More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC-MS/MS
More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC-MS/MS
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DOI：
10.1021/pr101060v
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发表时间：
2011-04-01
影响因子：
4.4
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
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作者：
Michalski, Annette;Cox, Juergen;Mann, Matthias
鸟枪法蛋白质组学需要从复杂的混合物中鉴定出尽可能多的肽。在这里，我们研究了在细胞裂解物的标准 LC 运行中，有多少肽可以通过高分辨率 MS 检测到，以及其中有多少肽可以通过数据依赖型 MS/MS 检测到。同位素簇由 MaxQuant 测定，并严格过滤肽的典型电荷状态和保留时间。这产生了超过 100 000 个可能的肽特征，其中只有约 16% 已成为 MS/MS 的目标。三个仪器属性决定了可识别的其他肽的比例：测序速度、灵敏度和前体离子分离。在我们的数据中，要针对所有肽特征，需要 25/s 的 MS/MS 扫描速率，但对于相当丰富的肽，该扫描速率会降至低于 17/s。灵敏度是一个更大的挑战，因为许多肽特征需要较长的 MS/MS 进样时间 (>250 ms)。然而，最大的限制是 MS/MS 选择窗口中目标肽离子强度的比例通常较低（“前体离子分数”或 PIF）。 PIP 中位数仅为 0.14，使得这些肽难以通过标准 MS/MS 方法进行识别。我们的结果有助于制定进一步提高鸟枪法蛋白质组学覆盖率的策略。
Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100 000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the "precursor ion fraction" or PIF). Median PIP is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.