measuring protein abundance and changes in protein
TRANSCRIPT
Measuring Protein Abundance and Changes in Protein Abundance with Targeted Quantitative Proteomics
Mike KinterOklahoma Medical Research FoundationAging and Metabolism Research Program
Caroline KinterJakub BunkHyerin KwakHaley LandrithWyatt Landrith
Common Proteomics WorkflowsA. Begin with discovery proteomics experiments
Need to validate those findings in a larger number of samplesFor example: Biomarker discovery
B. Begin with a hypothesis derived from other types of dataNeed to test the hypothesis by probing expression in a selected set of targetsFor example: Changes within a known biochemical pathway or protein complex
Western blot is the traditional tool Many strengths but also important weaknessesA good Western blot is accurate, relatively fast, has an easy to understand outputBut...One protein at a time, problems with many antibodies, hard to make new antibodies, often visual rather than numerical, hard to do large groups of samples, etc
1. How many of you use Western Blot in your work?
2. Where do you get your antibodies? Has anyone had a custom antibody made?
What's the point?Western blot is a targeted immunochemical method.The same thinking goes into a targeted mass spectrometry experiment.But,1) faster development2) multiplexed3) results are number4) validation is a part of method design5) once developed, they are good forever and can be transferred between la
Two Targeted Methods Used in the Kinter Laboratory
A) Selected Reaction Monitoring (SRM)ThermoScientific TSQ Quantiva (triple quadrupole instrument)
B) High resolution accurate mass Selected Ion Monitoring (HRAM or SIM)ThermoScientific QExactive Plus (orbitrap instrument)
Both methods digest the proteins to peptides with trypsinSelected peptides from each protein are Targeted as quantitative markers of the
parent protein
Both methods are fundamental liquid chromatography experimentsRetention time plus mass spectrometry is characteristic of identityChromatographic peak area is characteristic of amount
The role of the mass spectrometry is to enhance the Specificity and Sensitivityof detection
Universal Detection of All peptides Eluting from the HPLCMass spectra are used to reconstruct chromatograms
20 25 30 35 40 45 50 55 60 65Time, minutes
0
50
100 Total ion current chromatogram100% = 2.41E9
1100
Full scan mass spectrum #1301, RT = 43.9min100% = 1.02E8
300 400 500 600 700 800 900 1000m/z
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100
Rel
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nce,
%
655.37
666.35560.94
888.13840.91627.98
507.74 598.82737.39 940.96466.76
415.75343.72 949.16
640 642 644 646 648 6500
9 647.8542 (+0.0005 <1ppm)
648.3556
644.8380643.3729 645.3390 648.8574
640.6281
9% = 9.52E6
Rel
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unda
nce,
%
~3000 spectra
3D View Shows the Complexity
Show and tell purposes only. We don’t try to interpret this pattern.
Reminder: The mouse proteome is known – 29,769 proteins, 16M amino acids, ~2,000,000 peptides in a perfect trypsin digest
0
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100 Total ion current chromatogram100% = 2.41E9
Rel
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%
20 25 30 35 40 45 50 55 60 65
RT = 43.8minPeak area = 2457,495,861
RT = 48.4minPeak area = 2717,260,121
m/z 647.8537 ± 0.001SFLVGSAAQSLSK
m/z 839.4565 ± 0.001TQEFILNSPTVTSIK
20 25 30 35 40 45 50 55 60 65Time, minutes
Acox1 in liver
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100
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100%= 2.08E8
Specific LC-MS Data is extracted as mass chromatograms
Time, minutes
LC-full scan MS m/z 300-1100at R=280,000
Specific Detection is the Key
Detecting these two peptides is our assay for Acox1
Selected Ion Monitoring (SIM)
647.80@25 NCE100% = 2.20E7
200 300 400 500 600 700 800 900 1000 1100
m/z
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207.11
185.17
303.17 562.32
SFLVGSAAQSLSK from Acox1
y111060.60
y10947.52
y9848.45
y7704.40
y8791.43
y6633.36
b3348.19
y4434.26
b2235.11
Collision Induced Dissociation Spectra Give Peptide Sequences
- A key validation step. As a part of the development process, we record the CID spectra to prove correct detection of all peptides used in our assays.
