peptidomics technologies for human body fluids

Michael Schrader*and Peter Schulz-KnappeBioVisioN GmbH & Co.

KG, Feodor-Lynen-Str. 5,

D-30625 Hannover,

Germany.

*e-mail: m.schrader@

biovision.de

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Together with the establishment of the human genome

as the blueprint for human life, knowledge about the

functionally active substances in our body that have

evolved from the genome becomes more and more

important. It is therefore necessary that one aspect of

the human proteome project be directed towards

improving our understanding of the molecules that

make up our complex regulatory systems and, ultimately,

our life.

For decades, many research teams have searched for

the biochemical messenger molecules that organize the

myriad of regulatory processes in our body, such as

peptide hormones, neuropeptides, cytokines and enzyme

inhibitors.These molecules are necessary for the commu-

nication between all the different specialized cell types

that make up the subsystems of the whole body – fasci-

nating machinery that no engineer is able to duplicate.

The transport of these messengers is most often per-

formed through body fluids that enable communication

even between cells that are too remote to interact directly

or by migration.

There has been significant progression in the field of

proteomics since the research of Bayliss and Starling or

Banting and Best during the beginning of the last century,

and since the sequences of secretin and insulin (two of

the first prominent examples for the family of peptide

hormones) were determined decades later1. The tech-

nologies for isolation and identification that have evolved

have led to the discovery of several important classes of

peptide hormones, for example, gut hormones like

secretin and cholecystokinin, neuropeptides such as thy-

rotropin releasing hormone and leuteinizing hormone

releasing hormone, and the growth hormone somato-

statin1.These few examples demonstrate that peptides are

of paramount importance for many physiological

processes (Table 1). Since then, much progress has been

made; however, we have still to achieve a sound knowl-

edge about human peptides and their functions. One

reason is the lack of technologies that enable a compre-

hensive analysis of peptides.The classical discovery strate-

gies for peptides have been purifications from tissue

extracts guided by function, and almost always, many

steps are needed (which often take years) to generate a

new sequence.This can be attributed to the complexity of

biological sources, the small concentration of single

components and the overwhelming amounts of a few

housekeeping proteins.

With the establishment of tools for protein purifi-

cation (i.e. chromatography and electrophoresis) and

protein analysis [i.e. N-terminal chemical Edman

sequencing of almost pure proteins and peptides, and

mass spectrometry (MS) for identification and charac-

terization], the tools for peptide research have become

much more sophisticated. Moreover, the ability to

search expressed sequence tag (EST) and genome data-

bases has made the identification much easier.

Nevertheless, the subdiscipline of peptidomics is a still

growing field of research, although not very well

explored. From the estimated 30 000 genes in the

human genome, around three times the number of pro-

teins are expected to be identified, owing to variations

that occur during transcription and translation2. Further

post-translational and proteolytic processing3,4 should

lead to the discovery of several hundred thousand to

Peptidomics technologies forhuman body fluids

Michael Schrader and Peter Schulz-Knappe

Peptides play a central role in many physiological processes. In order to analysecomprehensively all peptides and small proteins of a whole organism or a subsystem(peptidome), the use of technologies other than 2D gel electrophoresis is necessary.Approaches that use liquid chromatography or affinity purification and massspectrometric identification have now been developed and applied successfully to theanalysis of human body fluids.

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TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001Review | A TRENDS Guide to Proteomics

millions of different peptides that exist in the human

body. A notable number of them should be of potential

use diagnostically or therapeutically; amyloid-β peptides

are proposed to be diagnostic markers for Alzheimer’s

disease5 and the recently discovered peptide hormone

resistin6 is assumed to be of relevance to obesity and

diabetes.

Extending proteomics with technologies forthe analysis of peptidesOver recent years, 2D gel electrophoresis in combi-

nation with MS has become the main proteomics

research tool7. This method analyses medium-sized

proteins that range from 10 to 200 kDa with an isoelec-

tric point (pI) between 4 and 10. However, smaller proteins

and native peptides are not yet covered by standard

proteomics methods8. As there is no clear-cut definition

of a peptide [from, for example, the International Union

of Pure and Applied Chemistry (IUPAC)], the term pep-

tide will be used throughout this article to refer to pep-

tides and small proteins of <1 kDa to molecules of

about 20 kDa. Owing to their physico-chemical proper-

ties and the shortcomings of gel technologies, only a

few faint spots that correspond to peptides of <20 kDa

are visible on a gel image. Development of proteomic

methods that deal with these smaller molecules has not

yet attracted a lot of attention, which does not correlate

with the importance of known and expected substances

in this range. For example, most of the important

biopharmaceutical products approved for therapeutic

applications are molecules within a molecular mass

range of 1–40 kDa (Ref. 9).

