landerson asms 2005v2 asms... · mass spectrometric quantitation of peptides and proteins using...

ASMS San Antonio 2005 9 June 2005

(c) Plasma Proteome Institute 1

PPI

Developing Peptide MRM-based Assays forCardiovascular Biomarker Proteins in PlasmaUsing a Hybrid Triple Quadrupole Linear Ion

Trap Mass Spectrometer

N. Leigh Anderson1, Christie L Hunter2,Gerald W. Becker3, and Andrew Breite3

1the Plasma Proteome Institute, DC2Applied Biosystems, CA

3Roche Applied Science, IN

ASMS June 2005 San Antonio

PPI

Biomarker PipelineDiscovery

1,000’sCandidate Validation

100’sClinical use

10’s

Ab arrays

LC-MS/MSImmunoassays

LC/LC-MS/MS2!-D gels

SELDI Patterns

PathwaysDNA arrays

Tissue arrays

…

Analytes:

Samples: 10-100’s 1000’s 1,000,000+



PPI

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

1.E+11

=

117 Proteins

Norm

al p

lasm

a co

nce

ntr

atio

n (

pg/m

l) 0 of 3 datasets1 of 3 datasets2 of 3 datasets3 of 3 datasets

Detected in:

From: Candidate-Based Proteomics in the Search for Biomarkers ofCardiovascular Disease, Leigh Anderson, J. Physiol., 563.1, 23-60 (2005)

Plasma Proteome Discovery Platforms Have LimitedSensitivity and Lack Comprehensiveness

PPI

Role for Candidate-Based Proteomics

• Multiplexed specific assays (candidate approach) can addressthree important drawbacks of biomarker discovery platformsfor the middle pipeline stage (verification/validation):

• Throughput• Sensitivity• Coverage (range of analytes)

• Candidate-based approaches forego most of the potential fordiscovery of new biomarkers, but retain some ability to“discover” optimal multi-analyte panels.

• Our purpose is to develop an MS-based candidate approach forhigh-throughput biomarker verification/validation in humanplasma and serum



PPI

Published List of 177 CVD CandidatesCandidate-Based Proteomics in the Search for Biomarkers of Cardiovascular Disease,

Leigh Anderson, J. Physiol 563.1, 23-60 (2005)

PPI

In Silico Selection of MRM Peptides

One or more tryptic peptides used asquantitative surrogates for theprotein (“monitor” peptide concept)

“Inside every bad protein there isat least one good peptide”

Began with 29,155 peptides from“mature” protein forms (21,609unique)

Downloaded SP annotation & computedparameters

Looked for occurrence in Pounds exptldata set

Ranked peptides on composite index ofdesirable properties



PPI

Initial Selection of 30 Targets

PPI

Additional MRM’s Developed Based on Experimental Data(35 Additional Proteins)

• Beavis GPM website (historical LC-MS/MS data on plasma)• Classical IDA analysis of plasma digest• MRM-triggered IDA to find additional proteins on CVD panel

that were not found by normal IDA

Final Method• 137 MRM’s• 17 stable isotope peptides as IS• 52 proteins monitored• 60 peptides, two transitions each• Includes 40 cardiovascular markers

Analytical Platform• ABI/Sciex 4000 QTRAP• 75u LC Packings C18 column, 250nl/min



PPI

Beavis’ GPM Useful in MRM Design

PPI

MRM-Triggered IDA to Develop PeptideMRM Transitions

(workflow now called MIDAS: MRM-Initiated Detection and Sequencing)

•Use script to build de novo MRM’s tosome plasma proteins

•MRM triggered IDA to confirm ID

•Use first and second y ion aboveprecursor mass



PPI

2+

Example Evidence for Ceruloplasmin (8 peptides found)

S/N 3σ = 930

PPI

MRM’s Selected for polySIS CVD_1 Peptides



PPI

Efficient Production of Stable Isotope-labeled Standards (SIS) as polySIS*

KATEHLSTLSEKNWGLSVYADKPETTKILGGHLDAKDTVQIHDITGKTVIGPDGHKQGFGNVATNTDGKEIGELYLPKTGLQEVEVKDDLYVSDAFHKIYHSHIDAPKETAASLLQAGYKITQVLHFTKFPEVDVLTKLGNQEPGGQTALKLSSPAVITDKQWAGLVEKIPPWEAPKLFLEPTQADIALLKSHAPEVITSSPLKIFYNQQNHYDGSTGKEHSSLAFWKVSVSQTSKESDTSYVSLKWELDLDIKSTVLTIPEIIIKLIENGYFHPVKASYPDITGEKDPPSDLLLLKALQDQLVLVAAKAEIEYLEK