- These fragmentation reactions, characteristic of the amino acid sequence, are used in the SRM experiments.
TQEFILNSPTVTSIK839.4565++Q [b2] - 230.1135+[1]E [b3] - 359.1561+[2]L [y10] - 1059.6045+[7]N [y9] - 946.5204+[8]S [y8] - 832.4775+[6]P [y7] - 745.4454+[4]V [y5] - 547.3450+[9]T [y4] - 448.2766+[3]S [y3] - 347.2289+[5]
SFLVGSAAQSLSK647.8537++F [b2] - 235.1077+[1]L [b3] - 348.1918+[3]L [y11] - 1060.5997+[8]V [y10] - 947.5156+[4]G [y9] - 848.4472+[2]S [y8] - 791.4258+[6]A [y7] - 704.3937+[9]A [y6] - 633.3566+[7]S [y4] - 434.2609+[5]
41 42 43 44 45 46 47 48 49 50 51 52 53Retention time, minutes
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L…
TQE…
Inte
nsity
(10^
6)Tandem Mass Spectrometry Is Used For SRM (aka PRM)
Specificity, Specificity, Specificity
Where Do the Assays Come From?Carefully designed based on things we already know about the protein:1) The amino acid sequence - protein sequence databases2) Peptides we know are detectable in a trypsin digestion of the protein – Discovery
data or the Peptide Atlas3) Known or calculated retention times – Discovery data or Sequence Specific
Retention Calculator4) mRNA expression data for different tissues to estimate expected amounts - BioGps
Reasonable to expect:Start of day, List of 5 proteins and a good sampleEnd of day, 5 working assays
AccessionPre AA Sequence
N Obs SSRT
N Gen Loc
N Samples
PAp00185196 R EIGTHKPLPGITVGDIGPK 260 31.97 1 19PAp00184451 K FGYEEMDNGYLK 325 27.46 1 12PAp00184963 K LVGGMVSYLNDLPSQR 310 37.63 1 10PAp00184669 R AAATFNPELITHILDGSPENTR 225 35.78 1 14PAp00183946 K TQEFILNSPTVTSIK 257 33.05 1 12PAp00184671 R INESIGQGDLSELPELHALTAGLK 260 40.88 1 11PAp00184375 R GLETTATYDPK 181 16.65 1 12PAp00184782 K TSNHAIVLAQLITR 240 42.23 1 8PAp00396807 K NLQAQVSHR 157 9.8 1 12PAp00184709 K YAQVKPDGTYVKPLSNK 179 23.71 1 10PAp00184787 R GECYGLHAFVVPIR 138 34.04 1 11PAp00184515 R YDGNVYENLFEWAK 177 43.27 1 8PAp00184177 R ILELLTVTRPNAVALVDAFDFK 199 58.37 1 7PAp00396347 K LFPLLATAYAFHFLGR 197 50.84 1 5PAp00184610 K DVTLGSVLGR 107 24.48 1 9PAp00184972 R SEPEPQILDFQTQQYK 126 31.04 1 8PAp00184433 R SFLVGSAAQSLSK 120 28.98 1 8PAp00350462 K EVAWNLTSVDLVR 161 38.37 1 6PAp00184146 R EIENLILNDPDFQHEDYNFLTR 107 46.02 1 8PAp00397265 R ASEAHCHYVTVK 79 12.97 1 9PAp00185015 R NLCLLYSLYGISQK 135 41.78 1 6PAp00185502 R EFGIADPEEIMWFK 75 42.51 1 7PAp00397716 K HLKPLQSK 30 15.6 1 7PAp00395910 K AFTTWTANAGIEECR 92 28.82 1 3PAp00398843 K IYDQVQSGK 44 10.9 1 5PAp00396838 K TEVHESYYK 39 12.19 1 5PAp00397332 R LVEIAAK 7 15.33 1 4
From the PeptideAtlas database*, mouse Acox1.