Our definition of peptides as small proteins with a mol-

ecular mass that is less than ~20 kDa has its implications,

because the physico-chemical properties of these

molecules are different from proteins. For example, the

mobility of peptides is higher than that of larger pro-

teins, which makes it very difficult to focus them in

gels, and their ability to bind the different stains used

in visualization procedures is small. Another important

factor is the stability of peptides, which tend not to

denature irreversibly. In addition, the folding behaviour

of peptides in solution, as well as during adsorption

and desorption processes, is mostly reversible.

However, their generally lower hydrophobicity enables

them to dissolve easily in aqueous systems without

the use of detergents. Therefore, the most prominent

separation technology for molecules of up to about

20 kDa is liquid chromatography (LC), in particular,

reversed-phase and ion-exchange chromatography8,10.

Another aspect of the lower hydrophobicity of smaller

peptides is that modifications to improve solubility,

such as glycosylation and phosphorylation, are less

common than in proteins. Alternatively, disulfide

bonds, which determine a specific spatial structure, are

often found (for example, in peptide hormones,

chemokines or defensins). Other modifications, such as

N-terminal pyroglutamylation or C-terminal amida-

tion, are introduced to protect these molecules from

proteolysis by amino- or carboxypeptidases during

their transport from the site of release to the site of

action. Consequently, different analytical strategies have

to be applied to peptides when compared with larger

proteins.

After successful extraction and purification of pep-

tides, the next important aspect is sequence identifi-

cation. Presently, most sequence information is

generated by MS followed by database comparison,

Table 1. Examples of peptides and small proteins in human body fluidsand their physiological relevance

Peptide Molecular mass (kDa) Physiological relevance

Thyrotropin-releasing hormone (TRH)33 0.4 Regulation of thyroid hormones

Oxytocin33 1.0 Contraction of the uterus and stimulation of lactation

Angiotensin II (Ref. 33) 1.0 Blood pressure regulation

Calcitonin33 3.5 Bone turnover

Cholecystokinin33 3.9 Flow of bile and exocrine pancreaticsecretion

Insulin33 5.8 Blood glucose regulation

Insulin-like growth factor I (Ref. 33) 7.5 Body growth

Parathyroid hormone33 9.4 Bone turnover

Serum-amyloid protein34 12 Inflammation



owing to the high-throughput nature of the technique

(when compared with Edman sequencing or amino acid

analysis11). Proteins are too large to be analysed directly

by MS, and hence, are usually converted into several pep-

tides by specific proteolytic enzymes, and identified by

comparison of the generated characteristic cleavage

pattern within databases. For peptides, the number of

possible specific internal cleavages is typically too small

for significant identification using databases; therefore,

peptides are identified by their characteristic fragment

pattern after collision-induced dissociation in MS–MS

experiments12. To circumvent the shortcomings of pro-

tein identification after separation by 2D gel electro-

phoresis, some effort has been made to apply LC to

enzymatically-treated samples, in order to convert pro-

teins to peptides. As shown successfully for yeast13, the

whole protein content of a source can be trypsinated and

submitted to multidimensional chromatography with

subsequent MS–MS analysis and identification. With the

improvements of instrument performance and software

for database searches14, peptides with molecular masses

of up to 10 kDa have already become accessible.

Although ion-trap instruments are widely used in

LC–MS–MS and direct MS–MS, identification of peptides

as enzymatic fragments of proteins, limited resolution of

higher charge states, restricted fragment ion resolution

and mass accuracy, make these instruments second

choice for analysis of (native) peptides that are greater

than 3–4 kDa. Hybrid quadrupole TOF mass spectrometers

are preferentially used because of their high mass accu-

racy and resolution11,15. An alternative approach is the

use of Fourier-transform ion cyclotron resonance MS

(Ref. 16), although this still lacks an adequate throughput

because of limited automization capability.

