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description
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NEW SOLUTIONS FOR HIGHLY
MULTIPLEXED PROTEIN ASSAYS VIA MRM
WebinarJune 04, 2012
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CAPRION PROTEOMICS• Leading proteomics-based service provider
- Biomarker and target discovery- Multiplexed MRM assays- Biomarker verification and validation
• Recently acquired ImmuneCarta• Multiparametric GLP flow cytometry for immune monitoring
• Located in: – Montreal, Canada– Menlo Park, California
• Founded in 2000• 62 employees (55 scientists)• Extensive experience
- Over 45 industry and government partners- Over 100 large scale studies completed (clinical and pre-clinical)- All major therapeutic areas and sample types- Expertise in study design, execution, statistical analysis,
biological insights and comprehensive reporting
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TECHNICAL APPROACHES IN MASS SPEC-BASED PROTEOMICS
Biomarker Discovery (LC-MS/MS)• Non-hypothesis based discovery approach• Label-free, gel-free quantitative mass
spectrometry• Shotgun sequencing• Profile 1000’s of proteins in 100’s samples• Identify differentially expressed proteins as
candidate targets and biomarkers
Multiplexed Assays (MRM)• Targeted quantification of up to 350 specific
proteins• Target list from MS, literature, transcriptomics• Rapid assay development • Ab-free or ab enrichment• Synthetic labeled standards for absolute
quantification
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Plasma, serum, CSF, urine,
other fluids
Deplete abundant proteins,
Antibody enrichment,
or exosome isolation
Enrich plasma membranes,Secreted proteins, phosphoproteins, other organelles or proteins
Trypsin digestion to peptides
ProteinSample
Cells and tissues
LC-MS analysis
PeptideSample
FFPE slides
Dissolve tissue sections
IDENTIFY MARKERS DIRECTLY IN FLUID OR IN THE SOURCE TISSUESAMPLE-SPECIFIC PROCESSING
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UNBIASED DISCOVERYLC-MS ANALYSIS AND MATCHING PEPTIDES ACROSS ALL SAMPLES
Peptidesamples
RP-HPLC andQTOF or Orbitrap Mass
Spectrometry
10,000-20,000 peptides observed, matched
and quantified
Retention time
Mass / charge ratio
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EXPRESSION PROFILING AND PROTEIN ID
Sequencing by LC-MS/MS
Identification of 500-2,500 proteins
Statistical analysis to identify differentially expressed peptides
Differentially expressed proteins in disease and drug treatment
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[0 - 0.1] [0.1 - 0.2] [0.2 - 0.3] [0.3 - 0.4] [0.4 - 0.5] [0.5 - 0.6] [0.6 - 0.7] [0.7 - 0.8] [0.8 - 0.9] [0.9 - 1.0]0%
5%
10%
15%
20%
25%
30%
35%
Median correlation coefficient
%Pr
otei
ns
76% of proteins with median r > 0.6
Dataset of 915 proteins (9,964 peptides) from cell lysate (cytosol) study
PEPTIDE EXPRESSION CORRELATION
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[0 - 0.1] [0.1 - 0.2] [0.2 - 0.3] [0.3 - 0.4] [0.4 - 0.5] [0.5 - 0.6] [0.6 - 0.7] [0.7 - 0.8] [0.8 - 0.9] [0.9 - 1.0]0%
5%
10%
15%
20%
25%
30%
35%
Median correlation coefficient
%Pr
otei
ns
Median r=0.96 (24 peptides)
PEPTIDE EXPRESSION CORRELATION: BEST CASE EXAMPLE
Comparison of 4 groups (n = 6)
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[0 - 0.1] [0.1 - 0.2] [0.2 - 0.3] [0.3 - 0.4] [0.4 - 0.5] [0.5 - 0.6] [0.6 - 0.7] [0.7 - 0.8] [0.8 - 0.9] [0.9 - 1.0]0%
5%
10%
15%
20%
25%
30%
35%
Median correlation coefficient
%Pr
otei
ns
Median r=0.75 (13 peptides)
PEPTIDE EXPRESSION CORRELATION: WEAKER BUT ROBUST EXAMPLE
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List of Candidate Proteins