AEIEYLEKALQDQLVLVAAKDPPSDLLLLKASYPDITGEKLIENGYFHPVKSTVLTIPEIIIKWELDLDIKESDTSYVSLKVSVSQTSKEHSSLAFWKIFYNQQNHYDGSTGKSHAPEVITSSPLKLFLEPTQADIALLKIPPWEAPKQWAGLVEKLSSPAVITDKLGNQEPGGQTALKFPEVDVLTKITQVLHFTKETAASLLQAGYKIYHSHIDAPKDDLYVSDAFHKTGLQEVEVKEIGELYLPKQGFGNVATNTDGKTVIGPDGHKDTVQIHDITGKILGGHLDAKNWGLSVYADKPETTKATEHLSTLSEK

L-selectinAngiotensinogenCoagulation factor VCholesteryl ester transfer proteinCoagulation factor XIII B chainCoagulation factor XIII A chainVitamin K-dependent protein CC-reactive proteinCoagulation factor IXBeta-2-glycoprotein IAdiponectinCoagulation factor XApolipoprotein(a)Coagulation factor XIIa heavy chainApolipoprotein EPlasminogenAlpha-2-antiplasminApolipoprotein B-100Complement C4 gamma chainProthrombinCeruloplasminAntithrombin-IIIComplement C3Alpha-1-antichymotrypsinFibrinogen beta chainFibrinogen alpha chainFibrinogen gamma chainHaptoglobin beta chainAlpha-1-acid glycoprotein 1Apolipoprotein A-I

Synthesize gene CVD_1a (codonoptimized)

Clone into pIVEX2.4dexpression vector

Express in Roche RTS E coli-basedcell-free system (15N,13C-Lys)

* Patent PendingpolySIS protein standard

PPI

Absolute Protein Quantitation in Relation topolySIS* Peptide Standards

ATEHLSTLSEKNWGLSVYADKPETTKILGGHLDAKDTVQIHDITGKTVIGPDGHKQGFGNVATNTDGKEIGELYLPKTGLQEVEVKDDLYVSDAFHKIYHSHIDAPKETAASLLQAGYKITQVLHFTKFPEVDVLTKLGNQEPGGQTALKLSSPAVITDKQWAGLVEKIPPWEAPKLFLEPTQADIALLKSHAPEVITSSPLKIFYNQQNHYDGSTGKEHSSLAFWKVSVSQTSKESDTSYVSLKWELDLDIKSTVLTIPEIIIKLIENGYFHPVKASYPDITGEKDPPSDLLLLKALQDQLVLVAAKAEIEYLEK

sL-Selectin (in sample)

* Patent Pending

WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYN

polySIS CVD_1 (lys-labeled standard)

+

LC-TQMS of AEIEYLEK

Digest Sample peptide

Labeled standard

polySIS MRM’s



PPI

Depleted PlasmaDigest of 0.01 uLplasma on column

Whole PlasmaDigest of 0.01 uLplasma on column

Subtraction of Top 6 Proteins(albumin, IgG, IgA, haptoglobin,transferrin and antitrypsin)

Using Agilent MARS Column

120 MRM’s covering 50 plasma proteins

PPI

Afamin peptides

Plasma

DepletedPlasma

LPNNVLQEK : 527.8/844.5 DADPDTFFAK : 563.8/825.4

S/N = 6

S/N = 28 S/N = 65

S/N = 7

Immunosubtraction of Top 6 ProteinsYields 5-9-fold Improved S/N



PPI

FibronectinSYTITGLQPGTDYK1543.8

Fibronectin, a protein of much lowernormal abundance (1.4µg/ml) could bemeasured using peptideSYTITGLQPGTDYK (selected insilico) using the transition772.4/680.3 with S/N of 170,suggesting an LLOQ of ~100ng/ml.

PPI

137 MRMs17 stable isotope peptides as IS52 proteins monitored60 peptides, two transitions each40 cardiovascular markers

In Depleted plasma,successfully monitored 48proteins: note two wereMRM to high abundanceproteins

Equivalent of 0.01 uL ofplasma on column

Current MRM Method



PPI

• Apex of the randomvs real distributionsare >2 orders ofmagnitude apart

• If we use a cutoff ofpeak areas for realsignal at 2e4, then only10% of MRM channelswill contain randomsignal

Designed vs “Random” MRM’sOccupancy of Plasma MRM Space Is Low

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

30.00%

35.00%

40.00%

0.0

E+

00

1.0

E+

02

3.2

E+

02

1.0

E+

03

3.2

E+

03

1.0

E+

04

3.2

E+

04

1.0

E+

05

3.2

E+

05

1.0

E+

06

3.2

E+

06

1.0

E+

07

3.2

E+

07

1.0

E+

08

Peak area bin (log)

Fre

qu

en

cy

Peak Area for real signal

Peak Area using mass defect

Peak area for second highest peak

Peak Area using random

SN=10

PPI

CV values of Peptide Quant

0

5

10

15

20

25

30

35

40

45

5 10 15 20 50 >50

CV Value

# P

epti

des

DP2 0.01uL plasma

Avg CV with IS 7%Avg CV overall for good peaks 9.5%

Reproducibility of MRM Panel inDepleted Plasma



PPI

0

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

0 10 20 30 40 50 60

Plasma Proteins

Am

ol/u

L p

lasm

a

Theoretical

MRM

L-selectin

IL-6

IL-8

Phospholipase A2

Apolipoprotein E

Dynamic Range of Current Method inDepleted Plasma: ~4.5 Orders

PPI

17,746

Dynamic Range of Current MethodDetection of L-selectin

Peak area ratio for the L-selectin monitor peptide(AEIEYLEK 497.8/794.4) andSIS (501.8/802.4) standardwas 0.216.

