title: Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.
abstract: There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
authors.forename: Susan E
authors.initials: SE
authors.lastname: Abbatiello
authors.affiliation: Buck Institute for Research on Aging, Novato, California 94945;
authors.forename: Birgit
authors.initials: B
authors.lastname: Schilling
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
authors.forename: D R
authors.initials: DR
authors.lastname: Mani
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, and the Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232;
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Zimmerman
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143;
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hall
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195;
authors.forename: Brendan
authors.initials: B
authors.lastname: MacLean
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143;
authors.forename: Matthew
authors.initials: M
authors.lastname: Albertolle
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143;
authors.forename: Simon
authors.initials: S
authors.lastname: Allen
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
authors.forename: Michael
authors.initials: M
authors.lastname: Burgess
authors.affiliation: Buck Institute for Research on Aging, Novato, California 94945;
authors.forename: Michael P
authors.initials: MP
authors.lastname: Cusack
authors.affiliation: Memorial Sloan-Kettering Cancer Center, New York, New York 10065;
authors.forename: Mousumi
authors.initials: M
authors.lastname: Gosh
authors.affiliation: Purdue University, West Lafayette, Indiana 47907;
authors.forename: Victoria
authors.initials: V
authors.lastname: Hedrick
authors.affiliation: Buck Institute for Research on Aging, Novato, California 94945;
authors.forename: Jason M
authors.initials: JM
authors.lastname: Held
authors.affiliation: Purdue University, West Lafayette, Indiana 47907;
authors.forename: H Dorota
authors.initials: HD
authors.lastname: Inerowicz
authors.affiliation: University of Victoria-Genome BC Proteomics Centre, Victoria, British Columbia V8Z 7X8 CAN;
authors.forename: Angela
authors.initials: A
authors.lastname: Jackson
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
authors.forename: Hasmik
authors.initials: H
authors.lastname: Keshishian
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Kinsinger
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, New York 10016;
authors.forename: John
authors.initials: J
authors.lastname: Lyssand
authors.affiliation: Argonne National Laboratory (currently at Northeastern University, Boston Massachusetts 02115;
authors.forename: Lee
authors.initials: L
authors.lastname: Makowski
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mesri
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
authors.forename: Henry
authors.initials: H
authors.lastname: Rodriguez
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, Maryland 20899;
authors.forename: Paul
authors.initials: P
authors.lastname: Rudnick
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, New York 10016;
authors.forename: Pawel
authors.initials: P
authors.lastname: Sadowski
authors.affiliation: National Institute of Statistical Sciences, Research Triangle Park, North Carolina 27709;
authors.forename: Nell
authors.initials: N
authors.lastname: Sedransk
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, and the Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232;
authors.forename: Kent
authors.initials: K
authors.lastname: Shaddox
authors.affiliation: Biostatistics Center, Massachusetts General Hospital, Boston, Massachusetts 02114;
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Skates
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
authors.forename: Eric
authors.initials: E
authors.lastname: Kuhn
authors.affiliation: University of Victoria-Genome BC Proteomics Centre, Victoria, British Columbia V8Z 7X8 CAN;
authors.forename: Derek
authors.initials: D
authors.lastname: Smith
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
authors.forename: Jeffery R
authors.initials: JR
authors.lastname: Whiteaker
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, and the Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232;
authors.forename: Corbin
authors.initials: C
authors.lastname: Whitwell
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
authors.forename: Shucha
authors.initials: S
authors.lastname: Zhang
authors.affiliation: University of Victoria-Genome BC Proteomics Centre, Victoria, British Columbia V8Z 7X8 CAN;
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143;
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Fisher
authors.affiliation: Buck Institute for Research on Aging, Novato, California 94945;
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, and the Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232;
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195;
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, New York 10016;
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Neubert
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
authors.affiliation: Purdue University, West Lafayette, Indiana 47907;
authors.forename: Fred E
authors.initials: FE
authors.lastname: Regnier
authors.affiliation: Memorial Sloan-Kettering Cancer Center, New York, New York 10065;
authors.forename: Paul
authors.initials: P
authors.lastname: Tempst
authors.affiliation: From the Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; scarr@broad.mit.edu.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D009363:Neoplasm Proteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D009369:Neoplasms / Q000097:blood / Q000378:metabolism*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D009363:Neoplasm Proteins; D010455:Peptides