MRM ASSAY DEVELOPMENT STRATEGY
UniProt_ID Protein Accession DescriptionPGRP2_HUMAN PGLYRP2 Q96PD5 peptidoglycan recognition protein 2KNG1_HUMAN KNG1 P01042 kininogen 1FIBB_HUMAN FGB P02675 fibrinogen beta chainFIBG_HUMAN FGG P02679 fibrinogen gamma chainKLKB1_HUMAN KLKB1 P03952 kallikrein B, plasma (Fletcher factor) 1URP2_HUMAN FERMT3 Q86UX7 fermitin family member 3PLMN_HUMAN PLG P00747 plasminogenCO6_HUMAN C6 P13671 complement component 6B2MG_HUMAN B2M P61769 beta-2-microglobulinMBL2_HUMAN MBL2 P11226 mannose-binding lectin (protein C ) 2, solubleHRG_HUMAN HRG P04196 histidine-rich glycoproteinVTDB_HUMAN GC P02774 group-specific component (vitamin D binding protein)ANT3_HUMAN SERPINC1 P01008 serpin peptidase inhibitor, clade C (antithrombin), member 1CO8B_HUMAN C8B P07358 complement component 8, beta polypeptideCFAB_HUMAN CFB P00751 complement factor BICAM1_HUMAN ICAM1 P05362 intercellular adhesion molecule 1
Highly multiplexed: Up to ~350 proteins
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MRM ASSAY DEVELOPMENT
List of Candidate Proteins
MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLKMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATA…
Select up to 5 peptides for each protein
UniProt_ID Protein Accession DescriptionPGRP2_HUMAN PGLYRP2 Q96PD5 peptidoglycan recognition protein 2KNG1_HUMAN KNG1 P01042 kininogen 1FIBB_HUMAN FGB P02675 fibrinogen beta chainFIBG_HUMAN FGG P02679 fibrinogen gamma chainKLKB1_HUMAN KLKB1 P03952 kallikrein B, plasma (Fletcher factor) 1URP2_HUMAN FERMT3 Q86UX7 fermitin family member 3PLMN_HUMAN PLG P00747 plasminogenCO6_HUMAN C6 P13671 complement component 6B2MG_HUMAN B2M P61769 beta-2-microglobulinMBL2_HUMAN MBL2 P11226 mannose-binding lectin (protein C) 2, solubleHRG_HUMAN HRG P04196 histidine-rich glycoproteinVTDB_HUMAN GC P02774 group-specific component (vitamin D binding protein)ANT3_HUMAN SERPINC1 P01008 serpin peptidase inhibitor, clade C (antithrombin), member 1CO8B_HUMAN C8B P07358 complement component 8, beta polypeptideCFAB_HUMAN CFB P00751 complement factor BICAM1_HUMAN ICAM1 P05362 intercellular adhesion molecule 1
Peptide SynthesisR
esolubilization and pooling of
peptides
Determination of best transitionsCE-Optimization
RT
Inte
nsity
Sample Analysis
RT
Inte
nsity
m/z
Inte
nsity
Selection of best 2 peptides and
transitions
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MRM SELECTS SPECIFIC PEPTIDE PEAKS FOR ANALYSIS
Tran
sitio
ns, i
n or
dere
d by
sch
edul
ed R
T
Retention Time
Less Intense More Intense
Discovery detects all peaks
MRM selects only specific peaks at
each retention time
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TOOLS FOR MRM AUTOMATION:ELUCIDATOR PEAK DETECTION AND QUANTIFICATION
Caprion has developed proprietary tools to automate MRM assays and improve results
Align one peptide peak across
hundreds of samples
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AVOIDING FALSE POSITIVES WITH SYNTHETIC PEPTIDE STANDARDS
Correct peak Interfering peak
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1498 PEPTIDES (~700 PROTEINS) IN THE SAME MRM ASSAY
# of transitions measured
Median Int CV of detected
transitions250 2.4500 3.1
1000 41500 5.22000 6.42500 7.6
• 1498 peptides spiked at 200 pmol/mL• MRM assay generated
• Transitions analyzed for reproducibility
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LINEARITY AND SENSITIVITY
Quantitative detection of up to ~350 proteins in each assay
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Options for MRM assays
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13C, 15N-LABELED STANDARDSRATIOMETRIC QUANTIFICATION
