Article

Design, Implementation, and Multi-Site Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in LC-MRM-MS.

The Broad Institute of MIT and Harvard, United States
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 05/2013; DOI: 10.1074/mcp.M112.027078
Source: PubMed

ABSTRACT Multiple reaction monitoring (MRM) mass spectrometry (MS) 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 eleven laboratories on a total of fifteen different instruments, enabled early diagnoses of LC and MS anomalies that indicated sub-optimal 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 (CV) <0.15, peak width CV <0.15, standard deviation of RT <0.15 min (9 sec) and the RT drift <0.5min (30 sec). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the utility and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to insure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread utilization of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

0 Followers
 · 
448 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: In the realm of clinical chemistry the field of clinical proteomics, i.e., the application of proteomic methods for understanding mechanisms and enabling diagnosis, prediction, measurement of activity, and treatment response in disease, is first and foremost a discovery and research tool that feed assay development downstream. Putative new assay candidates generated by proteomics discovery projects compete with well-established assays with known indications, well-described performance, and of known value in specific clinical settings. Careful attention to the many options available in the design, execution, and interpretation of clinical proteomics studies is thus necessary for translation into clinical practice. We here review and discuss important options associated with clinical proteomics endeavors stretching from the planning phases to the final use in clinical chemistry. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    PROTEOMICS - CLINICAL APPLICATIONS 02/2015; 9(1-2). DOI:10.1002/prca.201400137 · 2.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a1 ions. The advantageous generation of a1 ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported.
    Journal of the American Society for Mass Spectrometry 08/2014; 25(10). DOI:10.1007/s13361-014-0951-7 · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH MS2 data-independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CVinter <30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CVinter >30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell-extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers. This article is protected by copyright. All rights reserved.
    Electrophoresis 07/2014; 35(24). DOI:10.1002/elps.201400167 · 3.16 Impact Factor

Full-text

Download
50 Downloads
Available from
May 20, 2014