The existing databases, whether they cover proteins,

genomes or ESTs, are of great help in the acceleration and

automation of identification processes, although there is

still a lack of specific databases and software for searching

through for more peptide-specific information.At present,

post-translational modifications (PTMs), proteolytic pro-

cessing and amino acid variations, are recorded only as

annotations in databases and thus cannot be searched sys-

tematically.This prevents automatic identification of many

peptides, even though a good and representative MS–MS

spectrum might be available. This issue is especially

important for peptides and needs to be addressed in the

future.

Extracellular fluids in humansPhysiological and pathological changes are reflected in

the production and the metabolism of proteins and pep-

tides. Such changes are detectable in extracellular fluids,

which represent the major link between all cells, tissues

and organs of the human body3. Analysis by genomic or

transcriptomic methods is of little use, because body

fluids are a collection of substances from a variety of cell

types that arise as a consequence of transport or diffu-

sion and further processing. The development of pro-

teomic technologies to analyse the dynamics of these

systems is of significant interest, because these tech-

nologies represent the multitude of concerted biological

processes in a living organism7,8.

For the measurement of proteins within the range of

10–200 kDa, protocols using 2D gel electrophoresis are

well established7. Recently, a technology became avail-

able for the comprehensive analysis of peptides between

0.5 and 20 kDa in human body fluids, which play a pivotal

role in many physiological processes8. By identifying

specific changes in the peptide and protein composition

of human body fluids using quantitative differential display

methods, it has now become feasible to discover novel

biomarkers that indicate diseases and other conditions,

and to identify drug candidates and applications for drug

development.

Peptides in blood plasmaBlood is the major link between all cells, tissues and

organs of humans and contains the most representative

collection of peptides, proteins and protein fragments

that are produced in the entire body. Release of peptidic

or protein compounds into the extracellular space is one

of the most important mechanisms by which communi-

cation between cells and organs is established and main-

tained. Knowledge about the housekeeping plasma

proteins, such as albumin, fibrinogen, immunoglobulins

and others that are present in large concentrations, is well

established. However, the identification of peptides and

proteins with regulatory function is difficult because they

occur at low concentrations and within a complex mix-

ture of the main plasma proteins. Different strategies have

been applied to overcome this issue. These include

isolations that are directed by screening for a specific

biological activity, immunological detection, binding to

orphan receptors or chemical specificity, and have led to

the discovery of many important peptide hormones,

cytokines and growth factors8.

With the emergence of the Human Genome Project,

a comprehensive approach to unravelling this biologi-

cal source has become an issue of high medical impor-

tance.To this end, a basis for the systematic purification

of all peptides in blood plasma has been established

using haemofiltrate as a suitable source3. This blood

filtrate (with a cut-off at about 20–30 kDa) is generated

from individuals with end-stage renal disease and then



purified. The plasma protein content is reduced by a

factor of 1000, but regulatory compounds remain pre-

sent at their normal physiological concentrations.

Moreover, haemofiltrate contains little to no enzymatic

activity, and is quickly acidified and cooled to represent

a ‘frozen’ status of blood peptides. The availability of

haemofiltrate in large amounts has made possible the

establishment of a ‘peptide bank’ comprising a collection

of blood peptides in pre-fractionated batches from

several thousand litres of blood filtrate processed in a

reproducible manner. This material was systematically

analysed with MS methods17. Multi-dimensional maps

of more than 5000 distinct components were com-

piled using matrix-assisted laser desorption–ioniza-

tion (MALDI)–time-of-flight (TOF)–MS or on-line

LC–MS (Ref. 18), and subsequent identification by

tandem MS experiments has resulted in a database of

human blood peptides. Of 340 sequence entries regis-

tered in a first publication from 1999 (Ref. 19), 55%

were fragments of known plasma proteins, but a sub-

stantial part (12%) were members of peptide families

such as cytokines, defensins or peptide hormones –

some of them as-yet unknown compounds.

Interestingly, several of the discovered new molecules

occur at nanomolar concentrations and are thus very

abundant.

Other body fluidsSeveral other human body fluids are also rich in pep-

tides. Of particular interest for diagnostic purposes are

easily accessible sources, such as urine, lacrimal fluid or

saliva. For example, several biochemical markers in

urine are used to measure bone turnover20. Another

example is the successful application of surface-

enhanced laser desorption–ionization (SELDI) technolo-

gy for the identification of a defensin peptide of 3.4 kDa

in urine as a biomarker for transitional cell carcinoma of

the bladder21. In a systematic approach, peptides in

urine have been characterized and identified by using

LC–MS and Edman sequencing22. Recently, a LC–MS–MS

strategy has been used towards defining a urinary

proteome using unfractionated tryptic digests23.