Discovery peptides may not always be the best,Therefore a design process
Trouble amino acids:avoid Mvery careful with C, W, and DPCID trouble with H, KP, and RP
Other potential issues:- too hydrophilic- too large
Key thing we need is a sample where the peptides are detectable (tissue/sample library)
*Only a portion of the database entry is shown. Complete is 69 lines based on 6719 spectra entries.
Key Design Information is Available in the Peptide Atlas
Peptide Calculated m/z CalculatedRT , min
MeasuredRT, min
Peak area in development run
GLETTATYDPK 598.2957 33.2 33.4 1049,954,794
TQEFILNSPTVTSIK 839.4565 46.9 48.6 1747,752,569
SFLVGSAAQSLSK 647.8537 43.6 44.0 1794,373,485
SEPEPQILDFQTQQYK 975.9758 47.2 48.4 743,769,442
IYDQVQSGK 519.2667 28.5 33.2 182,973,346
LVEIAAK 372.2367 31.8 31.3 1193,401,108
EVAWNLTSVDLVR 750.4041 54.5 51.6 69,986,212
DVTLGSVLGR 508.7904 42.3 46.9 365,888,175
YDGNVYENLFEWAK 874.4018 58.7 59.7 765,539,816
Candidate Marker Peptides are Tested Experimentally
Looking for the best flyersLudwig C, Claassen M, Schmidt A, Aebersold R. Estimation of absolute protein quantities of unlabeled samples by
selected reaction monitoring mass spectrometry. Mol Cell Proteomics, 2012 11(3):M111.013987. PMID: 22101334
Validation stepsa) collision induced dissociation spectra, b) proper retention time, c) abundance/detectable in different tissues
Hypothesis: Increase Fatty Acid Processing Leads to Carbon Stress
Acadvl, Acadl, Acadm, Acads
H2O
β-Ketoacyl-CoA
CoASH
trans-D2-Enoyl-CoA
3-L-Hydroxyacyl-CoA
Fatty acyl-CoA (2 carbons shorter)
+ Acetyl-CoA
NAD+NADH + H+
FAD
Ech1
Acaa2
Eci2, Hadh, Hadha, Hadhb
FADH2
Fatty acyl-CoA
Krebs cycle
Long chain fatty acid
Acsl1
Fatty acyl-CoA
Fatty acyl-carnitine
Cpt1a, Cpt1bCo-ACarnitine
Slc25a20
Fatty acyl-carnitine
Fatty acyl-CoA
Co-ACarnitine
Cpt2
To test, we built a panel of assays
Genename Protein name
Genename Protein name
1 Abcd3 ATP-binding cassette sub-family D member 3 22 Eci1 enoyl-CoA delta isomerase 12 Acaa1ab 3-ketoacyl-CoA thiolase A 23 Eci2 enoyl-Coenzyme A delta isomerase 23 Acaa2 acetyl-Coenzyme A acyltransferase 2 24 Ehhadh peroxisomal bifunctional enzyme (liver) 4 Acad11 acetyl-Coenzyme A dehydrogenase family member 11 25 Fabp1 fatty acid binding protein 1 liver 5 AcadL long-chain specific acyl-CoA dehydrogenase 26 Fabp3 fatty acid-binding protein heart 6 AcadM medium-chain specific acyl-CoA dehydrogenase 27 Fabp4 fatty acid-binding protein adipocyte 7 AcadS short-chain specific acyl-CoA dehydrogenase 28 Hadh L-3-hydroxyacyl-Coenzyme A dehydrogenase short chain 8 AcadVL very long-chain specific acyl-CoA dehydrogenase 29 Hadha trifunctional protein alpha subunit 9 Acot13 acyl-coenzyme A thioesterase 13 30 Hadhb trifunctional protein beta subunit 10 Acox1 peroxisomal acyl-coenzyme A oxidase 1 31 HmgcL hydroxymethylglutaryl-CoA lyase11 Acsl1 long-chain-fatty-acid--CoA ligase 1 32 Hmgcs1 hydroxymethylglutaryl-CoA synthase12 Bdh1 D-beta-hydroxybutyrate dehydrogenase 33 Hmgcs2 