title: Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).
abstract: Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. susana@broad.mit.edu
authors.forename: Susan E
authors.initials: SE
authors.lastname: Abbatiello
authors.forename: D R
authors.initials: DR
authors.lastname: Mani
authors.forename: Birgit
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authors.forename: Lisa J
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authors.forename: Xingdong
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authors.initials: SC
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authors.forename: Terri
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authors.lastname: Addona
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authors.lastname: Allen
authors.forename: Nathan G
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authors.forename: Jason M
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authors.forename: H Dorota
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authors.initials: H
authors.lastname: Keshishian
authors.forename: Jong Won
authors.initials: JW
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authors.forename: John S
authors.initials: JS
authors.lastname: Lyssand
authors.forename: C Paige
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authors.lastname: Rudnick
authors.forename: Pawel
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authors.lastname: Sadowski
authors.forename: Kent
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authors.lastname: Shaddox
authors.forename: Derek
authors.initials: D
authors.lastname: Smith
authors.forename: Daniela
authors.initials: D
authors.lastname: Tomazela
authors.forename: Asa
authors.initials: A
authors.lastname: Wahlander
authors.forename: Sofia
authors.initials: S
authors.lastname: Waldemarson
authors.forename: Corbin A
authors.initials: CA
authors.lastname: Whitwell
authors.forename: Jinsam
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authors.lastname: You
authors.forename: Shucha
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authors.lastname: Zhang
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Kinsinger
authors.forename: Mehdi
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authors.lastname: Mesri
authors.forename: Henry
authors.initials: H
authors.lastname: Rodriguez
authors.forename: Christoph H
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authors.lastname: Borchers
authors.forename: Charles
authors.initials: C
authors.lastname: Buck
authors.forename: Susan J
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authors.lastname: Fisher
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.forename: Daniel
authors.initials: D
authors.lastname: Liebler
authors.forename: Michael
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authors.lastname: Maccoss
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Neubert
authors.forename: Amanda
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authors.lastname: Paulovich
authors.forename: Fred
authors.initials: F
authors.lastname: Regnier
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Skates
authors.forename: Paul
authors.initials: P
authors.lastname: Tempst
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authors.lastname: Wang
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mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D012984:Software; D013997:Time Factors
chemical_list: D010455:Peptides