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J Proteome Res DOI:10.1021/pr9006365

Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.

Publication TypeJournal Article
Year of Publication2010
AuthorsTabb, DL, Vega-Montoto, L, Rudnick, PA, Variyath, AMulayath, Ham, A-JL, Bunk, DM, Kilpatrick, LE, Billheimer, DD, Blackman, RK, Cardasis, HL, Carr, SA, Clauser, KR, Jaffe, JD, Kowalski, KA, Neubert, TA, Regnier, FE, Schilling, B, Tegeler, TJ, Wang, M, Wang, P, Whiteaker, JR, Zimmerman, LJ, Fisher, SJ, Gibson, BW, Kinsinger, CR, Mesri, M, Rodriguez, H, Stein, SE, Tempst, P, Paulovich, AG, Liebler, DC, Spiegelman, C
JournalJ Proteome Res
Volume9
Issue2
Pages761-76
Date Published2010 Feb 05
ISSN1535-3907
KeywordsChromatography, Liquid, Proteome, Reproducibility of Results, Tandem Mass Spectrometry
Abstract

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

URLhttp://dx.doi.org/10.1021/pr9006365
DOI10.1021/pr9006365
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/19921851?dopt=Abstract

Alternate JournalJ. Proteome Res.
PubMed ID19921851
PubMed Central IDPMC2818771
Grant ListU24 CA126485-02 / CA / NCI NIH HHS / United States
U24 CA126479-02 / CA / NCI NIH HHS / United States
U24 CA126485 / CA / NCI NIH HHS / United States
U24 CA126480 / CA / NCI NIH HHS / United States
U24 CA126476-02 / CA / NCI NIH HHS / United States
U24 126479 / / PHS HHS / United States
U24 CA126477-02 / CA / NCI NIH HHS / United States
U24 126480 / / PHS HHS / United States
U24 CA126477 / CA / NCI NIH HHS / United States
U24 CA126479 / CA / NCI NIH HHS / United States
U24 CA126480-02 / CA / NCI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
U24 126477 / / PHS HHS / United States