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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