Synthetic labeled standards can be used to quantify biomarkers over 4 orders of magnitude
Co-inject labeled synthetic peptides for better quantification
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QUANTIFICATION OF ENDOGENOUS PEPTIDES IN PLASMA: “PEPTIDOMICS”
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000Concentration
0.0e0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
Are
a
Calibration for 3325: y = 55.93506 x + 2277.88815 (r = 0.99974)
Protein precipitation followed by SPE cleanup of endogenous peptides 500 ul rat plasma LOQ 1-10 pg/ml (CV ~15%): Very sensitive approach
100 pg/ml
Remove all proteins,Analyze only native peptides (< 10,000 kDa)
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IMMUNOAFFINIY CAPTURE: QUANTIFYING PROTEINS IN THE LOW PG/ML RANGE
• Quick assay development– 2-3 weeks
• Parallel sample processing in 96 well-plates– 200 samples per person per 24 h
• MRM analysis time ~ 5-15min
• LLOD in the low pg/mL range
• Precision < 25 %
• Accuracy < 15 %
• Assay can be multiplexed 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200Concentration
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
Are
a
Calibration for LPQDLYHAR2: y = 32.23360 x + -4.72543 (r = 0.98832) (weighting: 1 / x)
Concentration of Cytokine in pg/mL
Peak
Are
a
0 10 20 40 60 80 100 120 140 160 180 200
Cytokine spiked at 10 pg/mL
Additionof beads
Binding Washing Trypsin digestion
LC-MRM-MSanalysis of s/n
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Tumor
ANALYSIS AND QUANTIFICATION OF FFPE SAMPLES
Normal
• Direct detection by MRM
• Quantitative assessment
• Detect and compare multiple candidate targets and control proteins
• 5 micron FFPE thin sections
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Case Study 1:
Non-Targeted Discovery in PlasmaFollowed by MRM
Markers of active TB in plasma and serum
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360 SAMPLE COMPARISON OF TB STATUS IN 3 INTERNATIONAL COHORTS
• US cohort is early stage disease• HIV background in South African cohort• Extrapulmonary TB in Brazil cohort• Samples from each site were analyzed independently
Group ConditionNumber of samples
US Brazil S. Africa Total
1 Active TB, HIV+ 40 40
2 Active TB, HIV- 23 42 65
3 LTBI, HIV+ 10 29 39
4 LTBI, HIV- 11 50 61
5 Asymptomatic, TST-, HIV+ 10 38 48
6 Asymptomatic, TST-, HIV- 10 50 60
7 Extrapulmonary TB, HIV- 47 47
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SUMMARY OF DISCOVERY RESULTS
• High abundance proteins depleted with IgY14/Supermix resin• Single LC-MS analysis per sample• Identified and quantified ~450 proteins• Compared active TB to other cohorts• Number of differentially expressed proteins:
US: 105Brazil: 146 S. Africa: 49Non-redundant total: 200
• Most differentially expressed proteins assigned to defined processes:Immune response ~50%Lipid metabolism ~20%Lung damage & wound healing ~20%
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ANALYSIS OF SAMPLES BY MRM
• Selected 89 differentially expressed proteins• 247 peptides were monitored• MRM assay run on 360 discovery samples• Markers from each site tested on all 3 sites
US BRAZIL S. AFRICA
DETECTED # % # % # % ASSAYED
TRANSITIONS
384
78%
342
70%
432 88% 494
PEPTIDES 192
78%
171
69%
235 95% 247
PROTEINS 83 93% 77 87
% 89 100% 89
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RESULTS OF MRM QUALIFICATION/VERIFICATION STUDY
54 proteins differentially expressed in at least one siteActive TB vs. Latent TB and Asymptomatic
– 34 US– 38 Brazil– 24 South Africa
13 proteins differentially expressed in all three sites– Immune response (3), lipid metabolism (4), lung damage (6)
