
0.044682023206968985
52.30432646809949
A0A0G2JL54
Related Proteins
Complement C4-B
7.302709069493522
7 0.0982410543092073
8
0.006079709370659259
187.59794049222188
P30041 Unrelated Proteins
Peroxiredoxin-6
51.78571428571429
9 0.9952623149688795
0.06390128968253962
25.01919029056794
D6RF35
Related and
Unrelated Proteins
Vitamin D-binding protein
30.252100840336134
8 0.3894954943731377
0.03878301320528211
52.98591682893214
P02790 Single
Protein Hemopexin
28.78787878787879
8 0.5199110829529336
0.04157795329670329
51.64327297890149
A0A075B6N9
Related Proteins
Ig mu chain C region
(Fragment)
27.81456953642384
10 0.8329807108324359
0.03578195247270388
49.40858023156175
P06727 Single
Protein Apolipoprotei
n A-IV 11.86868686
8686869 4
0.1897937907435858
2
0.017076502732240435
45.37146664308862
O95408 Single
Protein Beta globin (Fragment)
71.9298245614035
8 99.0 2.07525161818
78904 6.262154763
39843
A0A087WU08
Related Proteins
Haptoglobin 40.21352313
1672595 10
1.1544346900318838
0.040224712821190244
31.3878684863898
E9PFZ2
Related and
Unrelated Proteins
Ceruloplasmin 11.09936575
0528541 7
0.1828145730152692
7
0.003538597693273953
108.75141618854433
P32119
Related and
Unrelated Proteins
Peroxiredoxin-2
49.494949494949495
10 1.9935772947204895
0.1820579488262413
21.87823782783635
P30043 Unrelated Proteins
Flavin reductase (NADPH)
45.14563106796117
6 1.0691380811147897
0.14466019417475726
22.105426425215388
P04114 Related Proteins
Apolipoprotein B-100
1.9066403681788298
5 0.0234830276008564
1
0.0013677113604174149
515.282910917923
Q6J1Z9 Single
Protein
Hemoglobin alpha 1
(Fragment) 90.0 9 9.0
1.2347300461555972
9.585181362001366
Q86YL2 Single Hemoglobin 92.04545454 9 12.335214 0.80824663811 9.486065329
47
Protein alpha-1 chain (Fragment)
545455 32163324 36691 189616
Q9NQT3 Single
Protein
Alpha one globin
(Fragment)
89.01098901098901
9 12.33521432163324
0.7706121335604688
9.621118486961286
Q6MZU6
Related and
Unrelated Proteins
Putative uncharacteriz
ed protein DKFZp686C15
213
27.586206896551722
9 0.7012542798525889
0.056723493490963686
51.06619379122771
A0A075B6N8
Related and
Unrelated Proteins
Ig gamma-3 chain C region
(Fragment)
34.48275862068966
9 0.7508270317357244
0.02246440428258608
41.29932388898452
P01008 Related Proteins
Antithrombin-III
18.96551724137931
6 0.2798022139979537
0.012043308702791466
52.56886431029427
P07195 Related Proteins
L-lactate dehydrogenas
e B chain
20.35928143712575
5 0.4030037231905736
5
0.021908127208480566
36.61514296895986
B7ZKJ8
Related and
Unrelated Proteins
ITIH4 protein 9.304812834
224599 5
0.1397725198066268
6
0.006931692985677563
103.81622401008842
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
25.10729613733906
8 0.5671910803783076
0.040808139069134004
51.12147771750289
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
28.89518413597734
7 1.0805675382171707
0.037108399845293766
37.630638369583146
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
32.86118980169972
7 1.0805675382171707
0.02099582193724452
37.630638369583146
P01019 Related Proteins
Angiotensinogen
15.463917525773196
5 0.3894954943731377
0.042444444444444444
53.12051471946386
P00491 Related Proteins
Purine nucleoside
phosphorylase
35.98615916955018
6 0.637893706954064
0.01995386389850058
32.09715605245075
Q13228 Related Proteins
Selenium-binding
protein 1
15.889830508474576
5 0.2843862635897427
0.011899717514124292
52.35762502132521
Q86YQ1 Single
Protein
Hemoglobin alpha-2
(Fragment)
89.01098901098901
7 6.498942093324558
0.6348016129528891
9.665182424630526
Q9HAR8 Single
Protein
Mutant beta-globin
(Fragment) 100.0 5
45.4158883361278
1.6079404466501144
3.2907445311221695
Q6P5S8 Single
Protein IGK@ protein
42.79661016949153
6 1.0691380811147897
0.208164983164983
25.75683450608628
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
31.443298969072163
7 1.4484367468222272
0.18123535915743688
21.38873696621423
A0A087WT59
Related Proteins
Transthyretin 38.37837837
837838 5
1.2758459260747883
0.14069264069264076
20.132907042394372
P02787
Related and
Unrelated Proteins
Serotransferrin
9.025787965616045
5 0.1568875283162822
8
0.030758807588075893
77.01362996651181
P07738 Unrelated Bisphosphogl 11.58301158 2 0.1497569 0.02316602316 29.98651036
48
Proteins ycerate mutase
3011583 953977358 6023165 8450627
G3V3A0
Related and
Unrelated Proteins
Alpha-1-antichymotry
psin
14.634146341463413
3 0.3894954943731377
0.03414634146341463
23.39110667577842
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
2.339728591483388
4 0.0348320919317066
66
0.0013961359469750387
246.3157334321971
P60174 Related Proteins
Triosephosphate isomerase
12.237762237762238
2 0.1659144011798317
0.008741258741258742
30.771696101193438
Q86YQ4 Single
Protein
Alpha-1 globin
(Fragment)
90.9090909090909
5 3.2169650342858223
0.7371921390013633
9.476096235395074
E5RFL2 Related Proteins
Carbonic anhydrase 1 (Fragment)
60.16949152542372
6 3.6415888336127784
0.46892655367231645
13.248254100518784
A0A0G2JPD4
Related and
Unrelated Proteins
Ig gamma-4 chain C region
(Fragment)
20.489296636085626
6 0.637893706954064
0.04174777603349031
35.91786587959916
P02763 Related Proteins
Alpha-1-acid glycoprotein 1
33.33333333333333
6 0.9306977288832501
0.05607966457023061
23.49675931597699
A0A0C4DGL8
Related Proteins
Haptoglobin 7.204610951
008646 3
0.2252798573828647
0.013257575757575761
38.42734787385203
P13716 Related Proteins
Delta-aminolevulini
c acid dehydratase
22.424242424242426
5 0.4174741629268053
0.02878205128205128
36.27141416930379
P19652 Single
Protein Alpha-1-acid
glycoprotein 2 28.85572139
3034827 5
0.6876124757881477
0.06129126497224654
23.587631690019066
P01042 Related Proteins
Kininogen-1 5.124223602
484472 2
0.0690192046030009
3
0.012287334593572778
71.91215085210732
P08603
Related and
Unrelated Proteins
Complement factor H
3.4930950446791225
3 0.0563541037491872
57
0.003180039138943248
139.00469816836323
B2M1S7 Single
Protein
Beta-globin Showa
Yakushiji variant
(Fragment)
100.0 5 45.4158883361278
1.4656771799628925
4.4933946847851916
A0A087WV45
Related Proteins
Transthyretin 51.07913669
064749 4
1.0309176209047357
0.12826310380267214
15.110588167739953
P02787
Related and
Unrelated Proteins
Serotransferrin
6.59025787965616
4 0.1236548001387509
6
0.03455284552845528
77.01362996651181
A0A087WU08
Related Proteins
Haptoglobin 14.94661921
708185 4
0.3593563908785255
6
0.017666543982333454
31.3878684863898
E7EUT5 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e
25.769230769230766
4 0.4251026703029981
0.0581831831831832
27.853060805444024
C9JV77 Related Proteins
Alpha-2-HS-glycoprotein
24.184782608695652
5 0.5085907086001784
0.05133587786259543
39.386724601822
E9PGN7 Related Proteins
Plasma protease C1
9.208103130755065
4 0.216483949025851
0.016986410871302957
59.454555992296385
49
inhibitor
E9PFZ2
Related and
Unrelated Proteins
Ceruloplasmin 5.813953488
372093 4
0.1006941712522095
9
0.007793764988009592
108.75141618854433
P00352
Related and
Unrelated Proteins
Retinal dehydrogenas
e 1
7.984031936127744
3 0.1364636663857248
2
0.009830205540661306
54.82694780360638
P00558 Related Proteins
Phosphoglycerate kinase 1
8.393285371702639
2 0.0944997993195435
5
0.008393285371702638
44.58612676613191
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
29.381443298969074
4 0.6681005372000588
0.1753246753246753
21.38873696621423
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
15.979381443298967
4 0.6681005372000588
0.19832251082251087
21.38873696621423
C9JVG0 Related Proteins
Serotransferrin (Fragment)
26.865671641791046
4 1.0309176209047357
0.13772070626003205
14.680972242798342
A0A0A0MRQ5
Related and
Unrelated Proteins
Peroxiredoxin-1
29.896907216494846
3 0.8738174228603839
0.046907216494845375
10.669507125347321
P02652 Related Proteins
Apolipoprotein A-II
19.0 3 0.7782794100389228
0.05 11.16789735
900159
H0YAS8 Related Proteins
Clusterin (Fragment)
28.57142857142857
2 0.2589254117941673
0.04642857142857143
16.166165820637204
P19827 Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H1
5.26893523600439
3 0.0826367338740543
5
0.006743737957610789
101.32561491857365
B7ZKJ8
Related and
Unrelated Proteins
ITIH4 protein 4.278074866
31016 3
0.0816603471464902
9
0.004627249357326479
103.81622401008842
E9PIT3 Related Proteins
Prothrombin 8.919382504
288166 3
0.119902793272475
0.0026381129733085043
65.36674524764112
P00747 Related Proteins
Plasminogen 4.814814814
814815 2
0.0556729942333293
9
0.003536067892503536
90.5101571496777
A0A024R6I7
Related and
Unrelated Proteins
Alpha-1-antitrypsin
10.76555023923445
3 0.1993539462092342
0.09469696969696971
46.678988138410034
P02652 Related Proteins
Apolipoprotein A-II
28.999999999999996
2 0.4677992676220694
7 0.33
11.16789735900159
C9J5S7 Related Proteins
Peptidyl-prolyl cis-
trans isomerase
26.446280991735538
3 0.7782794100389228
0.07107438016528922
13.199402292131417
P62937 Related Proteins
Peptidyl-prolyl cis-
trans isomerase A
20.606060606060606
3 0.4125375446227544
0.02787878787878789
18.000885276520645
H0Y300 Related Proteins
Haptoglobin 8.823529411
764707 3
0.1699891065890659
6
0.006963788300835654
49.07441753459848
K7ERI9 Related Proteins
Apolipoprotein C-I
28.57142857142857
3 0.8738174228603839
0.15503875968992248
8.641669993226936
50
(Fragment)
A0A087WTE1
Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H2
2.751322751322751
2 0.0481131341546856
55
0.004768392370572207
106.58970315928195
B0YIW2 Related Proteins
Apolipoprotein C-III
29.059829059829063
2 0.4677992676220694
7
0.05555555555555555
12.80745671066277
C9JV37 Related Proteins
Prothrombin (Fragment)
10.802469135802468
2 0.1859710123376696
6
0.008992805755395683
35.90878157243862
B7UCU6
Related and
Unrelated Proteins
Gamma-globin chain (Fragment)
19.047619047619047
2 0.5199110829529336
0.6742857142857167
11.578876214589096
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
6.866952789699571
2 0.1188722115874221
2
0.037964774951076315
51.12147771750289
A0A0B4J231
Related and
Unrelated Proteins
Immunoglobulin lambda-
like polypeptide 5
13.023255813953488
2 0.2742749857031337
0.05243445692883893
23.135670373513683
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
2.525252525252525
2 0.0964781961431850
9
0.011363636363636364
45.11835747484806
P01009 Related Proteins
Alpha-1-antitrypsin
5.980861244019139
2 0.1288378916846890
7
0.027462121212121215
46.707019438538325
E7EPV7 Related Proteins
Alpha-synuclein
31.30434782608696
2 0.3593563908785255
6
0.0782608695652174
11.769249433169955
A0A075B6P5
Related and
Unrelated Proteins
Protein IGKV2-28
(Fragment)
30.833333333333336
2 1.1544346900318838
0.16279069767441862
12.948457741318867
P11166 Related Proteins
Solute carrier family 2,
facilitated glucose
transporter member 1
4.67479674796748
2 0.1364636663857248
2
0.021241830065359478
54.04853579695947
A0A087WTE1
Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H2
3.1746031746031744
2 0.0481131341546856
55
0.004768392370572207
106.58970315928195
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.04838709677419355
10.827649221794548
J3QRN2 Related Proteins
Beta-2-glycoprotein 1
(Fragment)
20.441988950276244
2 0.4677992676220694
7
0.019337016574585635
19.893845899589213
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
4.291845493562231
2 0.1188722115874221
2
0.00547945205479452
51.12147771750289
P02760 Related Proteins
Protein AMBP 8.806818181
818182 2
0.1325413151528118
7
0.005555555555555556
38.97398131913597
A0A0A0M Related Peroxiredoxin 16.95906432 3 0.4384498 0.03143274853 18.96367220
51
SI0 Proteins -1 (Fragment) 748538 8828766285
8011694 675963
Q6VFQ6 Single
Protein
Hemoglobin beta chain (Fragment)
71.42857142857143
4 20.544346900318832
6.7836734693878515
4.510410000496161
Appendix 3. All validated proteins in 7 days old blood sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
P01024 Unrelated Proteins
Complement C3
38.96572459410704
42 0.6726468435651758
0.011983136244567448
187.02987531966258
P01023 Single
Protein Alpha-2-
macroglobulin 43.01221166
892809 40
1.0892961308540396
0.018982332037160137
163.18789146924135
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
13.617220402433318
20 0.1867232263265372
6
0.0042177688439505485
246.3157334321971
A0A087WZE4
Related Proteins
Spectrin alpha chain,
erythrocytic 1
10.658436213991768
17 0.1284157410588553
4
0.0034657800100219667
280.9387841720546
C8C504 Single
Protein Beta-globin
95.23809523809523
23 33.14548873833603
1.0643221440986594
15.987342548192922
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.052355876457586464
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
22 0.8410273525621605
0.040973426126460585
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
