Measuring Protein Abundance and Changes in Protein Abundance with Targeted Quantitative Proteomics Mike Kinter Oklahoma Medical Research Foundation Aging and Metabolism Research Program Caroline Kinter Jakub Bunk Hyerin Kwak Haley Landrith Wyatt Landrith
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Measuring Protein Abundance and Changes in Protein Abundance with Targeted Quantitative Proteomics
Mike KinterOklahoma Medical Research FoundationAging and Metabolism Research Program
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
0
50
100
Rel
ativ
e ab
unda
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
ativ
e ab
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
50
100 Total ion current chromatogram100% = 2.41E9
Rel
ativ
e ab
unda
nce,
%
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
50
100
Rel
ativ
e ab
unda
nce,
%
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
0
50
100
Rel
ativ
e ab
unda
nce,
%
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.
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
-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
0
1
2
3
Prot
ein
abun
danc
e, re
lativ
e to
con
trol
A) Mitochondrial beta oxidation
*
* * **
* *
*# # ## ##
# # #
45% Fish oil diet60% Lard diet
0
2
4
6
8
10
Prot
ein
abun
danc
e, re
lativ
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con
trol B) Peroxisomal beta oxidation
*
*
* *
* *
*
*
# ## # # #
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