Number of Patients AUC of8 Protein
PanelStudy Site Active TB Latent TB Asymptomatic
U.S. 23 21 20 1.00Brazil 42 50 50 0.99S. Africa 40 29 38 0.98
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Case Study 2:
Targeted Discovery of Biomarkers by MRM in Plasma
Markers of H5N1 vaccine response
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DISCOVERY STUDY DESIGN: SAMPLES
NIAID-funded clinical trial involving 451 healthy adults• 291 samples from 97 patients receiving the highest dose (90 µg) of
inactivated subvirion influenza A (H5N1) vaccine:
1. Baseline (pre-vaccination)2. Samples taken 28 days post-dose 13. Samples taken 28 days post-dose 2
• Immunogenicity determined by hemagglutination inhibition (HAI) and microneutralization (MN)
• Response: increase in HAI or MN titer by 4-fold or more between the baseline and post-dose 2 values
GENDER SEASONAL FLU VACCINE RESPONSE NUMBER OF
PATIENTSFemale No NON-RESPONDER 7Female No RESPONDER 22Female Yes NON-RESPONDER 11Female Yes RESPONDER 11
Male No NON-RESPONDER 10Male No RESPONDER 13Male Yes NON-RESPONDER 13Male Yes RESPONDER 10
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ESTABLISHED IMMUNE RESPONSE MRM PANEL
• 336 proteins categorized according to the biological functions and pathways
• Protein classifications taken from the Ingenuity Pathway Analysis database
XIC of +MRM (2488 pairs): 321.180/275.100 amu Expected RT: 5.3 ID: HHGPTITAK_321.18_275.1 from Sample 8 (PSST00212J11101... Max. 9.0e4 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26 28Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
5.0e5
5.2e5
5.4e5
Inte
ns
ity, cp
s
5.19
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PROJECT STATUS
• 291 samples from the discovery study analyzed using the multiplexed MRM assay
• 101 immune response proteins tracked by MRM
• 20 differentially expressed proteins correlated with response to vaccine at 28 days following second inoculation
• AUC = 0.79
• Currently improving detection of immune response proteins
• Verification assay planned for different trials with earlier time points collected
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Case Study 3:
Discovery in the Source TissueFollowed by MRM in Plasma
a) Identify initial candidates in secretome of NSCLC tumors
b) Detect and quantify circulating markers in plasma
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DISTINGUISHING BENIGN FROM MALIGNANT LUNG NODULES
Problem:•150,000 lung nodules detected in the US each year – 60% are benign and 40% are malignant
•Standard of care is CT scans performed every 6 months•If the nodule enlarges, a biopsy may be ordered
Goal: •Develop blood-based test for early detection of small, malignant nodules to be used in conjunction with imaging
Approach:•Identify 347 candidate markers for testing in patient plasma
– Lung cancer tissue-based discovery studies– plasma membrane– secretory vesicles
– Transcriptomics– Literature review
•Establish MRM assay and screen patient plasma
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SECRETED PROTEINS: >1,000x MORE CONCENTRATED IN GOLGI THAN IN BLOOD
www.mie.utoronto.ca
Identify secreted proteins before release from the tissue
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LUNG TUMOR SECRETED PROTEINS
• Study Design– Collaboration with local hospitals– IRB approved, patient consented– 24 patients– Comparison of tumors and adjacent normal
• Results– 1196 proteins identified in lung secretome– 200 up-regulated in tumor
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“BODY ATLAS” APPROACH TO TISSUE-SPECIFIC SECRETED PROTEIN IDENTIFICATION