21 0.7906555296923772
0.20549480423022165
69.18136901532891
P02647 Related Proteins
Apolipoprotein A-I
56.92883895131086
19 2.1622776601683795
0.1652404941943857
30.758932500112536
A0A0K0K1H8
Related and
Unrelated Proteins
Epididymis secretory
sperm binding protein Li 71p
33.0945558739255
19 0.7398280529303642
0.03377540862939089
77.02963611452142
P02787
Related and
Unrelated Proteins
Serotransferrin
32.23495702005731
19 0.7398280529303642
0.024019311068415247
77.01362996651181
P02787
Related and
Unrelated Proteins
Serotransferrin
32.664756446991404
19 0.7398280529303642
0.02076727854809005
77.01362996651181
P02730
Related and
Unrelated Proteins
Band 3 anion transport protein
21.514818880351264
13 0.5632550875412283
0.03809714782962014
101.72741218512044
Q3LR79 Single
Protein
Hemoglobin beta
(Fragment)
94.28571428571428
16 38.810717055349734
1.1922836562172698
11.474842789202496
P02042 Related Proteins
Hemoglobin subunit delta
92.51700680272108
17 12.593563908785255
0.4981073880703037
16.045289860997062
A0A0C4D Related Serum 30.62913907 19 0.6940088 0.03937058809 69.18136901
52
GB6 and Unrelated Proteins
albumin 2847682 022878714 203235 532891
P04040 Related Proteins
Catalase 33.77609108
159393 13
0.6907141034735806
0.03618560134024048
59.71875531355211
I1VZV6 Single
Protein Hemoglobin
alpha 1 92.95774647
887323 15
13.251026703029977
2.3448754749739096
15.269957551186833
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
18 1.2908676527677732
0.11001750049354113
45.11835747484806
P02675 Unrelated Proteins
Fibrinogen beta chain
44.806517311608964
14 0.7433288221999881
0.04154553627081096
55.89226078115152
P02671 Single
Protein Fibrinogen alpha chain
22.170900692840647
14 0.4364925262166947
7
0.020800481827672152
94.91441497589778
Q4TZM4 Single
Protein
Hemoglobin beta chain (Fragment)
90.0990099009901
14 24.118864315095795
1.2600347778541152
11.015638867792866
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
29.545454545454547
17 1.1877616239495525
0.09846991913892551
45.11835747484806
P16157 Single
Protein Ankyrin-1
6.273258904837853
8 0.1058999718195106
1
0.004302780918875907
206.1369244395263
Q9BWU5 Single
Protein
Mutant hemoglobin beta chain (Fragment)
87.61904761904762
15 22.101297000831593
0.8998818548406703
11.493947863407726
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
16 1.0892961308540396
0.05707182473359479
45.11835747484806
A0A087WTM7
Related and
Unrelated Proteins
Apolipoprotein B-100
1.3812154696132597
4 0.0196629977857685
2
0.0014637904468412942
489.52739493234196
D9YZU8 Single
Protein Hemoglobin,
gamma A 89.11564625
850339 12
5.309573444801933
0.24460958668441302
16.11827504126487
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
40.19138755980861
13 1.1983926488622894
0.1048145667463854
46.678988138410034
A0A0G2JL54
Related Proteins
Complement C4-B
7.597173144876326
7 0.0982410543092073
8
0.0030211561064216264
187.59794049222188
P00918
Related and
Unrelated Proteins
Carbonic anhydrase 2
54.61538461538461
11 1.5550970903525068
0.09428895946363199
29.227922186630536
C9JC84 Related Proteins
Fibrinogen gamma chain
36.659436008676785
11 0.7782794100389228
0.0442509226131786
52.30432646809949
A0A075B6N9
Related Proteins
Ig mu chain C region
(Fragment)
25.386313465783665
9 0.7252105499420405
0.03894164602257084
49.40858023156175
P30041 Unrelated Proteins
Peroxiredoxin-6
51.78571428571429
9 0.9952623149688795
0.050053146258503364
25.01919029056794
P06727 Single
Protein Apolipoprotei
n A-IV 17.17171717
171717 5
0.2426236719115049
0.01762295081967213
45.37146664308862
O95408 Single
Protein Beta globin (Fragment)
70.17543859649122
7 55.23413251903491
2.700371221023367
6.26215476339843
A0A087WU08
Related Proteins
Haptoglobin 37.01067615
658363 9
0.9952623149688795
0.0317071552722898
31.3878684863898
53
P07195 Related Proteins
L-lactate dehydrogenas
e B chain
26.047904191616766
7 0.6065060038537282
0.017937068820461054
36.61514296895986
P00491 Related Proteins
Purine nucleoside
phosphorylase
44.982698961937714
7 0.7782794100389228
0.019352446861097383
32.09715605245075
P60174 Single
Protein Triosephosphate isomerase
17.832167832167833
3 0.2589254117941673
0.01223776223776224
30.771696101193438
P32119
Related and
Unrelated Proteins
Peroxiredoxin-2
49.494949494949495
10 1.9935772947204895
0.16804455706894716
21.87823782783635
A0A075B6N8
Related and
Unrelated Proteins
Ig gamma-3 chain C region
(Fragment)
34.48275862068966
9 0.7508270317357244
0.02822759059122695
41.29932388898452
P02790 Single
Protein Hemopexin
28.78787878787879
8 0.5199110829529336
0.038049450549450525
51.64327297890149
Q6J1Z9 Single
Protein
Hemoglobin alpha 1
(Fragment) 90.0 9 9.0
1.0792442122953434
9.585181362001366
Q6MZU6
Related and
Unrelated Proteins
Putative uncharacteriz
ed protein DKFZp686C15
213
27.586206896551722
9 0.7012542798525889
0.04995489772862896
51.06619379122771
P30043 Unrelated Proteins
Flavin reductase (NADPH)
50.0 7 1.3357214690901222
0.09879796578825704
22.105426425215388
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
25.107296137339056
8 0.5671910803783076
0.10269080234833662
51.12147771750289
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
32.29461756373937
8 1.3101297000831598
0.019618994126092033
37.630638369583146
B7ZKJ8
Related and
Unrelated Proteins
ITIH4 protein 6.310160427
807486 4
0.110336318167638
0.0052485004284490155
103.81622401008842
Q86YQ1 Single
Protein
Hemoglobin alpha-2
(Fragment)
87.91208791208791
6 4.623413251903491
0.6500600240096058
9.665182424630526
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
31.443298969072163
7 1.4484367468222272
0.13544067764846984
21.38873696621423
A0A087WT59
Related Proteins
Transthyretin 37.83783783
783784 4
0.9306977288832501
0.12127396413110697
20.132907042394372
P02787
Related and
Unrelated Proteins
Serotransferrin
9.025787965616045
6 0.1911031332830068
8
0.024999999999999998
77.01362996651181
D6RBJ7
Related and
Unrelated Proteins
Vitamin D-binding protein
30.747126436781606
5 0.3433993325989002
0.013553639846743288
38.77189341127183
P02763 Related Proteins
Alpha-1-acid glycoprotein 1
40.79601990049751
7 1.1544346900318838
0.05678845961864828
23.49675931597699
E9PFZ2 Related
and Ceruloplasmin
4.968287526427061
3 0.0746078283213174
0.0061950439648281366
108.75141618854433
54
Unrelated Proteins
P01019 Related Proteins
Angiotensinogen
5.154639175257731
2 0.1406249238513208
6
0.016666666666666666
53.12051471946386
P04114 Related Proteins
Apolipoprotein B-100
0.591715976331361
2 0.0093278530753431
18
8.104384471999352E-4
515.282910917923
Q86YQ4 Single
Protein
Alpha-1 globin
(Fragment)
90.9090909090909
5 3.2169650342858223
0.8393980340312006
9.476096235395074
Q9HAR8 Single
Protein
Mutant beta-globin
(Fragment)
96.7741935483871
4 20.544346900318832
2.4040342060153015
3.2907445311221695
E5RFL2 Related Proteins
Carbonic anhydrase 1 (Fragment)
60.16949152542372
6 3.6415888336127784
0.4572033898305086
13.248254100518784
A0A0G2JPD4
Related and
Unrelated Proteins
Ig gamma-4 chain C region
(Fragment)
20.489296636085626
6 0.637893706954064
0.034402332361516005
35.91786587959916
P02787
Related and
Unrelated Proteins
Serotransferrin
6.73352435530086
5 0.1568875283162822
8
0.030216802168021674
77.01362996651181
P19652 Single
Protein Alpha-1-acid
glycoprotein 2 21.39303482
5870647 4
0.5199110829529336
0.05419222903885479
23.587631690019066
P13716 Related Proteins
Delta-aminolevulini
c acid dehydratase
22.12121212121212
5 0.4174741629268053
0.02967948717948718
36.27141416930379
A0A0C4DGL8
Related Proteins
Haptoglobin 6.916426512
9683 2
0.1450475699382818
3
0.00606060606060606
38.42734787385203
E9PGN7 Related Proteins
Plasma protease C1
inhibitor
9.392265193370166
4 0.216483949025851
0.007893685051958433
59.454555992296385
P07738 Unrelated Proteins
Bisphosphoglycerate mutase
10.424710424710424
2 0.1497569953977358
0.025096525096525095
29.986510368450627
B2M1S7 Single
Protein
Beta-globin Showa
Yakushiji variant
(Fragment)
100.0 4 20.544346900318832
1.6641534391534425
4.4933946847851916
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
79.43925233644859
5 1.8480358684358014
0.5171784601691172
11.711667188377447
A0A087WV45
Related Proteins
Transthyretin 51.07913669
064749 4
1.0309176209047357
0.10558410414525522
15.110588167739953
C9JV77 Related Proteins
Alpha-2-HS-glycoprotein
21.467391304347828
4 0.3894954943731377
0.037849872773536894
39.386724601822
G3V3A0
Related and
Unrelated Proteins
Alpha-1-antichymotry
psin
9.268292682926829
2 0.2451970847350328
8
0.026829268292682926
23.39110667577842
P01042 Related Proteins
Kininogen-1 4.347826086
9565215 2
0.0690192046030009
3
0.012287334593572778
71.91215085210732
P00568 Related Proteins
Adenylate kinase
24.742268041237114
3 0.2589254117941673
0.014175257731958761
21.621307247002466
55
isoenzyme 1
P08603
Related and
Unrelated Proteins
Complement factor H
2.518277822908205
2 0.0372250954070572
1
0.0029354207436399216
139.00469816836323
M0R009 Related Proteins
Alpha-1B-glycoprotein (Fragment)
10.130718954248366
2 0.1859710123376696
6
0.01507537688442211
33.43379931857088
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
29.381443298969074
4 0.6681005372000588
0.17965367965367965
21.38873696621423
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
15.979381443298967
4 0.6681005372000588
0.19182900432900435
21.38873696621423
A0A0B4J231
Related and
Unrelated Proteins
Immunoglobulin lambda-
like polypeptide 5
32.093023255813954
4 0.6237767391887217
0.06905430711610487
23.135670373513683
E7EUT5 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e
25.76923076923077
4 0.4251026703029981
0.05292792792792794
27.853060805444024
C9JVG0 Related Proteins
Serotransferrin (Fragment)
26.865671641791046
4 1.0309176209047357
0.12263242375601918
14.680972242798342
A0A0A0MRQ5
Related and
Unrelated Proteins
Peroxiredoxin-1
38.144329896907216
4 1.3101297000831598
0.039846179021436744
10.669507125347321
P01008 Related Proteins
Antithrombin-III
7.543103448275862
2 0.0857111194022042
2
0.00646551724137931
52.56886431029427
Q13228 Related Proteins
Selenium-binding
protein 1
10.805084745762713
3 0.1620301363862613
0.0069209039548022615
52.35762502132521
P26447 Single
Protein Protein S100-
A4 18.81188118
811881 2
0.3593563908785255
6
0.034653465346534656
11.720732061372276
A0A087WTE1
Related Proteins
Inter-alpha-trypsin
inhibitor heavy chain
H2
2.751322751322751
2 0.0481131341546856
55
0.006130790190735695
106.58970315928195
P02749 Related Proteins
Beta-2-glycoprotein 1
19.420289855072465
4 0.3111339374215642
6
0.010338164251207732
38.27265933588615
P00558 Related Proteins
Phosphoglycerate kinase 1
8.393285371702639
2 0.0944997993195435
5
0.008393285371702638
44.58612676613191
A0A024R6I7
Related and
Unrelated Proteins
Alpha-1-antitrypsin
10.76555023923445
3 0.1993539462092342
0.05445075757575755
46.678988138410034
P02652 Related Proteins
Apolipoprotein A-II
28.999999999999996
2 0.4677992676220694
7
0.2866666666666666
11.16789735900159
C9J5S7 Related Proteins
Peptidyl-prolyl cis-
trans isomerase
26.446280991735538
3 0.7782794100389228
0.038016528925619845
13.199402292131417
E7EPV7 Related Proteins
Alpha-synuclein
31.30434782608696
2 0.3593563908785255
0.07391304347826087
11.769249433169955
56
6
H0Y300 Related Proteins
Haptoglobin 8.597285067
873303 2
0.110336318167638
0.004456824512534818
49.07441753459848
E9PIT3 Related Proteins
Prothrombin 6.346483704
97427 2
0.0784173969290393
7
0.00558659217877095
65.36674524764112
B7UCU6
Related and
Unrelated Proteins
Gamma-globin chain (Fragment)
19.047619047619047
2 0.5199110829529336
0.6580952380952414
11.578876214589096
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
6.866952789699571
2 0.1188722115874221
2
0.03757338551859099
51.12147771750289
A0A0B4J231
Related and
Unrelated Proteins
Immunoglobulin lambda-
like polypeptide 5
13.023255813953488
2 0.2742749857031337
0.05280898876404492
23.135670373513683
P01009 Related Proteins
Alpha-1-antitrypsin
5.980861244019139
2 0.1288378916846890
7
0.026515151515151516
46.707019438538325
A0A075B6P5
Related and
Unrelated Proteins
Protein IGKV2-28
(Fragment)
30.833333333333336
2 1.1544346900318838
0.10852713178294572
12.948457741318867
C9JTX5
Related and
Unrelated Proteins
Actin, cytoplasmic 1
(Fragment) 33.75 2
0.9306977288832501
0.046875 8.528166114
106645
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.04838709677419355
10.827649221794548
E9PFZ2
Related and
Unrelated Proteins
Ceruloplasmin 3.276955602
536998 2
0.0491397291363098
2
0.002997601918465228
108.75141618854433
P00352
Related and
Unrelated Proteins
Retinal dehydrogenas
e 1
5.389221556886228
2 0.0890229622637301
9
0.005361930294906166
54.82694780360638