Given 1,300 amol SIS loading(one copy of this peptide permolecule of intact L-selectin)= 280 amol L-selectin per0.01ul injection.

Molecular weight of plasma L-selectin is ~35,000, giving ameasured concentration of980 ng/ml vs published normalvalue of 670 ng/ml.

The dynamic range betweenalbumin (~55mg/ml), and L-selectin is ~4.5 orders ofmagnitude



PPI

SISCAPA*: A Method Combining The Specificity of MS Detectionwith Sensitivity of Antibody Capture

(SISCAPA = Stable Isotope Standards with Capture by Anti-Peptide Antibody)

* patent pending

Mass Spectrometric Quantitation of Peptides and Proteins Using StableIsotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA).Anderson, N.L., et al, Journal of Proteome Research, 3: 235-44 (2004).

PPI

Strategies for Progressive Increases inSensitivity of LC-MS Peptide Quantitation

Our approach generates acoherent, layered seriesof methods based on asingle analytical platform(TQMS), with an explicitsensitivity vs costtradeoff



PPI

Conclusions• Large numbers of candidate biomarkers already exist to jumpstart

verification/validation, and from which improved panels could beconstructed

• MRM assays of monitor peptides offer a potentially rapid path toverification/validation with less cost and effort than sandwichimmunoassays

• Optimal MRM design makes use of both in silico and experimental data

• Current MRM’s appear able to access the top 5 logs of plasma proteinabundance, and cover everything visible on a plasma 2-D gel

• Novel paths to creation of internal standards (e.g., polySIS proteins) canfacilitate assays development

• Marker panel development and validation in large sample sets (e.g.,epidemiological studies) now appears feasible

PPI

AcknowledgementsSISCAPA Experiments

Bob Olafson, Derryl Hardy, UVIC-Genome B.C. Proteomics Centre

Terry Pearson, Lee Haines, Departmentof Biochemistry and Microbiology,University of Victoria, B.C, Canada

John Rush, Cell SignalingPlasma Proteome Database

Malu Polanski (PPI)Richard Fagan, Anna Lobley,

Inpharmatica Ltd., LondonRembert Pieper, Tina Gatlin, present

address: The Institute for GenomicResearch

Radhakrishna S. Tirumalai, Timothy D.Veenstra, Mass Spectrometry Center,U. S. National Cancer Institute

Joshua N. Adkins, Joel G. Pounds,Biological Sciences Department,Pacific Northwest NationalLaboratory

www.plasmaproteome.org

polySIS labeled peptide standardsJerry Becker, Andrew Breite,

Roche Protein Expression Group,Indianapolis

MRM Assay DevelopmentChristie Hunter, Applied

Biosystems, Foster CityMALDI TOF/TOF

Glen Hortin, Alan Remaley, SteveDrake, Gerry Hoehn, ClinicalChemistry Laboratory, NIHClinical Center

GraphicsArkitek Studios, Seattle



PPI

Recent Relevant PapersThe human plasma proteome: History, character, and diagnostic prospects. Anderson, N.L. and Anderson,

N.G., Molecular and Cellular Proteomics, 1.11, 845-867 (2002)

The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Pieper, R., et al Proteomics3(7): 1345-64. (2003).

Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensivesurvey of the human plasma proteome. Pieper, R., Su, Q., Gatlin, C. L., Huang, S. T., Anderson, N. L.,Steiner, S. Proteomics 3(4): 422-32 (2003).

Therapeutic potential of the plasma proteome. Lathrop, J.T., Anderson, N.L., Anderson, N.G., and Hammond,D.J. Current Opinion in Mol. Therapeutics 5:250-257 (2003).

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Captureby Anti-Peptide Antibodies (SISCAPA). Anderson, N.L., Anderson, N.G., Haines, L.R., Hardie, D.B.,Olafson. R.W., and Pearson, T.W. Journal of Proteome Research, 3: 235-44 (2004).

NHLBI Clinical Proteomics Working Group Report. Granger, C.B., Van Eyk, J.E., Mockrin, S.C., and Anderson,N.L., on behalf of the Working Group Members. Circulation 109: 1697-703 (2004).

Candidate-Based Proteomics in the Search for Biomarkers of Cardiovascular Disease, Leigh Anderson, J.Physiol., 563.1, 23-60 (2005)

landerson asms 2005v2 asms... · mass spectrometric quantitation of peptides and proteins using...

Documents