Rationale• Identify proteins secreted
by each tissue to identify the uniquely and commonly secreted
• Aid in candidate prioritization
Method• Isolate secretory vesicles
from appropriately preserved normal human tissue
• Extract secreted proteins for MS analysis
• Minimum 6 subjects per tissue, low abundance tissue was pooled
Heart749 proteins
Liver 1010 proteins
Kidney1244 proteins
Muscle653 proteinsFat883 proteins
Pancreas856 proteins
Lung1196 proteinsBreast713 proteins
Kidney1509 proteins
Prostate1534 proteins
Bladder739 proteins
Colon1549 proteins
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• Development of MRM assay– Predict best 10 peptides/protein, synthesize– Determine empirically the best 2, monitor 2 transitions each
• Pre-verification study– Depleted plasma from 20 cancer and 20 control subjects
• Results– 64 targeted proteins (71%) successfully detected in cancer and
control samples– Candidate classifiers identified
90-PROTEIN LUNG CANCER MRM PANEL
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CANDIDATE CLASSIFIERS IDENTIFIEDa) b)
c) d)
LDHB (LIAPVAEEEATVPNNK) TBB3 (ALTVPELTQQMFDAK)
IF4A1 (MFVLDEADEMLSR)
Nor
mal
ized
Inte
nsity
0.025
0.020
0.015
0.010
0.005
CANCER NORMAL
0.005
0.004
0.003
0.002
0.001
0.006
CANCER NORMAL
CANCER NORMAL
Nor
mal
ized
Inte
nsity
0.0000.000
0.005
0.004
0.003
0.002
0.001
0.006
Nor
mal
ized
Inte
nsity
0.000
LDHA (LVIITAGAR)
CANCER NORMAL
0.05
0.04
0.03
0.02
0.01
0.06
Nor
mal
ized
Inte
nsity
0.00
a) b)
c) d)
LDHB (LIAPVAEEEATVPNNK) TBB3 (ALTVPELTQQMFDAK)
IF4A1 (MFVLDEADEMLSR)
Nor
mal
ized
Inte
nsity
0.025
0.020
0.015
0.010
0.005
CANCER NORMAL
0.005
0.004
0.003
0.002
0.001
0.006
CANCER NORMAL
CANCER NORMAL
Nor
mal
ized
Inte
nsity
0.0000.000
0.005
0.004
0.003
0.002
0.001
0.006
Nor
mal
ized
Inte
nsity
0.000
LDHA (LVIITAGAR)
CANCER NORMAL
0.05
0.04
0.03
0.02
0.01
0.06
Nor
mal
ized
Inte
nsity
0.00
Current study: Full set of 347 candidate markers and more samples (>600) from multiple sources
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Current Studies:
Integrated Diagnostics Alzheimer’s Panel
AD Biomarker Discovery in Plasma
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ALZHEIMER’S DISEASE DIAGNOSTIC DISCOVERY
• Initial study incorporated 50 candidate markers, tested on 60 samples – AD patients– healthy controls
• Currently testing an expanded panel of 350 proteins on 300 samples– AD patients– MCI– healthy controls– other dementias)
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MRM PANEL FROM INITIAL STUDY ACHIEVES 80% SENSITIVITY AND SPECIFICITY
AUC is 0.80 4 proteins (8 peptides) with p-value ≤ 0.0081.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Sen
sitiv
ity
01–Specificity
1.00.90.80.70.60.50.40.30.20.1
Individual ProteinsMultivariate Majority Rule Biomarker
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Off the Shelf Assays:
Plasma Proteins
CSF Proteins
Secreted Proteins, Various Tissues
Rat Toxicology
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126 DETECTED PROTEINS IN MRM RAT TOX PANEL
KidneyAHSGALBAMBPAQP1ASS1B2MCALB1CLUCST3CTSL1EGFENO1FABP3GCLMGSTA1GSTM1GSTP1HAVCR1HGDHIST2H2AA3
LiverA1I3A1MACTBALADALBALPLANXA3ANXA4ANXA5APOA1APOEAPOHBCKDHAC3C4CA2CATCOMTCTSDFAH
VascularA2MCAV1CRPHPICAM1ORM1PLATSAASELETHBDTHBS1VCAM1VEGFA
HeartCKBENO1FABP3LDHAMBS100A9
BrainAPOA1APOBBDNFCRPD3ZNQ7 FGAFGBFGGIL6MMP9Pappalysin-3PARK7SERPINA3LTGFB1TNFVCAM1VEGFA
Skeletal muscleFABP3GOT1MBTNNI2
FETUBGCGDAGGT1GGT5GOT1GPD1GSNGSSGSTA1HPHPDHPXHSPA8HSPD1LAP3MAT2AMDH1MDH2MUP
HSPA5LCN2MGEA5MUPPBLDPHBRBP4SELENBP1SPP1UMODVEGFA
PON1PSMA4PYGLRBP4RGNSERPINA1SERPINA3LSERPINC1SORDSUCLG1SULT1A1TFTHBS1TTRUrocanase