P01024 Unrelated Proteins
Complement C3
38.96572459410704
42 0.6726468435651758
0.011983136244567448
187.02987531966258
P01023 Single
Protein Alpha-2-
macroglobulin 43.01221166
892809 40
1.0892961308540396
0.018982332037160137
163.18789146924135
P11277
Related and
Unrelated Proteins
Spectrin beta chain,
erythrocytic
13.617220402433318
20 0.1867232263265372
6
0.0042177688439505485
246.3157334321971
A0A087WZE4
Related Proteins
Spectrin alpha chain,
erythrocytic 1
10.658436213991768
17 0.1284157410588553
4
0.0034657800100219667
280.9387841720546
C8C504 Single
Protein Beta-globin
95.23809523809523
23 33.14548873833603
1.0643221440986594
15.987342548192922
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.052355876457586464
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
22 0.8410273525621605
0.040973426126460585
69.18136901532891
A0A0C4DGB6
Related and
Serum albumin
35.264900662251655
21 0.7906555296923772
0.20549480423022165
69.18136901532891
57
Unrelated Proteins
(A0A0C4DGB6_HUMAN)
P02647 Related Proteins
Apolipoprotein A-I
56.92883895131086
19 2.1622776601683795
0.1652404941943857
30.758932500112536
A0A0K0K1H8
Related and
Unrelated Proteins
Epididymis secretory
sperm binding protein Li 71p
33.0945558739255
19 0.7398280529303642
0.03377540862939089
77.02963611452142
Appendix 4. All validated proteins in one day old saliva sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
A8K2U0 Unrelated Proteins
Alpha-2-macroglobulin-like protein 1
7.2214580467675376
7 0.1609534723271461
9
0.004905847373637266
161.00406439261246
P04745 Related Proteins
Alpha-amylase 1
35.61643835616438
12 0.7378008287493754
0.011299818465682208
57.73091248903519
P04745 Related Proteins
Alpha-amylase 1
26.614481409001954
10 0.5848931924611136
0.032871872666955196
57.73091248903519
H0YKL9 Related Proteins
Annexin (Fragment)
46.36363636363636
4 1.0309176209047357
0.09402597402597403
12.05628484306039
H0YL33 Related Proteins
Annexin (Fragment)
42.95774647887324
4 0.4924955450518296
0.04144869215291754
16.422326680576667
P04083 Related Proteins
Annexin A1 35.54913294
797688 9
0.5264179671752336
0.09116088631984576
38.689979125461925
P04083 Related Proteins
Annexin A1 22.54335260
115607 7
0.3894954943731377
0.06519749518304435
38.689979125461925
P04083 Related Proteins
Annexin A1 8.381502890
17341 3
0.1513953993264474
0.012524084778420038
38.689979125461925
P07355 Related Proteins
Annexin A2 34.80825958
702065 9
0.4896248995834391
3
0.01449155564199811
38.579814745475446
Q96DR5 Single
Protein
BPI fold-containing
family A member 2
24.899598393574298
5 0.7782794100389228
0.01979166666666667
26.99474719539754
Q8TDL5 Single
Protein
BPI fold-containing
family B member 1
33.057851239669425
11 1.06198600950222
0.0333488064813366
52.40847237175294
Q8N4F0 Single
Protein
BPI fold-containing
family B member 2
19.65065502183406
5 0.4677992676220694
7
0.05413832199546482
49.14172224203288
F8W148 Related Proteins
Carbonic anhydrase 6 (Fragment)
19.25133689839572
2 0.4677992676220694
7
0.04205607476635514
21.0494281284372
P08311 Related Proteins
Cathepsin G 27.05882352
9411764 6
0.4839817889675653
0.11816993464052285
28.819069590686258
B4DQ50 Related Proteins
cDNA FLJ56823,
highly similar to Protein-glutamine gamma-
glutamyltransferase E (EC 2.3.2.13)
9.75609756097561
5 0.2648552168552962
0.01735749630486472
58.757343311769674
58
A8K2N8 Related Proteins
cDNA FLJ77935,
highly similar to Homo sapiens
guanylate binding protein family,
member 6 (GBP6), mRNA
7.424960505529226
3 0.0978437670592489
1
0.006497175141242938
72.31878032663606
A0A087WVQ9
Related and
Unrelated Proteins
Elongation factor 1-alpha
1
5.442176870748299
2 0.0841458689358347
6
0.004076086956521739
47.85292586470423
Q08380 Related Proteins
Galectin-3-binding protein
7.17948717948718
3 0.1620301363862613
0.003386004514672686
65.28928569959861
F8WE04
Related and
Unrelated Proteins
Heat shock protein beta-1
13.978494623655912
2 0.2216773489967920
3
0.045045045045045036
20.39351236294829
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
27.762039660056658
8 1.3101297000831598
0.0873453603579128
37.630638369583146
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
23.79603399433428
8 1.3101297000831598
0.07897715952109273
37.630638369583146
A0A0G2JMB2
Related and
Unrelated Proteins
Ig alpha-2 chain C region
(Fragment)
19.705882352941178
4 0.5505157798326246
0.05065502183406112
36.48504939159779
A0A087WV47
Related and
Unrelated Proteins
Ig gamma-1 chain C region
9.656652360515022
3 0.1835067295194687
8
0.01187214611872146
51.12147771750289
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
79.43925233644859
5 1.8480358684358014
0.2401869158878504
11.711667188377447
A1A4E9 Unrelated Proteins
Keratin 13 43.23144104
803493 25
2.4034148825183075
0.05969603479713928
49.55539934926395
O76011 Related Proteins
Keratin, type I cuticular Ha4
6.192660550458716
2 0.1158839925077483
9
0.009316770186335406
49.39222835129151
O76013 Related Proteins
Keratin, type I cuticular Ha6
30.406852248394006
11 0.7343248017724648
0.05603970482487109
52.21397052719633
P13645 Single
Protein
Keratin, type I cytoskeletal
10
40.06849315068493
23 2.0857568527160524
0.04090458865775523
58.79169361540863
P13646 Related Proteins
Keratin, type I cytoskeletal
13
52.40174672489083
30 3.3480834671045114
0.14208560162064462
49.55737861381936
P02533 Single
Protein
Keratin, type I cytoskeletal
14
58.26271186440678
30 2.5938136638046276
0.07079219228164929
51.52938407421822
P08779 Single
Protein
Keratin, type I cytoskeletal
16
64.27061310782241
30 3.2169650342858223
0.0670095521349188
51.236202521320465
F5GWP8 Related Keratin, type I 26.36103151 10 0.6876124 0.01977262894 40.26237172
59
and Unrelated Proteins
cytoskeletal 17
8624642 757881477 1683384 672955
C9JM50
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
19 (Fragment)
9.693877551020408
2 0.2451970847350328
8
0.024092970521541936
20.83054632446172
P35527 Single
Protein Keratin, type I cytoskeletal 9
14.285714285714285
5 0.2589254117941673
0.011071987480438188
62.02681468242336
Q9NSB2 Related Proteins
Keratin, type II cuticular
Hb4
38.83333333333333
17 0.7782794100389228
0.02703092965937998
64.80146264176814
P04264 Related Proteins
Keratin, type II cytoskeletal
1
32.142857142857146
26 1.5118864315095801
0.07067735734666779
65.99900087210398
P04264 Related Proteins
Keratin, type II cytoskeletal
1
31.987577639751557
24 1.3400803470013467
0.01855447460828886
65.99900087210398
P35908 Related Proteins
Keratin, type II cytoskeletal 2 epidermal
16.588419405320813
11 0.476490881614259
0.009021635107839432
65.39321717101762
Q01546 Single
Protein
Keratin, type II cytoskeletal
2 oral
21.786833855799376
15 0.7154378963428789
0.029619499569468553
65.80008965822827
P12035 Single
Protein
Keratin, type II cytoskeletal
3
12.10191082802548
10 0.4773776525985111
7
0.012150872780268033
64.37756213683015
F8VX05 Related Proteins
Keratin, type II cytoskeletal
4
53.84615384615384
6 2.511191734215131
0.09299219299219298
7.783852222922241
P19013 Related Proteins
Keratin, type II cytoskeletal
4
57.303370786516865
44 4.411695265464636
0.3026775414591496
57.249828579886504
P19013 Related Proteins
Keratin, type II cytoskeletal
4
7.116104868913857
6 0.2589254117941673
0.01755477939357032
57.249828579886504
F8VV57 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
37.121212121212125
4 1.3101297000831598
0.0689922480620155
12.14979750828232
F8W0C6 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
48.60335195530727
13 2.1622776601683795
0.03806504199904183
20.643608929250135
P13647 Related Proteins
Keratin, type II cytoskeletal
5
34.91525423728814
24 1.3101297000831598
0.010840742651444782
62.33997694347283
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
47.16312056737589
32 2.0538555088334154
0.023024637601976136
60.00827426891604
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
9.397163120567376
4 0.1497569953977358
0.016474654377880184
60.00827426891604
P04259
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6B
17.5531914893617
13 0.5738730431619035
0.007594720056995347
60.03029098160933
P48668
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6C
53.72340425531915
31 1.949150729647068
0.016751938655459726
59.98830556904431
H0YIC5 Related Keratin, type 14.73684210 6 0.4677992 0.02236525754 31.27068623
60
Proteins II cytoskeletal 73 (Fragment)
5263156 6762206947
0057658 2512684
Q8N1N4 Single
Protein
Keratin, type II cytoskeletal
78
42.88461538461539
19 1.0733215734859551
0.01583255815452162
56.83049222411559
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
16.954022988505745
9 0.2875262659803554
7
0.005995712461229703
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
11.350574712643677
6 0.1835067295194687
8
0.004732894819101716
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
5.890804597701149
4 0.1188722115874221
2
0.002134646962233169
76.57688676417338
P30740
Related and
Unrelated Proteins
Leukocyte elastase inhibitor
27.440633245382585
8 0.5505157798326246
0.0156984126984127
42.714702418590555
P24158 Related Proteins
Myeloblastin 8.203125 2 0.3335214321633239
7
0.011986301369863013
27.789271098985488
P05164 Single
Protein Myeloperoxid
ase 9.932885906
040267 6
0.1721022975334802
8
0.004480796586059744
83.81483871714372
C3PTT6
Related and
Unrelated Proteins
Pancreatic adenocarcino
ma upregulated
factor
47.95918367346939
6 1.6826957952797255
0.06527777777777778
21.55300886308947
P01833 Single
Protein
Polymeric immunoglobul
in receptor
21.596858638743456
13 0.4405773606943357
3
0.011679751638768032
83.23165254290421
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.07526881720430108
10.827649221794548
Q08188 Single
Protein
Protein-glutamine gamma-
glutamyltransferase E
28.282828282828284
15 0.7012542798525889
0.01309080795901598
76.58358425055039
P02787
Related and
Unrelated Proteins
Serotransferrin
13.753581661891118
7 0.2263306841775629
7
0.0021151116273067498
77.01362996651181
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
18 1.2908676527677732
0.025039988456087847
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
16 1.0892961308540396
0.022638280649507472
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
28.282828282828284
13 0.8197008586099834
0.020433838047474402
45.11835747484806
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.44370860927152
21 0.7906555296923772
0.024472496230653194
69.18136901532891
61
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.013126754666477575
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
21 0.7906555296923772
0.031193520323226573
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
30.629139072847686
19 0.6940088022878714
0.014158728420040324
69.18136901532891
C9JDS9 Related Proteins
Tubulin alpha-4A chain
(Fragment)
23.717948717948715
2 0.4677992676220694
7
0.012962962962962963
17.65980705160292
Appendix 5. All validated proteins in one week old saliva sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
P60709
Related and
Unrelated Proteins
Actin, cytoplasmic 1
17.333333333333336
4 0.2915496650148839
0.012000000000000002
41.709730139029176
C9JTX5
Related and
Unrelated Proteins
Actin, cytoplasmic 1
(Fragment) 33.75 2
0.9306977288832501
0.015625 8.528166114
106645
I3L1U9 Related Proteins
Actin, cytoplasmic 2
(Fragment)
21.02803738317757
3 0.4125375446227544
0.017912772585669784
23.82995491331281
I3L1U9
Related and
Unrelated Proteins
Actin, cytoplasmic 2
(Fragment)
14.018691588785046
2 0.2589254117941673
0.053738317757009345
23.82995491331281
I3L4N8
Related and
Unrelated Proteins
Actin, cytoplasmic 2
(Fragment)
9.50413223140496
2 0.2451970847350328
8
0.008264462809917356
26.917436121169494
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
20.813397129186605
6 0.4384498882876628
5
0.013778409090909095
46.678988138410034
A8K2U0 Unrelated Proteins
Alpha-2-macroglobulin-like protein 1
8.872077028885833
8 0.1859710123376696
6
0.0028525378376716335
161.00406439261246
A8K2U0
Related and
Unrelated Proteins
Alpha-2-macroglobulin-like protein 1
6.121045392022008
5 0.1124904621939377
7
0.003014535844070036
161.00406439261246
P04745 Related Proteins
Alpha-amylase 1
38.16046966731898
17 1.1877616239495525
0.018212733605126877
57.73091248903519
P04745 Related Proteins
Alpha-amylase 1
29.9412915851272
15 0.9952623149688795
0.023635227502940503
57.73091248903519
H0YKL9 Related Proteins
Annexin (Fragment)
54.54545454545454
6 1.8942661247167512
0.05628492719401809
12.05628484306039
H0YL33 Related Proteins
Annexin (Fragment)
45.07042253521127
8 1.2275429519995575
0.04700542024485685
16.422326680576667
H0YL33 Related Proteins
Annexin (Fragment)
42.95774647887324
6 0.8233480008684411
0.055046948356807464
