Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.
Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.
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DOI：
10.1021/pr9006365
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发表时间：
2010-02-05
影响因子：
4.4
通讯作者：
Spiegelman, Cliff
中科院分区：
文献类型：
--
作者：
Tabb, David L.;Vega-Montoto, Lorenzo;Rudnick, Paul A.;Variyath, Asokan Mulayath;Ham, Amy-Joan L.;Bunk, David M.;Kilpatrick, Lisa E.;Billheimer, Dean D.;Blackman, Ronald K.;Cardasis, Helene L.;Carr, Steven A.;Clauser, Karl R.;Jaffe, Jacob D.;Kowalski, Kevin A.;Neubert, Thomas A.;Regnier, Fred E.;Schilling, Birgit;Tegeler, Tony J.;Wang, Mu;Wang, Pei;Whiteaker, Jeffrey R.;Zimmerman, Lisa J.;Fisher, Susan J.;Gibson, Bradford W.;Kinsinger, Christopher R.;Mesri, Mehdi;Rodriguez, Henry;Stein, Stephen E.;Tempst, Paul;Paulovich, Amanda G.;Liebler, Daniel C.;Spiegelman, Cliff
液相色谱 - 串联质谱（LC - MS/MS）蛋白质组学仪器的复杂性带来了许多可能的变异源。肽段的数据依赖性采集是发现蛋白质组学核心的一个随机因素。尽管这种变异影响肽段的鉴定，但蛋白质组学鉴定远非完全随机。在这项研究中，我们分析了来自美国国家癌症研究所（NCI）癌症临床蛋白质组技术评估的实验室间数据集，以检验肽段和蛋白质鉴定的重复性和再现性。所包含的数据涵盖了在四台赛默飞世尔（Thermo）LTQ和四台轨道阱（Orbitrap）仪器上进行的144次LC - MS/MS实验。样本包括酵母裂解物、NCI - 20定义的动态范围蛋白质混合物以及西格玛（Sigma）UPS 1定义的等摩尔蛋白质混合物。我们的一些发现强化了传统观点，例如蛋白质的重复性和再现性高于肽段。然而，从数据中得到的大多数经验教训更为微妙。轨道阱被证明能够具有更高的重复性和再现性，但异常的性能偶尔会抵消这些优势。即使是最简单的蛋白质酶解所产生的肽离子也比单次实验中LC - MS/MS所能鉴定的要多。我们观察到，技术重复样本对的肽段列表重叠率为35% - 60%，这给出了这些实验中肽段水平重复性的范围。样本复杂性似乎并不影响肽段鉴定的重复性，即使鉴定出的光谱数量变化了一个数量级。对蛋白质光谱计数的统计分析表明，轨道阱在技术重复样本间具有更高的稳定性，这使得它们在生物标志物候选物发现方面优于LTQ仪器。最具重复性的肽段是那些对应于常规胰蛋白酶切割位点的肽段、那些产生强烈质谱信号的肽段以及那些由产生许多不同肽段的蛋白质所产生的肽段。同一类型不同仪器之间的再现性比单台仪器上技术重复样本的重复性低几个百分点。这些发现强调了将重复性作为分析技术的基本特征进行评估的重要性。
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.
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影响因子：
7.4
作者：
Tabb, DL;MacCoss, MJ;Yates, JR
通讯作者：
Yates, JR
影响因子：
48
作者：
Bammler, T;Beyer, RP;Zarbl, H
通讯作者：
Zarbl, H
影响因子：
7.4
作者：
Liu, HB;Sadygov, RG;Yates, JR
通讯作者：
Yates, JR
影响因子：
3.4
作者：
Omenn, GS;States, DJ;Hanash, SM
通讯作者：
Hanash, SM
DOI：
10.1074/mcp.m900223-mcp200
发表时间：
2010-02
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Rudnick PA;Clauser KR;Kilpatrick LE;Tchekhovskoi DV;Neta P;Blonder N;Billheimer DD;Blackman RK;Bunk DM;Cardasis HL;Ham AJ;Jaffe JD;Kinsinger CR;Mesri M;Neubert TA;Schilling B;Tabb DL;Tegeler TJ;Vega-Montoto L;Variyath AM;Wang M;Wang P;Whiteaker JR;Zimmerman LJ;Carr SA;Fisher SJ;Gibson BW;Paulovich AG;Regnier FE;Rodriguez H;Spiegelman C;Tempst P;Liebler DC;Stein SE
通讯作者：
Stein SE