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ASMS San Antonio 2005 9 June 2005
(c) Plasma Proteome Institute 1
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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
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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+
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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
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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
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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)
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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
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Initial Selection of 30 Targets
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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
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Beavis’ GPM Useful in MRM Design
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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
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2+
Example Evidence for Ceruloplasmin (8 peptides found)
S/N 3σ = 930
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MRM’s Selected for polySIS CVD_1 Peptides
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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
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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
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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
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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
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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.
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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
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• 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
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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
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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
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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
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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).
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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
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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
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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
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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)