16.422326680576667
P04083 Related Annexin A1 40.46242774 12 0.7575106 0.04241729689 38.68997912
62
Proteins 566474 248547918 7065616 5461925
P04083 Related Proteins
Annexin A1 22.54335260
115607 7
0.3894954943731377
0.047663776493256274
38.689979125461925
P04083 Related Proteins
Annexin A1 10.98265895
9537572 5
0.2648552168552962
0.021290944123314063
38.689979125461925
P07355 Related Proteins
Annexin A2 32.44837758
1120944 10
0.5570684047537309
0.010374816748621512
38.579814745475446
P25705 Related Proteins
ATP synthase subunit alpha, mitochondrial
6.329113924050633
2 0.0722672220103233
2
0.007383966244725738
59.713593485620585
Q562R1 Single
Protein Beta-actin-like
protein 2 11.17021276
5957446 4
0.2826498305280605
0.007092198581560282
41.97596729003312
Q8TDL5 Single
Protein
BPI fold-containing
family B member 1
32.64462809917355
12 1.202201949987375
0.018371755419948198
52.40847237175294
Q8N4F0 Single
Protein
BPI fold-containing
family B member 2
21.397379912663755
6 0.5848931924611136
0.046712018140589576
49.14172224203288
P08311 Related Proteins
Cathepsin G 30.58823529
411765 7
0.5848931924611136
0.024463118580765653
28.819069590686258
B3KSL2
Related and
Unrelated Proteins
cDNA FLJ36533 fis,
clone TRACH200442
8, highly similar to
Lactotransferrin (EC 3.4.21.-)
(Fragment)
27.83357245337159
16 0.5671910803783076
0.007601771062670419
76.70396132609598
B4E1U2 Related Proteins
cDNA FLJ53421,
highly similar to Homo sapiens
guanylate binding protein family,
member 6 (GBP6), mRNA
26.490066225165563
9 0.318256738556407
0.004984721161191751
69.15420835722563
B0AZP3 Related Proteins
cDNA, FLJ79483,
highly similar to Homo sapiens
guanylate binding protein family,
member 6 (GBP6), mRNA
19.589257503949447
7 0.2362470354499415
2
0.004180076044482824
72.28891128123597
P01024
Related and
Unrelated Proteins
Complement C3
9.320505111244739
9 0.1165349973889215
9
0.003083679590270928
187.02987531966258
F8WE04
Related and
Unrelated Proteins
Heat shock protein beta-1
18.27956989247312
3 0.3503140378698730
3
0.02195945945945946
20.39351236294829
P01876 Related
and Ig alpha-1
chain C region 16.71388101
983003 4
0.5199110829529336
0.010011954572624028
37.630638369583146
63
Unrelated Proteins
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
60.747663551401864
4 1.3101297000831598
0.026479750778816202
11.711667188377447
P14923 Related Proteins
Junction plakoglobin
14.630872483221477
6 0.2409377607517195
8
0.005118694362017805
81.69273021332828
A1A4E9 Unrelated Proteins
Keratin 13 8.515283842
79476 6
0.3417128354799117
0.02486452671610999
49.55539934926395
Q15323 Related Proteins
Keratin, type I cuticular Ha1
8.653846153846153
3 0.1993539462092342
0.004275227877712349
47.20705069770084
O76013 Related Proteins
Keratin, type I cuticular Ha6
36.402569593147746
16 1.2275429519995575
0.019729003815018072
52.21397052719633
P13645 Single
Protein
Keratin, type I cytoskeletal
10
59.24657534246576
38 5.4344382633289925
0.06138780152088599
58.79169361540863
K7EMD9 Related Proteins
Keratin, type I cytoskeletal
13 (Fragment)
23.636363636363633
5 0.7301957388458942
0.14370296370296348
19.20172933573258
P13646
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
13
58.29694323144105
36 4.833879397552119
0.07084789166092717
49.55737861381936
P02533 Single
Protein
Keratin, type I cytoskeletal
14
66.52542372881356
40 4.50478980785497
0.040893038124746706
51.52938407421822
P08779 Single
Protein
Keratin, type I cytoskeletal
16
66.38477801268499
34 4.108969774506928
0.08616791555915368
51.236202521320465
F5GWP8
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
17
32.378223495702
11 0.7782794100389228
0.0122549521647342
40.26237172672955
C9JM50
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
19 (Fragment)
9.693877551020408
2 0.2451970847350328
8
0.009566326530612245
20.83054632446172
P35527 Single
Protein Keratin, type I cytoskeletal 9
46.06741573033708
31 3.1686938347033538
0.032695992229512326
62.02681468242336
Q9NSB2 Related Proteins
Keratin, type II cuticular
Hb4 49.0 22
1.1063445423241212
0.018366257401291743
64.80146264176814
P04264 Related Proteins
Keratin, type II cytoskeletal
1
44.409937888198755
44 3.752510411057904
0.07001459599988424
65.99900087210398
P04264 Related Proteins
Keratin, type II cytoskeletal
1
44.25465838509317
42 3.4274518434944925
0.019124388122095014
65.99900087210398
P35908 Single
Protein
Keratin, type II cytoskeletal 2 epidermal
52.895148669796555
34 2.334854751235186
0.01867898503002453
65.39321717101762
Q01546 Single
Protein
Keratin, type II cytoskeletal
2 oral
25.54858934169279
18 0.9109529749704406
0.022339240977388604
65.80008965822827
P12035 Single
Protein
Keratin, type II cytoskeletal
3
17.515923566878982
15 0.7957144943716399
0.017491630746450677
64.37756213683015
F8VX05 Related Proteins
Keratin, type II cytoskeletal
4
53.84615384615384
8 4.3366992312063095
0.1666819291819291
7.783852222922241
64
P19013 Related Proteins
Keratin, type II cytoskeletal
4
57.67790262172285
50 5.812920690579613
0.11313900535763279
57.249828579886504
F8VV57 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
37.121212121212125
4 1.3101297000831598
0.05232558139534884
12.14979750828232
F8W0C6 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
50.279329608938546
15 2.7750532053243937
0.03977384552889407
20.643608929250135
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
49.75124378109453
15 2.5938136638046276
0.053792105802683364
23.09540181798727
H0YIN9 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
17.857142857142858
5 0.5848931924611136
0.0076402905484538024
21.951672834646317
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
47.695035460992905
38 2.7649358067924683
0.02982048393288893
60.00827426891604
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
9.397163120567376
4 0.1497569953977358
0.01528417818740398
60.00827426891604
P04259
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6B
17.5531914893617
16 0.7475284000076838
0.008287214379493335
60.03029098160933
P48668
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6C
52.659574468085104
34 2.2745491628777286
0.016313025069223844
59.98830556904431
Q8N1N4 Single
Protein
Keratin, type II cytoskeletal
78 30.0 13
0.6468978654828685
0.013024097049226038
56.83049222411559
C9JCF5
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
33.51648351648351
4 0.5199110829529336
0.031004709576138153
20.330700568898102
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
54.310344827586206
31 1.3880794998622172
0.00921900328794767
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
25.57471264367816
15 0.5237960002208242
0.014518519682884447
76.57688676417338
P30740 Unrelated Proteins
Leukocyte elastase inhibitor
40.89709762532981
13 1.0395004690050569
0.030679745635627996
42.714702418590555
Q9HC84 Related Proteins
Mucin-5B 0.711558486
6365846 3
0.0251593260625877
5
0.0011342407259140647
595.9601181160408
P08246 Single
Protein Neutrophil
elastase 23.59550561
7977526 4
0.4924955450518296
0.010646108663729809
28.499788498823822
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.04838709677419355
10.827649221794548
Q08188 Single
Protein
Protein-glutamine gamma-
glutamyltransferase E
5.194805194805195
2 0.0734188827013531
0.005351681957186544
76.58358425055039
A0A087W Related Serum 22.22222222 6 0.3182567 0.00910006449 45.11835747
65
WT3 and Unrelated Proteins
albumin 222222 38556407 7791766 484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
8.080808080808081
2 0.0964781961431850
9
0.003787878787878788
45.11835747484806
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
18.70860927152318
7 0.2143317475937911
4
0.0038583588045508575
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
16.225165562913908
7 0.2143317475937911
4
0.008825246221769395
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
2.4834437086092715
2 0.0570520810009829
1
0.0028973509933774835
69.18136901532891
C9JDS9 Related Proteins
Tubulin alpha-4A chain
(Fragment)
32.69230769230769
3 0.7782794100389228
0.023478835978835978
17.65980705160292
A0A0A0MR02
Related Proteins
Voltage-dependent
anion-selective channel
protein 2 (Fragment)
14.539007092198581
3 0.2915496650148839
0.01590594744121715
30.328869561053356
Appendix 6. All validated proteins in three weeks old saliva sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
B0AZS6 Related Proteins
14-3-3 protein
zeta/delta
22.023809523809522
2 0.2915496650148839
0.026785714285714284
19.060395339266766
P60709
Related and
Unrelated Proteins
Actin, cytoplasmic 1
17.6 5 0.37685716485275833
0.006828282828282827
41.709730139029176
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
38.995215311004785
14 1.3357214690901222
0.034123676985892866
46.678988138410034
P01023 Single
Protein
Alpha-2-macroglobuli
n
5.563093622795115
5 0.09647819614318509
0.005975924333619948
163.18789146924135
A8K2U0 Single
Protein
Alpha-2-macroglobulin-like protein
1
20.97661623108666
23 0.6329126856447997
0.012726190530940045
161.00406439261246
O43707 Related Proteins
Alpha-actinin-4
10.867178924259058
7 0.16939481814731705
0.004791916766706683
104.78847372042017
P04745 Related Proteins
Alpha-amylase 1
45.79256360078278
18 1.2908676527677732
0.017815754826975377
57.73091248903519
P04745 Related Proteins
Alpha-amylase 1
37.573385518590996
16 1.0892961308540396
0.037563825063825114
57.73091248903519
D6RA82 Related Proteins
Annexin 15.49295774
6478872 4
0.2589254117941673
0.013791079812206574
32.098530647992916
H0YKL9 Related Proteins
Annexin (Fragment)
54.54545454545454
5 1.4244620170823286
0.23088023088023074
12.05628484306039
66
H0YM50 Related Proteins
Annexin (Fragment)
42.74193548387097
14 1.5808615404180748
0.05414790023084957
28.35243878541001
P04083 Related Proteins
Annexin A1 40.75144508
67052 13
0.8420699693267162
0.111610010092669
38.689979125461925
P04083 Related Proteins
Annexin A1 29.19075144
5086704 10
0.5998587196060581
0.0944382849007126
38.689979125461925
P04083 Related Proteins
Annexin A1 10.98265895
9537572 5
0.2648552168552962
0.04609826589595377
38.689979125461925
P07355 Related Proteins
Annexin A2 36.87315634
218289 11
0.6275654480267538
0.05662621980945771
38.579814745475446
P06576 Related Proteins
ATP synthase subunit beta, mitochondrial
10.01890359168242
4 0.20679264063932856
0.008451656524678837
56.524605849238945
Q562R1 Single
Protein Beta-actin-
like protein 2 11.17021276
5957448 3
0.20526093687084246
0.010638297872340422
41.97596729003312
Q96DR5 Single
Protein
BPI fold-containing
family A member 2
17.269076305220885
3 0.4125375446227544
0.02964743589743589
26.99474719539754
Q8TDL5 Single
Protein
BPI fold-containing
family B member 1
39.25619834710744
13 1.3519526350709588
0.017428669055175074
52.40847237175294
Q8N4F0 Single
Protein
BPI fold-containing
family B member 2
21.39737991266375
7 0.7113283041617808
0.05046971169420148
49.14172224203288
F8W148 Related Proteins
Carbonic anhydrase 6 (Fragment)
19.25133689839572
2 0.46779926762206947
0.056074766355140186
21.0494281284372
E9PQ63 Related Proteins
Carbonyl reductase [NADPH] 1
15.168539325842698
2 0.35935639087852556
0.01 19.00973458
34827
P08311 Related Proteins
Cathepsin G 41.17647058
823529 9
0.8077686769634342
0.055639589169000914
28.819069590686258
B4DQ50 Related Proteins
cDNA FLJ56823,
highly similar to Protein-glutamine gamma-
glutamyltransferase E (EC
2.3.2.13)
6.378986866791745
3 0.1513953993264474
0.00755735492577598
58.757343311769674
P01024 Unrelated Proteins
Complement C3
8.057726999398678
8 0.10294331179161298
0.0035953427196365063
187.02987531966258
P12532 Single
Protein
Creatine kinase U-type, mitochondrial
8.393285371702639
2 0.08571111940220422
0.006527415143603133
47.00720625799705
Q9UGM3 Single
Protein
Deleted in malignant
brain tumors 1 protein
1.0360547036883547
2 0.03272814726171824
0.0040650406504065045
260.5688045493978
P15924 Single
Protein Desmoplakin
3.6224312086381047
6 0.03124778549427165
0.002487652090109626
331.5687042418961
Q16610 Single
Protein
Extracellular matrix
protein 1
10.37037037037037
3 0.14815362149688283
0.00629555997349238
60.635275149574795
F8WE04
Related and
Unrelated Proteins
Heat shock protein beta-
1
18.27956989247312
3 0.35031403786987303
0.052364864864864864
20.39351236294829
67
P62805 Related Proteins
Histone H4 41.74757281
553398 4
0.46779926762206947
0.03802588996763755
11.360381729559025
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
21.246458923512748
6 0.8738174228603839
0.01536245600053131
37.630638369583146
A0A0G2JMB2
Related and
Unrelated Proteins
Ig alpha-2 chain C region
(Fragment)
19.705882352941178
4 0.5505157798326246
0.024599708879184848
36.48504939159779
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