hydroxymethylglutaryl-CoA synthase13 Cat catalase 34 Hsd17b4 peroxisomal multifunctional enzyme type 2 14 Cpt1a carnitine O-palmitoyltransferase 1 liver 35 Lonp1 lon peptidase 115 Cpt1b carnitine palmitoyltransferase 1b, muscle 36 Lonp2 lon peptidase 216 Cpt2 carnitine O-palmitoyltransferase 2 37 Pecr peroxisomal trans-2-enoyl-CoA reductase (liver) 17 Crat carnitine O-acetyltransferase 38 Slc25a20 mitochondrial carnitine/acylcarnitine carrier protein 18 Crot carnitine O-octanoyltransferase 39 Bsa bovine serum albumin 19 Decr1 2,4-dienoyl-CoA reductase 20 Ech1 enoyl coenzyme A hydratase 1 21 Echs1 enoyl Coenzyme A hydratase short chain 1
Repeat as Needed to Build the Assay You WantOur Beta Oxidation Enzyme Panel
Acox1 SFLVGSAAQSLSK, m/z 647.8537
Acadvl GIVNEQFLLQR, m/z 658.8697
What Do the Data Look Like? Chromatograms from Selected Ion Monitoring
Controldiet
45% Larddiet
45% Fish Oildiet
What Do the Data Look Like?A Large Spreadsheet (Ugggh)
x17 x28 x16 x29 x30 x18 y49 y39 y48 y34 y38 y50 z70 z57 z58 z56 z69 z68s63008 s63010 s63007 s63011 s63012 s63009 s63017 s63015 s63016 s63013 s63014 s63018 s63024 s63020 s63021 s63019 s63023 s63022control 45% lard 45% fish oil
Bsa in at 8pmol/60µg total proteinLVTDLTK 3.888E+09 4.256E+09 4.362E+09 4.242E+09 4.404E+09 3.673E+09 4.028E+09 4.146E+09 3.782E+09 4.710E+09 4.032E+09 3.821E+09 3.580E+09 3.970E+09 4.193E+09 4.078E+09 3.993E+09 3.890E+09YLYEIAR 4.756E+09 4.714E+09 4.923E+09 4.852E+09 5.149E+09 4.445E+09 5.097E+09 5.043E+09 4.569E+09 4.574E+09 4.658E+09 4.978E+09 4.854E+09 5.272E+09 4.900E+09 4.493E+09 4.917E+09 4.362E+09LVNELTEFAK 5.194E+09 5.641E+09 5.915E+09 5.348E+09 5.648E+09 5.482E+09 5.766E+09 5.989E+09 5.119E+09 5.437E+09 5.532E+09 5.309E+09 5.917E+09 5.815E+09 5.143E+09 4.681E+09 5.410E+09 5.224E+09geomean 4.580E+09 4.837E+09 5.027E+09 4.793E+09 5.040E+09 4.473E+09 4.910E+09 5.003E+09 4.456E+09 4.893E+09 4.701E+09 4.657E+09 4.685E+09 4.956E+09 4.728E+09 4.410E+09 4.736E+09 4.459E+09
Acox1SFLVGSAAQSLSK 2.253E+07 2.497E+07 1.619E+07 2.057E+07 2.755E+07 2.899E+07 4.517E+07 5.922E+07 4.318E+07 4.042E+07 3.278E+07 4.512E+07 4.984E+08 5.212E+08 4.992E+08 4.669E+08 5.537E+08 5.093E+08TQEFILNSPTVTSIK 1.998E+07 2.066E+07 1.633E+07 2.036E+07 2.684E+07 2.521E+07 4.991E+07 6.028E+07 4.148E+07 4.782E+07 3.740E+07 4.299E+07 5.815E+08 5.073E+08 5.043E+08 4.319E+08 5.859E+08 6.092E+08geomean 2.122E+07 2.271E+07 1.626E+07 2.047E+07 2.719E+07 2.703E+07 4.748E+07 5.975E+07 4.232E+07 4.397E+07 3.501E+07 4.404E+07 5.384E+08 5.142E+08 5.017E+08 4.491E+08 5.695E+08 5.570E+08
pmol/100µg total P 0.062 0.062 0.043 0.057 0.072 0.080 0.129 0.159 0.126 0.120 0.099 0.126 1.529 1.380 1.412 1.354 1.600 1.662
average 0.063 average 0.126 average 1.49sd 0.013 sd 0.019 sd 0.13
rsd 20% rsd 15% rsd 8%r/control 2.02 r/control 23.8