Specific peptides in saliva have been linked to diseases

of the teeth (e.g. caries)24, and human breast milk is a

rich source of many peptide growth hormones and

antibiotics25.

Less accessible liquid subsystems in the body, such as

cerebrospinal or synovial fluid, are the first sources to be

screened in order to understand local physiological

processes and diseases. Cerebrospinal fluid is rich in neu-

ropeptides and, with its relevance to neurodegenerative

diseases5, small amounts (less than 1 ml) have already

been analysed. Comprehensive methods have been used

to show a specific peptide pattern8 and many peptide

components have been generated by specific proteolytic

processing26. For synovial fluid, a few characteristic pep-

tide biomarkers are already known20 but a comprehensive

peptidomic analysis is still lacking.

Quantitative approachesQuantitative analysis of selected componentsA very promising feature of peptidomic analysis is that

several methods for quantitative analysis are already

TRENDS in Biotechnology

Similarities and differences

Peptide mass fingerprint

96 mass spectra per sample

Differential displayof multiple samples

Transformationof data

Peptide extraction andseparation of proteins

HPLC separation

MALDI–MS

Clinical sample

Figure 1. Schematic representation of the underlying process for adifferential display of complex peptide mixtures

Peptides are extracted from a clinical sample and separated into 96 fractions using reversed-phasechromatography. All fractions are measured by matrix-assisted laser desorption–ionization(MALDI)–time-of-flight (TOF)–mass spectrometry (MS) and data are transformed into a 2D gel-likepicture by means of a specialized software. Typically, 1000–5000 components in the range of0.5–15.0 kDa are visualized and defined by the three dimensions: (1) chromatographic fraction number;(2) mass per charge ratio; and (3) intensity from MS data. Differential comparison of the resultingpeptide mass fingerprint is achieved by comparing the components detected by calculating signalheights and/or areas, using appropriate internal and external calibration and standardization procedures.



available. Besides the immunoassay-based measurement

with radioimmunoassay (RIA) or enzyme-linked

immunosorbent assay (ELISA), MS can now be used to

determine the amounts of specific peptides with the

modern instruments that are capable of both improving

the purification and performing the quantification. The

initial purification can be performed by affinity27,28 or

chromatographic24 purification, with the analysis per-

formed either on- or off-line. It has been shown that the

quantitative measurement can be based not only on

electrospray MS (Ref. 29), but also on MALDI–TOF–MS

(Refs 27,28) detection methods. The quantification is

performed by comparison with external27 or inter-

nal28,29 references. A less specific method using diode-

array detection during reversed-phase chromatography

has been described for the quantification of several sali-

vary peptides30.

Differential display of complex mixturesA few years ago, two initial publications appeared that

clearly demonstrated the capability of MALDI–TOF–MS

in semiquantitative analysis of complex peptide

mixtures31,32. Although a differential display had been

shown only for a few different spectra of rat neurointer-

mediate lobes31 and the haemolymph of Drosophila32, it

became clear that an application for other biological

sources, such as body fluids, could become possible.With

the development of a robust and reproducible separation

technology for peptides in complex biological sources by

adsorption or affinity purification on a chip21, chro-

matography yielding a 3D display8 (Fig. 1) and more

sophisticated data-mining tools, the comprehensive

discovery of human peptides relevant as biomarkers or

drug targets has just begun.

ProspectsThe application of peptidomic approaches to analyse

human body fluids will complement efforts to study gene

expression using proteomic or transcriptomic technologies.

It will serve as a tool to study gene expression products in

a relevant and complex class of clinically interesting

molecules, with the potential for application in clinical

analysis and drug development.

The knowledge gained from analysis of the peptide

content in human body fluids will have a direct impact

on the diagnosis of many diseases. In the future, the old

paradigm of one disease related to a single gene and

diagnosed by a single marker or treated by a single

drug, will no longer be valid. The identification of

interacting gene expression networks and the measure-

ment of marker patterns must be achieved in order to

differentiate between diseases. Using such tools, it will

be possible to distinguish between different types or

individual variations of a disease, such as tumours and

their ability to undergo metastases. Finally, peptidomes

from body fluids will contribute to the human proteome

initiative, and will thus become an important tool in

pharmaceutical development.

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peptidomics technologies for human body fluids

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