79.43925233644859
5 1.8480358684358014
0.1459501557632398
11.711667188377447
D6RD17 Related Proteins
Immunoglobulin J chain
(Fragment)
13.924050632911392
2 0.2589254117941673
0.014227642276422764
18.110050897845266
P14923 Related Proteins
Junction plakoglobin
25.503355704697984
14 0.6548170999431815
0.01536694018148024
81.69273021332828
O76013 Related Proteins
Keratin, type I cuticular Ha6
55.03211991434689
21 1.8610255691043776
0.0609852152082874
52.21397052719633
P13645 Single
Protein
Keratin, type I cytoskeletal
10
48.97260273972603
26 2.5743040182210506
0.061645886221465454
58.79169361540863
K7EMD9 Related Proteins
Keratin, type I cytoskeletal
13 (Fragment)
18.787878787878785
4 0.5505157798326246
0.06070707070707076
19.20172933573258
P13646
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
13
40.61135371179039
20 1.6639858572587722
0.027271583980691398
49.55737861381936
K7ERE3
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
13
31.08433734939759
16 1.4040991835099716
0.02068739243662859
45.23223677712046
P02533 Single
Protein
Keratin, type I cytoskeletal
14
55.50847457627119
24 1.7825594022071245
0.06805756935821794
51.52938407421822
P08779 Single
Protein
Keratin, type I cytoskeletal
16
68.92177589852008
33 3.86967525
1658631 0.07589838127
230322 51.23620252
1320465
F5GWP8
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
17
7.736389684813753
2 0.11033631
8167638 0.00900532132
623823 40.26237172
672955
F5GWP8
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
17
8.022922636103152
3 0.16998910658906596
0.011246418338108884
40.26237172672955
F5GWP8
Related and
Unrelated Proteins
Keratin, type I cytoskeletal
17
22.636103151862464
8 0.5199110829529336
0.01448122558314239
40.26237172672955
P35527 Related Proteins
Keratin, type I cytoskeletal 9
34.02889245585875
14 0.9054607179632472
0.02699504460067839
62.02681468242336
Q9NSB2 Related Proteins
Keratin, type II cuticular
Hb4
63.83333333333333
33 2.0569907009353776
0.06387756583233463
64.80146264176814
P04264 Related Proteins
Keratin, type II cytoskeletal
1
36.33540372670807
33 2.21878751
1821236 0.09234496109
924112 65.99900087
210398
P04264 Related Keratin, type 36.18012422 31 1.99863134 0.03715559188 65.99900087
68
Proteins II cytoskeletal 1
360248 85755667 736077 210398
P35908 Single
Protein
Keratin, type II cytoskeletal 2 epidermal
32.70735524256651
22 1.1800253234900517
0.021890729594864256
65.39321717101762
Q01546 Single
Protein
Keratin, type II cytoskeletal
2 oral
32.9153605015674
27 1.6416483203860923
0.04003354097912183
65.80008965822827
P12035 Single
Protein
Keratin, type II cytoskeletal
3
14.649681528662422
12 0.5973122800602544
0.041079397861950924
64.37756213683015
P19013 Related Proteins
Keratin, type II cytoskeletal
4
51.49812734082397
31 2.28599324
7600655 0.14461868300
500147 57.24982857
9886504
F8VV57 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
37.121212121212125
4 1.3101297000831598
0.12286821705426357
12.14979750828232
F8W0C6 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
48.60335195530726
12 1.8942661247167512
0.07975541948714984
20.643608929250135
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
49.75124378109453
14 2.3000347911252845
0.03732172646381498
23.09540181798727
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
47.340425531914896
34 2.2745491628777286
0.03514860914390704
60.00827426891604
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
9.397163120567376
4 0.1497569953977358
0.024014336917562724
60.00827426891604
P04259
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6B
17.5531914893617
14 0.6297508346206442
0.010502703516885679
60.03029098160933
P48668
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6C
53.723404255319146
34 2.2745491628777286
0.016279644616316077
59.98830556904431
Q8N1N4 Single
Protein
Keratin, type II cytoskeletal
78
15.192307692307693
8 0.35935639087852556
0.012384434406173537
56.83049222411559
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
25.28735632183908
14 0.4816025176256622
0.010914261146381048
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
15.086206896551724
9 0.28752626598035547
0.015634960880904082
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
10.057471264367816
6 0.18350672951946878
0.007262588943623421
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
8.620689655172413
4 0.11887221158742212
0.005923458725182864
76.57688676417338
P30740 Unrelated Proteins
Leukocyte elastase inhibitor
44.06332453825858
14 1.1544346900318838
0.04148739114915583
42.714702418590555
Q9HC84 Single Mucin-5B 1.614022908 6 0.05095164 0.00172137710 595.9601181
69
Protein 7122526 3813099246
16813453 160408
P24158 Related Proteins
Myeloblastin 27.734375 4 0.7782794100389228
0.05165525114155255
27.789271098985488
P05164 Single
Protein Myeloperoxid
ase 4.832214765
100671 3
0.08263673387405435
0.005926979611190138
83.81483871714372
P08246 Single
Protein Neutrophil
elastase 35.58052434
456929 7
1.015337685941733
0.05328298720369201
28.499788498823822
P80188 Related Proteins
Neutrophil gelatinase-associated lipocalin
12.121212121212121
2 0.22167734899679203
0.012269938650306749
22.57371613770579
C3PTT6
Related and
Unrelated Proteins
Pancreatic adenocarcino
ma upregulated
factor
47.95918367346939
6 1.6826957952797255
0.09078282828282824
21.55300886308947
P01833 Single
Protein
Polymeric immunoglobu
lin receptor
5.49738219895288
3 0.08789095479256037
0.004553734061930784
83.23165254290421
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.12365591397849462
10.827649221794548
P06702 Related Proteins
Protein S100-A9
26.31578947368421
2 0.4251026703029981
0.05263157894736842
13.233508230835282
Q08188 Single
Protein
Protein-glutamine gamma-
glutamyltransferase E
14.141414141414142
7 0.2814223889440839
0.013810009222853255
76.58358425055039
P29508 Related Proteins
Serpin B3 11.53846153
846154 4
0.216483949025851
0.013677324916518003
44.536544995847535
A0A024QZX5
Related Proteins
Serpin B6 9.210526315
789473 2
0.11887221158742212
0.005263157894736842
42.99625360584759
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
34.343434343434346
12 0.7378008287493754
0.01192559335721838
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
34.84848484848485
12 0.7378008287493754
0.010662967094592107
45.11835747484806
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
28.14569536423841
15 0.5160826983319609
0.013175929060987523
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
27.81456953642384
14 0.47460159321419093
0.03359869768349508
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
28.14569536423841
13 0.4342554407501811
0.027316182997497662
69.18136901532891
P68363
Related and
Unrelated Proteins
Tubulin alpha-1B chain
9.090909090909092
3 0.19377664171443643
0.00975975975975976
50.119604615111854
C9JDS9 Related Proteins
Tubulin alpha-4A chain
(Fragment)
19.230769230769234
2 0.46779926762206947
0.0308641975308642
17.65980705160292
A0A075B736
Related and
Tubulin beta-8 chain
3.43980343980344
2 0.1497569953977358
0.0035211267605633804
45.66683080042066
70
Unrelated Proteins
A0A0A0MR02
Related Proteins
Voltage-dependent
anion-selective channel
protein 2 (Fragment)
15.24822695035461
3 0.2915496650148839
0.027662517289073305
30.328869561053356
Q9Y277 Single
Protein
Voltage-dependent
anion-selective channel
protein 3
10.60070671378092
2 0.1659144011798317
0.008264462809917354
30.639285645312086
P25311 Single
Protein Zinc-alpha-2-glycoprotein
21.140939597315437
4 0.32194114846602906
0.010889774236387784
34.23710469367686
Appendix 7. All validated proteins in fresh semen sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
P10323 Single
Protein Acrosin
8.788598574821853
2 0.1497569953977358
0.004424778761061947
45.817403494908454
A6NL76
Related and
Unrelated Proteins
Actin, alpha skeletal muscle
4.724409448818897
2 0.2022644346174129
5
0.008858267716535433
28.147013604276893
F5H7P4
Related and
Unrelated Proteins
A-kinase anchor
protein 3 (Fragment)
19.1358024691358
3 0.4677992676220694
7
0.011316872427983538
18.2717306218222
Q5JQC9 Related Proteins
A-kinase anchor
protein 4
31.850117096018735
23 0.6559515234819178
0.010743683417059502
94.4165133446506
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
27.51196172248804
9 0.7252105499420405
0.011496663059163059
46.678988138410034
P03973 Single
Protein Antileukoprot
einase 20.45454545
4545457 2
0.2742749857031337
0.011363636363636364
14.315908944800055
A1YZ08 Related Proteins
Beta-globin (Fragment)
16.19047619047619
2 0.6681005372000588
0.02222222222222222
11.411847146306945
J3KNB4 Related Proteins
Cathelicidin antimicrobial
peptide
29.47976878612717
5 0.5085907086001784
0.025144508670520236
19.578268250841486
E5RG36 Related Proteins
Clusterin (Fragment)
14.634146341463413
3 0.7782794100389228
0.027777777777777776
9.318919755181668
H0YAS8 Related Proteins
Clusterin (Fragment)
42.142857142857146
5 0.7782794100389228
0.045782312925170064
16.166165820637204
H0YC35 Related Proteins
Clusterin (Fragment)
13.028169014084508
3 0.2689610031679221
5
0.021985815602836876
33.50827632671542
H0YC35 Related Proteins
Clusterin (Fragment)
17.6056338028169
3 0.2689610031679221
5
0.009361702127659573
33.50827632671542
P12277
Related and
Unrelated Proteins
Creatine kinase B-type
6.036745406824147
2 0.1253355826007649
4
0.00487012987012987
42.617318186065454
P54107 Related Cysteine-rich 23.69477911 5 0.7301957 0.02294520547 28.46233643
71
Proteins secretory protein 1
6465863 388458942 9452058 95561
P81605 Single
Protein Dermcidin 20.0 2
0.4251026703029981
0.05714285714285714
11.27682755014452
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
25.37313432835821
5 0.7782794100389228
0.039271708683473384
22.426332974690606
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
28.35820895522388
5 0.7782794100389228
0.039271708683473384
22.426332974690606
P56851 Unrelated Proteins
Epididymal secretory
protein E3-beta
31.292517006802722
3 0.4677992676220694
7
0.015873015873015876
17.572542210621645
Q16610 Single
Protein
Extracellular matrix protein
1
42.96296296296296
17 1.1877616239495525
0.011345410946334811
60.635275149574795
P02751 Related Proteins
Fibronectin 3.855825649
6228 7
0.0947774484233141
5
0.001420512820512821
262.4601018836126
P02751 Related Proteins
Fibronectin 1.466890192
7912825 3
0.0395704655308148
9
9.435897435897435E-4
262.4601018836126
K7EJY8 Related Proteins
Galectin-3-binding protein
(Fragment)
15.972222222222221
2 0.5848931924611136
0.0273972602739726
15.600550849698703
Q08380 Related Proteins
Galectin-3-binding protein
20.68376068376068
8 0.4924955450518296
0.012289046544125555
65.28928569959861
P20142 Single
Protein Gastricsin
5.154639175257731
2 0.3593563908785255
6
0.04054054054054054
42.39860601325266
A0A0A0MTN3
Related Proteins
Glutathione S-transferase
Mu 3
19.81132075471698
4 0.4454397707459274
4
0.010422282120395327
24.898433659591365
P21266 Related Proteins
Glutathione S-transferase
Mu 3
15.11111111111111
3 0.2915496650148839
0.009185185185185183
26.542137718578985
E7EUT5 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e
10.76923076923077
2 0.1937766417144364
3
0.006756756756756757
27.853060805444024
O14556 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e, testis-specific
11.519607843137255
3 0.1787686347935873
0.00853413654618474
44.47278994794948
G3V1N2
Related and
Unrelated Proteins
HCG1745306, isoform CRA_a
8.181818181818182
2 0.6681005372000588
0.045454545454545456
11.940248402513872
P54652 Related Proteins
Heat shock-related 70 kDa
protein 2
4.694835680751173
2 0.0570520810009829
1
0.0024671052631578946
69.97786578851715
F8W6P5 Related
and Unrelated
Hemoglobin subunit beta (Fragment)
24.444444444444443
2 0.6681005372000588
0.08148148148148147
9.664011956204076
72
Proteins
P16104 Related Proteins
Histone H2AX 22.37762237
7622377 2
0.2022644346174129
5
0.027972027972027972
15.13541509128964
Q96A08 Single
Protein Histone H2B
type 1-A 27.55905511
8110237 5
0.53174046370208
0.017716535433070862
14.158740271128844
P62805 Related Proteins
Histone H4 29.12621359
2233007 3
0.3335214321633239
7
0.022653721682847898
11.360381729559025
P01876
Related and
Unrelated Proteins
Ig alpha-1 chain C region
5.382436260623229
2 0.2328467394420661
3
0.006276150627615063
37.630638369583146
A0A0G2JN06
Related and
Unrelated Proteins
Ig gamma-2 chain C region
(Fragment)
7.975460122699387
2 0.1787686347935873
0.004347826086956522
35.87775615263485
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
47.66355140186916
3 0.8738174228603839
0.040498442367601244
11.711667188377447
C9J4N6 Related Proteins
Isocitrate dehydrogenas
e [NADP] cytoplasmic (Fragment)
14.012738853503185
2 0.2328467394420661