ttest 9.8E-05 ttest 9.5E-07AcadvlIFEGANDILR 1.262E+09 1.410E+09 1.411E+09 1.406E+09 1.447E+09 1.524E+09 2.075E+09 1.963E+09 1.716E+09 1.667E+09 1.744E+09 1.848E+09 9.502E+08 8.587E+08 1.072E+09 8.724E+08 9.480E+08 9.087E+08GIVNEQFLLQR 1.288E+09 1.439E+09 1.437E+09 1.470E+09 1.435E+09 1.556E+09 2.232E+09 2.143E+09 1.875E+09 1.695E+09 1.893E+09 2.003E+09 2.286E+09 2.179E+09 2.474E+09 1.902E+09 2.159E+09 2.002E+09geomean 1.275E+09 1.424E+09 1.424E+09 1.438E+09 1.441E+09 1.540E+09 2.152E+09 2.051E+09 1.794E+09 1.681E+09 1.817E+09 1.924E+09 1.474E+09 1.368E+09 1.629E+09 1.288E+09 1.431E+09 1.349E+09pmol/100µg total P 3.70 3.92 3.77 3.99 3.80 4.58 5.83 5.45 5.35 4.57 5.14 5.49 4.18 3.67 4.58 3.89 4.02 4.02
average 3.96 average 5.31 average 4.06sd 0.32 sd 0.43 sd 0.31
rsd 8% rsd 8% rsd 8%r/control 1.34 r/control 1.026
ttest 1.4E-04 ttest 0.590
-Statistically significant differences at 30%-Acox1 is low abundance protein in the heart
for comparison Gapdh = 30pmol/100µg total protein (~500x measured in the same run)
-18 samples = 25hrs LC-MS time + data analysis time
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A) Mitochondrial beta oxidation
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45% Fish oil diet60% Lard diet
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45% Fish oil diet60% Lard dietControl = 1.0 Control = 1.0
Significant Increases in ß-Oxidation Enzyme Expression
Finally, How Do You Work With Us?
heart 1cm
1cm
1cmliver
culturedcells
Sample Homogenates
Biological Experimentmultiple treatments x
multiple replicates
In-Gel Digestion
AnalysisData Processing
Acetone PrecipitationGel Electrophoresis
Volume equivalent to 20-100µg total protein
Step 1. Talk by email.- What type of samples do you have?- Experimental design (n=5 or more)?- What do you want measured?- Do you have a tested homogenization protocol?
Step 2. Develop any new assays.Step 3. Test pilot samples if needed.Step 4. Go!
1. Specificity of the analysis is the key2. Two mass spectrometric approaches to achieving this specificity
High resolution accurate mass selected ion monitoring (SIM)Tandem mass spectrometry selected reaction monitoring (SRM)
3. These assays are built based on known peptides exclusively linked to the protein being measured
4. The building process inherently verifies that the correct peptides are being detected.
5. Once built, an assay is good forever and may be transferred between laboratories.
6. With the high resolution accurate mass approach (SIM) data can be reinterrogated anytime after they are acquired.
Summary of Our Targeted Quantitative Proteomics Methods
Questions??
J.J. ThomsonNobel Prize 1906 in Physics for the conduction of electricity by gases - discovery of the electron (corpusles)
1913 paper ‘Rays of positive electricity’ widely cited as the beginning of mass spectrometry
(Seven trainees and his son also won Nobel prizes)
‘The parabola due to the heavier gas is always much fainter than that due to the lighter, so that probably the heavier gas forms only a small percentage of the mixture.’
Isotope abundance: Ne22 9.3%Ne20 90.5%
Mass Spectrometry has Always Been Used for Quantitative Analyses