3
0.008492569002123142
17.708279879975482
O75874 Related Proteins
Isocitrate dehydrogenas
e [NADP] cytoplasmic
14.492753623188406
5 0.2532543355087804
0.005744125326370757
46.62951442889963
A0A0B4J1X3
Related and
Unrelated Proteins
Kallikrein 3, (Prostate specific
antigen), isoform CRA_f
46.666666666666664
3 1.6826957952797255
0.052222222222222225
9.939295244151824
P29622
Related and
Unrelated Proteins
Kallistatin 4.683840749
414521 2
0.1188722115874221
2
0.003978779840848806
48.51115813677777
Q15323 Related Proteins
Keratin, type I cuticular Ha1
40.38461538461539
17 1.8013567611988681
0.016917374451138015
47.20705069770084
O76009 Related Proteins
Keratin, type I cuticular Ha3-I
4.702970297029703
3 0.1835067295194687
8
0.0023765432098765433
45.91139143371252
Q14525 Related Proteins
Keratin, type I cuticular Ha3-
II
31.683168316831683
16 1.5719138090593452
0.012657806754216351
46.18453981219836
O76011 Related Proteins
Keratin, type I cuticular Ha4
21.55963302752294
13 1.0395004690050569
0.01149717324066475
49.39222835129151
O76013 Related Proteins
Keratin, type I cuticular Ha6
4.496788008565311
4 0.2216773489967920
3
0.0019430697180057791
52.21397052719633
O76014 Related Proteins
Keratin, type I cuticular Ha7
5.79064587973274
2 0.1220184543019633
4
0.00469283276450512
49.71517718694274
P13645 Single
Protein
Keratin, type I cytoskeletal
10
59.93150684931506
29 3.1401995764592234
0.047906600693310125
58.79169361540863
P02533 Single
Protein
Keratin, type I cytoskeletal
14
34.74576271186441
17 1.0644996558525563
0.022440496169737353
51.52938407421822
P08779 Single Keratin, type I 34.67230443 19 1.4879023 0.02172154965 51.23620252
73
Protein cytoskeletal 16
97463 672388363 5470452 1320465
K7EPJ9 Related Proteins
Keratin, type I cytoskeletal
17 (Fragment)
13.852813852813853
3 0.3335214321633239
7
0.014896867838044309
24.850220348241024
P35527 Single
Protein Keratin, type I cytoskeletal 9
28.08988764044944
12 0.7378008287493754
0.047004586441206156
62.02681468242336
P78386 Related Proteins
Keratin, type II cuticular
Hb5
37.08086785009862
25 1.392147081462638
0.010629797754568116
55.766472822378276
O43790
Related and
Unrelated Proteins
Keratin, type II cuticular
Hb6
25.720164609053498
18 0.9952623149688795
0.009028859142731487
53.46629878272636
P04264 Related Proteins
Keratin, type II cytoskeletal
1
46.58385093167702
39 2.9810717055349722
0.0386707662314013
65.99900087210398
P04264 Related Proteins
Keratin, type II cytoskeletal
1
46.42857142857142
37 2.7087774117744745
0.01770754838608496
65.99900087210398
P35908 Single
Protein
Keratin, type II cytoskeletal 2 epidermal
38.96713615023474
23 1.2586355207213766
0.014882862439378827
65.39321717101762
F8W0C6 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
11.1731843575419
2 0.1937766417144364
3
0.02304964539007092
20.643608929250135
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
10.945273631840797
2 0.1859710123376696
6
0.009271822704658523
23.09540181798727
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
25.37313432835821
5 0.53174046370208
0.013092656376238469
23.09540181798727
P02538
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
6A
23.936170212765955
14 0.6297508346206442
0.005963741688562697
60.00827426891604
P04259 Single
Protein
Keratin, type II cytoskeletal
6B
37.765957446808514
23 1.2309243744089828
0.008459523616154664
60.03029098160933
O95678
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
75
5.9891107078039925
3 0.110336318167638
0.004112006137322593
59.52403085076316
P0C5Y4 Single
Protein
Keratin-associated protein 1-4
18.181818181818183
2 9.0 1.25 12.31459956
1097713
A0A087WY89
Related Proteins
Keratin-associated protein 4-1
23.62204724409449
2 99.0 0.20512820512
820515 13.23078217
2692193
Q9BYR4 Single
Protein
Keratin-associated protein 4-3
20.51282051282051
2 1.5118864315095801
0.020588235294117647
20.487390980336997
C9JCF5
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
38.46153846153846
6 0.8738174228603839
0.028268777925371327
20.330700568898102
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
59.339080459770116
38 1.9067965371321285
0.013106851572035795
76.57688676417338
E7EQB2 Related
and Lactotransferrin (Fragment)
28.16091954022988
17 0.6118219775228428
0.008434298498947139
76.57688676417338
74
Unrelated Proteins
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
24.71264367816092
16 0.5671910803783076
0.010177119543455395
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
14.942528735632186
8 0.2518750258625291
0.00974821906363488
76.57688676417338
F5H245 Related Proteins
L-lactate dehydrogenas
e
18.553459119496853
5 0.3894954943731377
0.008194514554938583
34.78345530992753
F5GZ11 Related Proteins
L-lactate dehydrogenas
e C chain
25.748502994011975
4 0.6681005372000588
0.0124750499001996
18.273454494679733
A0A0G2JN42
Related Proteins
Mucin-6 4.100041000
410004 7
0.1504548126912206
3
0.004102170231202491
256.87106254481546
C9JR96 Related Proteins
Neprilysin (Fragment)
7.725321888412018
2 0.1787686347935873
0.003787878787878788
26.206501468672535
P23284 Related Proteins
Peptidyl-prolyl cis-trans
isomerase B
19.90740740740741
4 0.3219411484660290
6
0.014274691358024692
23.72753554152188
G3V264 Related Proteins
Plasma serine protease inhibitor
(Fragment)
15.11627906976744
3 0.5399265260594921
0.025362318840579712
19.00183930625142
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
19.704433497536947
8 0.5347813444464111
0.012889781159011926
45.6456892396854
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
5.665024630541872
2 0.1130421932675225
2
0.009861932938856018
45.6456892396854
P13796
Related and
Unrelated Proteins
Plastin-2 2.870813397
1291865 2
0.0670112884883351
5
0.0012626262626262627
70.24389151037927
P12273 Single
Protein
Prolactin-inducible protein
65.75342465753425
9 2.651741272548377
0.12902261361165474
16.561795793109898
C9JIZ6
Related and
Unrelated Proteins
Prosaposin 18.02656546
4895636 8
0.4924955450518296
0.005188124632569078
58.402024132169025
C9JIZ6 Related Proteins
Prosaposin 10.62618595
825427 5
0.2843862635897427
0.004774968972499836
58.402024132169025
A0A0J9YXB8
Related and
Unrelated Proteins
Prosaposin (Fragment)
22.15568862275449
4 0.6237767391887217
0.011477045908183632
18.84223226985116
M0QZF9 Related Proteins
Prostate-specific antigen
(Fragment)
28.488372093023255
4 1.0309176209047357
0.07951070336391435
19.077636924772218
M0QZF9 Related Proteins
Prostate-specific antigen
(Fragment)
25.0 2 0.4251026703029981
0.011467889908256881
19.077636924772218
P15309 Single Prostatic acid 21.76165803 10 0.8632463 0.02812852981 44.53747290
75
Protein phosphatase 1088082 119315603 392307 479376
P33764 Single
Protein Protein S100-
A3 34.65346534
653465 3
0.9952623149688795
0.03300330033003301
11.705427107785646
P49221
Related and
Unrelated Proteins
Protein-glutamine gamma-
glutamyltransferase 4
28.50877192982456
13 0.5738730431619035
0.005911085256414506
77.09605137624143
P49221
Related and
Unrelated Proteins
Protein-glutamine gamma-
glutamyltransferase 4
25.146198830409354
12 0.5199110829529336
0.005976360669002827
77.09605137624143
A6NCN2 Single
Protein
Putative keratin-87
protein
43.92156862745098
16 1.5719138090593452
0.02454025852217254
29.098648422651973
P04279 Single
Protein Semenogelin-
1 46.96969696
969697 21
1.209338675153079
0.02094908501664457
52.099881419104314
Q02383
Related and
Unrelated Proteins
Semenogelin-2
47.93814432989691
23 1.0073891800546302
0.02278506623973967
65.40541810235962
Q02383
Related and
Unrelated Proteins
Semenogelin-2
25.601374570446733
14 0.5283067326587692
0.023977254131893274
65.40541810235962
C9JVG0 Related Proteins
Serotransferrin (Fragment)
15.671641791044777
2 0.4251026703029981
0.016853932584269662
14.680972242798342
P02787
Related and
Unrelated Proteins
Serotransferrin
9.169054441260744
6 0.1911031332830068
8
0.004521228545618789
77.01362996651181
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
16 1.0892961308540396
0.031570906409462265
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
16 1.0892961308540396
0.014730420537636993
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
29.545454545454547
12 0.7378008287493754
0.06611060095908577
45.11835747484806
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
24 0.946061794205562
0.021300016170924474
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
24 0.946061794205562
0.013899067497554938
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
23 0.8928161540172448
0.009830790547386548
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
30.629139072847682
21 0.7906555296923772
0.019144742655375656
69.18136901532891
P35326 Related Proteins
Small proline-rich protein
43.05555555555556
2 0.9306977288832501
0.023148148148148147
7.959674810476288
76
2A
P11169 Related Proteins
Solute carrier family 2,
facilitated glucose
transporter member 3
5.846774193548387
2 0.1450475699382818
3
0.0030737704918032786
53.88925360580755
O00391 Related Proteins
Sulfhydryl oxidase 1
7.228915662650602
4 0.1188722115874221
2
0.0029467232437529463
82.525505695686
P60174 Related Proteins
Triosephosphate isomerase
8.041958041958042
2 0.1659144011798317
0.005244755244755245
30.771696101193438
Q13748 Related Proteins
Tubulin alpha-3C/D chain
17.77777777777778
6 0.4251026703029981
0.0106425702811245
49.92760276463705
P04350
Related and
Unrelated Proteins
Tubulin beta-4A chain
5.63063063063063
2 0.1406249238513208
6
0.006230529595015576
49.5539008224435
P25311 Single
Protein Zinc-alpha-2-glycoprotein
29.86577181208054
8 0.7475284000076838
0.02192657939669893
34.23710469367686
Appendix 8. All validated proteins in one week old semen sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
B0AZS6 Related Proteins
14-3-3 protein zeta/delta
18.452380952380953
2 0.2915496650148839
0.008928571428571428
19.228432227324365
E5RGE1 Related Proteins
14-3-3 protein zeta/delta (Fragment)
44.230769230769226
3 1.6826957952797255
0.052884615384615384
5.900867956717122
A6NL76
Related and
Unrelated Proteins
Actin, alpha skeletal muscle
4.330708661417323
2 0.2022644346174129
5
0.005905511811023622
28.343056640344084
I3L1U9
Related and
Unrelated Proteins
Actin, cytoplasmic 2
(Fragment)
3.7383177570093453
2 0.2589254117941673
0.004672897196261682
23.941979505351206
I3L1U9
Related and
Unrelated Proteins
Actin, cytoplasmic 2
(Fragment)
14.018691588785046
2 0.2589254117941673
0.005841121495327103
23.941979505351206
O75969
Related and
Unrelated Proteins
A-kinase anchor
protein 3
3.9859320046893316
3 0.0715193052376064
1
0.005015959872321022
95.79756464191061
Q5JQC9 Related Proteins
A-kinase anchor
protein 4
31.733021077283368
24 0.692666615037876
0.01645890406275211
95.71879922709695
G3V3A0
Related and
Unrelated Proteins
Alpha-1-antichymotry
psin
9.268292682926829
2 0.2451970847350328
8
0.012195121951219513
23.587149711845623
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
45.45454545454545
14 1.3357214690901222
0.012158375084511445
47.029064988530074
P01009 Related Proteins
Alpha-1-antitrypsin
5.980861244019139
2 0.1288378916846890
7
0.0037878787878787876
47.05709628865835
P06733 Related Alpha-enolase 6.221198156 2 0.0890229 0.00345622119 47.50339920
77
and Unrelated Proteins
682028 6226373019
81566822 969828
D6RBL5 Related Proteins
Annexin 6.538461538
461539 2
0.1601553017399717
2
0.005128205128205128
29.557028662930108
E9PHT9 Related Proteins
Annexin 14.72392638
0368098 2
0.2451970847350328
8
0.008179959100204498
18.35249589254833
H0YKS4 Related Proteins
Annexin (Fragment)
15.340909090909092
2 0.2328467394420661
3
0.008522727272727272
19.72811294758306
A1YZ08 Related Proteins
Beta-globin (Fragment)
16.19047619047619
2 0.6681005372000588
0.030952380952380953
11.481862516330946
C9JE88 Related Proteins
Carboxypeptidase E
(Fragment)
17.687074829931973
2 0.3894954943731377
0.008503401360544218
16.655435195112936
J3KNB4 Related Proteins
Cathelicidin antimicrobial
peptide
35.83815028901734
6 0.637893706954064
0.04672447013487476
19.732302064894288
C9JH19 Related Proteins
Cathepsin D (Fragment)
7.023411371237458
2 0.2022644346174129
5
0.006818181818181818
32.94940452458041
E7ERK6 Related Proteins
Clusterin (Fragment)
10.294117647058822
2 0.2022644346174129
5
0.03592814371257485
23.89271049633231
H0YAS8 Related Proteins
Clusterin (Fragment)
42.142857142857146
5 0.7782794100389228
0.08129251700680269
16.348205782699605
H0YC35 Related Proteins
Clusterin (Fragment)
17.6056338028169
3 0.2689610031679221
5
0.0198581560283688
33.858353176835415
P01034 Unrelated Proteins
Cystatin-C 36.98630136
9863014 4
0.8478497974222909
0.024543378995433792
15.915108539527141
P54107 Related Proteins
Cysteine-rich secretory protein 1
34.53815261044177
8 1.4040991835099716
0.059776581865622944
28.7283948456473
P81605 Single
Protein Dermcidin 20.0 2
0.4251026703029981
0.1357142857142857
11.374849068178118
P27487 Single
Protein Dipeptidyl
peptidase 4 3.655352480
417754 3
0.1052951411260216
7
0.005817335660267597
89.11868106265338
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
25.37313432835821
5 0.7782794100389228
0.05460317460317456
22.706394454786608
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
28.35820895522388
5 0.7782794100389228
0.04872082166199813
22.706394454786608
P56851 Unrelated Proteins
Epididymal secretory
protein E3-beta
12.925170068027212
2 0.2915496650148839
0.017006802721088437
17.796591394698446
A0A087WY45
Related Proteins
Epididymal sperm-binding
protein 1
21.363636363636363
4 0.7190722018585745
0.01648841354723708
25.912657899809762
Q16610 Single
Protein
Extracellular matrix protein
1
10.37037037037037
5 0.2589254117941673
0.012160371106693173
61.09737659173322
P02751 Related Proteins
Fibronectin 8.298407376
362114 16
0.2299505200127773
8
0.003948484848484847
265.13468901853
78
P02751 Related Proteins
Fibronectin 3.730092204
5264038 8
0.1090313431155935
9
0.003977289377289378
265.13468901853
H3BQN4 Related Proteins
Fructose-bisphosphate
aldolase
4.43213296398892
2 0.1188722115874221
2
0.004155124653739612
39.581448596571214
Q08380 Related Proteins
Galectin-3-binding protein
15.213675213675213
7 0.4196258964747483
5
0.009185209072342257
66.04545169585785
A0A087WT12
Related Proteins
Glutathione peroxidase
8.583690987124463
2 0.1029433117916129
8
0.006437768240343348
27.07075946024807
A0A0A0MTN3
Related Proteins
Glutathione S-transferase
Mu 3
34.43396226415094
6 0.7378008287493754
0.010413093196112066
25.038464399639373
P21266 Related Proteins
Glutathione S-transferase
Mu 3
22.22222222222222
4 0.4065272421052370
5
0.00726984126984127
26.696171532631798
E7EUT5 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e
20.384615384615383
3 0.3043213867190053
0.008258258258258258
28.11911921153523
O14556 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e, testis-specific
20.588235294117645
6 0.3894954943731377
0.01641566265060241
44.83686987207431
A0A087WU08
Related Proteins
Haptoglobin 7.829181494
661921 2
0.1659144011798317
0.006072874493927126
31.653926892481003
G3V1N2
Related and
Unrelated Proteins
HCG1745306, isoform CRA_a
8.181818181818182
2 0.6681005372000588
0.11363636363636363
12.08027914256187
P07900
Related and
Unrelated Proteins
Heat shock protein HSP
90-alpha
8.19672131147541
5 0.1124904621939377
7
0.003784421116689487
85.19481044264995
F8W6P5
Related and
Unrelated Proteins
Hemoglobin subunit beta (Fragment)
24.444444444444443
2 0.6681005372000588
0.06666666666666668
9.734027326228073
F8W6P5
Related and
Unrelated Proteins
Hemoglobin subunit beta (Fragment)
21.11111111111111
2 0.6681005372000588
0.03333333333333333
9.734027326228073
P68871
Related and
Unrelated Proteins
Hemoglobin subunit beta
14.965986394557824
2 0.3894954943731377
0.008503401360544218
16.05830554829089
E9PEW8
Related and
Unrelated Proteins
Hemoglobin subunit delta (Fragment)
16.346153846153847
2 0.5848931924611136
0.019230769230769232
11.422787674368216
P02042 Related Proteins
Hemoglobin subunit delta
9.523809523809524
2 0.3593563908785255
6
0.015306122448979591
16.129308305025862
P16104 Related Proteins
Histone H2AX 22.37762237
7622377 2
0.2022644346174129
5
0.038461538461538464
15.233436609323244
Q96A08 Single Histone H2B 35.43307086 7 0.8165997 0.04628796400 14.41079560
79
Protein type 1-A 614173 883753275 4499436 3215245
P62805 Related Proteins
Histone H4 50.48543689
320388 5
0.6155980984398741
0.044336569579288033
11.388387877568626
A0A0G2JN06
Related and
Unrelated Proteins
Ig gamma-2 chain C region
(Fragment)
11.65644171779141
3 0.2798022139979537
0.007971014492753625
36.40987296481729
A0A0G2JN06
Related and
Unrelated Proteins
Ig gamma-2 chain C region
(Fragment)
7.975460122699387
2 0.1787686347935873
0.01594202898550725
36.40987296481729
A0A075B6N8
Related and
Unrelated Proteins
Ig gamma-3 chain C region
(Fragment)
5.570291777188329
2 0.1325413151528118
7
0.003272727272727272
41.873449923181354
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
79.43925233644859
5 1.8480358684358014
0.048909657320872275
11.935716372454246
A0A075B6L0
Related and
Unrelated Proteins
Ig lambda-3 chain C regions
(Fragment)
28.30188679245283
2 0.5848931924611136
0.012578616352201257
11.535570877437527
A0A0B4J231
Related and
Unrelated Proteins
Immunoglobulin lambda-like polypeptide 5
13.023255813953488
2 0.2742749857031337
0.007490636704119849
23.485747223633687
O75874 Related Proteins
Isocitrate dehydrogenas
e [NADP] cytoplasmic
5.797101449275362
2 0.0944997993195435
5
0.0039164490861618795
46.937582057005244
P13645 Single
Protein
Keratin, type I cytoskeletal
10
47.26027397260274
24 2.2406960357871677
0.04110284571876937
60.02396412783115
P02533 Single
Protein
Keratin, type I cytoskeletal
14
19.06779661016949
9 0.4677992676220694
7
0.013770903330440724
52.25754392246785
P08779 Single
Protein
Keratin, type I cytoskeletal
16
26.004228329809727
11 0.6949881513903466
0.013477315849512467
52.09039003561333
P35527 Single
Protein Keratin, type I cytoskeletal 9
46.3884430176565
24 2.019951720402016
0.04271799445788091
63.24508212084106
P04264 Related Proteins
Keratin, type II cytoskeletal
1
43.32298136645963
41 3.27335754210932
0.04726986085696493
67.25927753253609
P04264 Related Proteins
Keratin, type II cytoskeletal
1
43.7888198757764
41 3.27335754210932
0.045027708390597214
67.25927753253609
P04264 Related Proteins
Keratin, type II cytoskeletal
1
45.03105590062112
41 3.27335754210932
0.02709048865965556
67.25927753253609
P35908 Related Proteins
Keratin, type II cytoskeletal 2 epidermal
31.29890453834116
19 0.9602329826462779
0.02388981589445987
66.56947538742092
F8W0C6 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
10.614525139664805
2 0.1937766417144364
3
0.00911854103343465
20.797642743302937
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
29.850746268656714
6 0.6681005372000588
0.013638158414277821
23.22142948403047
P02538 Related
and Keratin, type II cytoskeletal
12.943262411347517
7 0.2766169490574077
0.008008076153237445
60.8484587092041
80
Unrelated Proteins
6A 5
H0YIC5 Related Proteins
Keratin, type II cytoskeletal 73 (Fragment)
5.964912280701754
2 0.1364636663857248
2
0.01809954751131222
31.676775378651893
O95678
Related and
Unrelated Proteins
Keratin, type II cytoskeletal
75
5.9891107078039925
3 0.110336318167638
0.004163150492264417
60.47623988308964
P49223 Single
Protein
Kunitz-type protease
inhibitor 3
10.112359550561797
2 0.5848931924611136
0.03932584269662921
10.300968613824361
E7ER44
Related and
Unrelated Proteins
Lactotransferrin
22.88135593220339
16 0.5505157798326246
0.005016411911613034
78.61991452957133
C9JCF5
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
37.91208791208791
5 0.6876124757881477
0.03177152517811858
20.4147190129269
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
58.90804597701149
39 1.9895770856517885
0.02092331723906281
77.27704046441345
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
25.718390804597703
17 0.6118219775228428
0.013826185775666008
77.27704046441345
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
24.71264367816092
16 0.5671910803783076
0.01129316686614199
77.27704046441345
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
14.942528735632186
8 0.2518750258625291
0.011376777943715057
77.27704046441345
F5H155 Related Proteins
L-lactate dehydrogenas
e 24.0 4
0.4924955450518296
0.008941798941798942
24.856767372954035
F5H245 Related Proteins
L-lactate dehydrogenas
e
14.465408805031446
4 0.3010252169108314
0.009098939929328623
35.16153830805714
F5GZ11 Related Proteins
L-lactate dehydrogenas
e C chain
20.35928143712575
3 0.4677992676220694
7
0.022027373823781008
18.469497530746935
A0A0G2JN42
Related Proteins
Mucin-6 8.446084460
844608 18
0.4339094903163154
0.01029776627682042
261.3940554483679
P59665 Related Proteins
Neutrophil defensin 1
20.212765957446805
3 1.1544346900318838
0.12433862433862433
10.236187843494735
Q5T4C6
Related and
Unrelated Proteins
Outer dense fiber protein 2
(Fragment)
10.731707317073171
2 0.1188722115874221
2
0.01707317073170732
23.519176908481832
P23284 Related Proteins
Peptidyl-prolyl cis-trans
isomerase B
39.351851851851855
8 0.7475284000076838
0.01466600529100529
23.853563207565088
A0A0A0MRQ5
Related and
Unrelated Proteins
Peroxiredoxin-1
19.587628865979383
2 0.5199110829529336
0.015463917525773196
10.767528643380924
P00558 Related Proteins
Phosphoglycerate kinase 1
6.474820143884892
2 0.0944997993195435
0.0035971223021582736
44.92220054224713
81
5
P07205 Related Proteins
Phosphoglycerate kinase 2
4.796163069544365
2 0.0926008611173783
1
0.008393285371702638
45.08940048266703
G3V264 Related Proteins
Plasma serine protease inhibitor
(Fragment)
15.11627906976744
2 0.3335214321633239
7
0.010869565217391304
19.26789771234262
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
17.733990147783253
7 0.4547578108480476
7
0.027574668920822755
46.17780605186783
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
5.665024630541872
2 0.1130421932675225
2
0.00690335305719921
46.17780605186783
P13796
Related and
Unrelated Proteins
Plastin-2 15.15151515
1515152 7
0.2548492322523297
0.004365079365079364
70.6919898785329
B4DV12
Related and
Unrelated Proteins
Polyubiquitin-B
23.52941176470588
2 0.2328467394420661
3
0.004357298474945533
17.288250487698054
P12273 Single
Protein
Prolactin-inducible protein
67.8082191780822
9 2.651741272548377
0.13393944335725155
16.617808089129095
M0QZF9 Related Proteins
Prostate-specific antigen
(Fragment)
45.93023255813954
5 1.4244620170823286
0.07479248580166008
19.245673812829825
M0R1F0 Related Proteins
Prostate-specific antigen
(Fragment)
39.69465648854962
7 1.0805675382171707
0.03593736593736593
28.937531405477543
P15309 Single
Protein Prostatic acid phosphatase
34.715025906735754
13 1.2456979955397744
0.03839685075640131
44.9575651249378
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.016129032258064516
10.883661517813751
P49221
Related and
Unrelated Proteins
Protein-glutamine gamma-
glutamyltransferase 4
27.192982456140353
14 0.6297508346206442
0.007661527070861417
77.82421122449104
P49221
Related and
Unrelated Proteins
Protein-glutamine gamma-
glutamyltransferase 4
27.631578947368425
14 0.6297508346206442
0.0045987399345673915
77.82421122449104
P20337 Unrelated Proteins
Ras-related protein Rab-
3B
8.67579908675799
2 0.2115276586285883
6
0.005707762557077625
24.98016280066708
P04279 Related Proteins
Semenogelin-1
9.090909090909092
3 0.119902793272475
0.0059781488352916926
52.870050489368396
Q02383
Related and
Unrelated Proteins
Semenogelin-2
18.04123711340206
8 0.2742749857031337
0.01161225658648339
66.48365480072933
P02787
Related and
Unrelated Proteins
Serotransferrin
11.031518624641834
8 0.2626001098748562
7
0.008519339738851928
77.58775600070864
82
P02787
Related and
Unrelated Proteins
Serotransferrin
4.584527220630372
4 0.1236548001387509
6
0.005555555555555556
77.58775600070864
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
19 1.3988329190194904
0.023499754725918646
45.34240665892489
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
18 1.2908676527677732
0.02053831534976497
45.34240665892489
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
29.545454545454547
17 1.1877616239495525
0.08613473346650724
45.34240665892489
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
23 0.8928161540172448
0.019978244272235022
69.55945201345854
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
23 0.8928161540172448
0.013898044857081628
69.55945201345854
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.007684772421369718
69.55945201345854
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
30.629139072847682
20 0.7416619158620705
0.017509927256889657
69.55945201345854
Q9HBV2 Single
Protein
Sperm acrosome
membrane-associated protein 1
8.503401360544217
2 0.2328467394420661
3
0.011904761904761904
32.45897068733914
P60174 Related Proteins
Triosephosphate isomerase
15.034965034965033
3 0.2589254117941673
0.00815850815850816
30.981742211265434
Q13748 Related Proteins
Tubulin alpha-3C/D chain
20.22222222222222
7 0.5117750706156623
0.008813826735513482
50.20766424473307
Q14508 Single
Protein
WAP four-disulfide core
domain protein 2
25.806451612903224
3 1.371373705661655
0.014784946236559139
13.109904292093264
P25311 Single
Protein Zinc-alpha-2-glycoprotein
41.946308724832214
10 1.0092330025650473
0.02529089989249352
34.50316309976807
Appendix 9. All validated proteins in two weeks old semen sample.
Accession
Protein Inference
Class
Protein name Sequence
Coverage (%)
#Validated Peptides
emPAI
NSAF
MW (kDa)
Q5JQC9 Related Proteins
A-kinase anchor
protein 4
13.348946135831381
10 0.24519708473503288
0.00573272699185108
94.4165133446506
A0A024R6I7
Related Proteins
Alpha-1-antitrypsin
23.205741626794257
8 0.6237767391887217
0.009970238095238095
46.678988138410034
P15144 Related
and Unrelated
Aminopeptidase N
2.5853154084798344
2 0.05925372517728888
5
0.002074688796680498
109.47088086489971
83
Proteins
O95412 Related Proteins
Beta globin (Fragment)
56.666666666666664
2 9.0 0.30555555555
555547 3.105616870
99756
C8C504 Single
Protein Beta-globin
95.23809523809523
17 12.593563908785255
0.09113337337605304
15.987342548192922
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
31.443298969072163
6 1.1544346900318838
0.039015151515151524
21.38873696621423
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
15.979381443298967
4 0.6681005372000588
0.044101731601731614
21.38873696621423
E5RFE7 Related Proteins
Carbonic anhydrase 1 (Fragment)
17.010309278350515
3 0.46779926762206947
0.026623376623376625
21.38873696621423
E5RFL2 Related Proteins
Carbonic anhydrase 1 (Fragment)
53.38983050847458
4 1.7825594022071245
0.0713276836158192
13.248254100518784
P04040 Related Proteins
Catalase 4.174573055
028463 2
0.08414586893583476
0.003092783505154639
59.71875531355211
J3KNB4 Related Proteins
Cathelicidin antimicrobial
peptide
31.213872832369944
5 0.5085907086001784
0.04007707129094412
19.578268250841486
H0YAS8 Related Proteins
Clusterin (Fragment)
42.142857142857146
5 0.7782794100389228
0.04180272108843538
16.166165820637204
H0YC35 Related Proteins
Clusterin (Fragment)
17.6056338028169
3 0.26896100316792215
0.013049645390070926
33.50827632671542
P54107 Related Proteins
Cysteine-rich secretory protein 1
34.53815261044177
6 0.9306977288832501
0.025913242009132424
28.4623364395561
P81605 Single
Protein Dermcidin
22.727272727272727
3 0.7012542798525889
0.16904761904761906
11.27682755014452
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
25.37313432835821
5 0.7782794100389228
0.04492997198879551
22.426332974690606
E7EMS2
Related and
Unrelated Proteins
Epididymal secretory protein E1
28.35820895522388
5 0.7782794100389228
0.04492997198879551
22.426332974690606
P02751 Related Proteins
Fibronectin 6.789606035
205365 11
0.15291622965094254
0.0023473748473748475
262.4601018836126
H0Y7Z1
Related and
Unrelated Proteins
Fibronectin (Fragment)
5.802357207615594
4 0.11735910194850852
0.0021684223495348977
120.69924410608232
Q08380 Related Proteins
Galectin-3-binding protein
9.914529914529915
4 0.22167734899679203
0.010346124905944318
65.28928569959861
A0A0A0MTN3
Related Proteins
Glutathione S-transferase
Mu 3
9.433962264150944
2 0.20226443461741295
0.007075471698113208
24.898433659591365
O14556 Related Proteins
Glyceraldehyde-3-
phosphate dehydrogenas
e, testis-specific
6.127450980392156
2 0.11588399250774839
0.004518072289156626
44.47278994794948
G3V1N2
Related and
Unrelated Proteins
HCG1745306, isoform CRA_a
27.27272727272727
4 1.7825594022071245
0.07575757575757575
11.940248402513872
G3V1N2 Related HCG1745306, 8.181818181 2 0.66810053 0.29545454545 11.94024840
84
and Unrelated Proteins
isoform CRA_a
818182 72000588 454547 2513872
P07900
Related and
Unrelated Proteins
Heat shock protein HSP
90-alpha
3.415300546448088
2 0.04356262977539105
0.0021299254526091584
84.60668133444835
Q4TZM4
Related and
Unrelated Proteins
Hemoglobin beta chain (Fragment)
18.81188118811881
2 0.5848931924611136
0.03630363036303629
11.015638867792866
P02042 Related Proteins
Hemoglobin subunit delta
92.51700680272108
15 9.0 0.08311330198
635113 16.04528986
0997062
P62805 Related Proteins
Histone H4 38.83495145
631068 4
0.46779926762206947
0.023462783171521034
11.360381729559025
A0A0G2JN06
Related and
Unrelated Proteins
Ig gamma-2 chain C region
(Fragment)
7.975460122699387
2 0.1787686347935873
0.007608695652173913
35.87775615263485
A0A075B6N8
Related and
Unrelated Proteins
Ig gamma-3 chain C region
(Fragment)
6.896551724137931
2 0.13254131515281187
0.008484848484848484
41.29932388898452
A0A075B6H6
Related and
Unrelated Proteins
Ig kappa chain C region
(Fragment)
49.532710280373834
3 0.8738174228603839
0.02959501557632399
11.711667188377447
A0A0B4J1X3
Related and
Unrelated Proteins
Kallikrein 3, (Prostate specific
antigen), isoform CRA_f
46.666666666666664
3 1.6826957952797255
0.06555555555555556
9.939295244151824
P13645 Single
Protein
Keratin, type I cytoskeletal
10
45.37671232876712
22 1.9382253839710089
0.03855687187377902
58.79169361540863
P02533 Single
Protein
Keratin, type I cytoskeletal
14
15.889830508474576
8 0.40652724210523705
0.014081559621608156
51.52938407421822
P08779 Single
Protein
Keratin, type I cytoskeletal
16
19.45031712473573
9 0.5399265260594921
0.014800206568499246
51.236202521320465
P35527 Related Proteins
Keratin, type I cytoskeletal 9
38.04173354735153
21 1.6302679918953817
0.06886960919822949
62.02681468242336
Q9NSB2 Related Proteins
Keratin, type II cuticular
Hb4 8.0 4
0.14504756993828183
0.0036613942589338016
64.80146264176814
P04264 Related Proteins
Keratin, type II cytoskeletal
1
42.39130434782609
42 3.4274518434944925
0.04218357074981934
65.99900087210398
P04264 Related Proteins
Keratin, type II cytoskeletal
1
42.85714285714286
42 3.4274518434944925
0.046667875682554795
65.99900087210398
P04264 Related Proteins
Keratin, type II cytoskeletal
1
44.099378881987576
42 3.4274518434944925
0.019762046086142234
65.99900087210398
P35908 Single
Protein
Keratin, type II cytoskeletal 2 epidermal
45.22691705790297
24 1.3400803470013467
0.017632989006432412
65.39321717101762
H0YI76 Related Proteins
Keratin, type II cytoskeletal 5 (Fragment)
24.378109452736318
5 0.53174046
370208 0.01677327647
4769016 23.09540181
798727
P02538 Related Keratin, type 13.82978723 8 0.32194114 0.00964113105 60.00827426
85
and Unrelated Proteins
II cytoskeletal 6A
4042552 846602906 9537131 891604
P04259 Related Proteins
Keratin, type II cytoskeletal
6B
14.361702127659573
8 0.32194114846602906
0.007883559093236512
60.03029098160933
P49223 Single
Protein
Kunitz-type protease
inhibitor 3
24.719101123595504
4 1.5118864315095801
0.040262172284644196
10.244956317805164
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
56.75287356321839
35 1.6719557914574237
0.018069572959375415
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
24.71264367816092
16 0.5671910803783076
0.010202868370256859
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
14.942528735632186
8 0.2518750258625291
0.0077747695941888335
76.57688676417338
E7EQB2
Related and
Unrelated Proteins
Lactotransferrin (Fragment)
9.913793103448276
5 0.15073576110506148
0.006490603904397009
76.57688676417338
F5H155 Related Proteins
L-lactate dehydrogenas
e 24.0 4
0.4924955450518296
0.008518518518518519
24.576705892858026
F5H245 Related Proteins
L-lactate dehydrogenas
e
6.918238993710692
2 0.14062492385132086
0.00530035335689046
34.78345530992753
P00338 Related Proteins
L-lactate dehydrogenas
e A chain
6.626506024096386
2 0.12533558260076494
0.005611672278338945
36.66536097963206
A0A0G2JN42
Related Proteins
Mucin-6 11.56211562
1156211 20
0.4924955450518296
0.009394621720425095
256.87106254481546
P59665 Related Proteins
Neutrophil defensin 1
20.212765957446805
3 1.1544346900318838
0.06878306878306878
10.194178621480336
P32119
Related and
Unrelated Proteins
Peroxiredoxin-2
23.232323232323232
4 0.5505157798326246
0.026219512195121957
21.87823782783635
G3V264 Related Proteins
Plasma serine protease inhibitor
(Fragment)
15.11627906976744
2 0.33352143216332397
0.014492753623188406
19.00183930625142
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
19.458128078817733
7 0.45475781084804767
0.01819878839109609
45.6456892396854
P05154
Related and
Unrelated Proteins
Plasma serine protease inhibitor
5.665024630541872
2 0.11304219326752252
0.010848126232741619
45.6456892396854
P13796
Related and
Unrelated Proteins
Plastin-2 2.870813397
1291865 2
0.06701128848833515
0.0025252525252525255
70.24389151037927
B4DV12
Related and
Unrelated Proteins
Polyubiquitin-B
23.52941176470588
2 0.23284673944206613
0.004901960784313725
17.204232043669254
86
P12273 Single
Protein
Prolactin-inducible protein
67.8082191780822
9 2.65174127
2548377 0.11627799521
635135 16.56179579
3109898
M0QZF9 Related Proteins
Prostate-specific antigen
(Fragment)
28.488372093023255
4 1.0309176209047357
0.08103975535168192
19.077636924772218
M0QZF9 Related Proteins
Prostate-specific antigen
(Fragment)
25.0 2 0.4251026703029981
0.022935779816513763
19.077636924772218
P15309 Single
Protein Prostatic acid phosphatase
19.4300518134715
7 0.5459277364194774
0.03256643481362582
44.53747290479376
P05109 Single
Protein Protein S100-
A8 23.65591397
8494624 2
0.3894954943731377
0.021505376344086023
10.827649221794548
P49221
Related and
Unrelated Proteins
Protein-glutamine gamma-
glutamyltransferase 4
10.818713450292398
6 0.23284673944206613
0.006176620724859624
77.09605137624143
Q02383
Related and
Unrelated Proteins
Semenogelin-2
8.075601374570446
4 0.12883789168468907
0.006300114547537228
65.40541810235962
P02787
Related and
Unrelated Proteins
Serotransferrin
12.034383954154727
8 0.26260010987485627
0.004137953284294748
77.01362996651181
P02787
Related and
Unrelated Proteins
Serotransferrin
5.014326647564469
3 0.0913767146512734
0.0014905149051490515
77.01362996651181
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
40.909090909090914
18 1.2908676527677732
0.020301118239581258
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
39.39393939393939
16 1.0892961308540396
0.024466815542912388
45.11835747484806
A0A087WWT3
Related and
Unrelated Proteins
Serum albumin
29.545454545454547
14 0.9054607179632472
0.08490954892470043
45.11835747484806
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
23 0.8928161540172448
0.01785082171675831
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
33.443708609271525
23 0.8928161540172448
0.018516638484375028
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
35.264900662251655
22 0.8410273525621605
0.007941904415063799
69.18136901532891
A0A0C4DGB6
Related and
Unrelated Proteins
Serum albumin
30.629139072847682
20 0.7416619158620705
0.014051571625427894
69.18136901532891
87