Quantifying the Impact of Chimera MS/MS Spectra on Peptide Identification in Large-Scale Proteomics Studies
Quantifying the Impact of Chimera MS/MS Spectra on Peptide Identification in Large-Scale Proteomics Studies
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DOI：
10.1021/pr1003856
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发表时间：
2010-08-01
影响因子：
4.4
通讯作者：
Old, William M.
中科院分区：
文献类型：
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作者：
Houel, Stephane;Abernathy, Robert;Old, William M.
通过LC/MS/MS在复杂混合物中进行蛋白质鉴定的复杂因素是“嵌合体”光谱的问题，其中具有相似质量和保留时间的两个或更多个前体离子通过MS/MS共测序。嵌合体光谱显示由于来自污染母体的不可识别的碎片离子而降低的分数。然而，LC/MS/MS数据集中嵌合体的程度及其对蛋白质鉴定工作流程的影响尚未完全了解。我们报告嵌合体计数器，一个软件程序，检测嵌合体的数据集上收集的Orbitrap/LTQ仪器。对从定义明确的肽MS/MS光谱对创建的合成嵌合体的评价显示，当污染碎片离子强度超过目标碎片离子强度的20%时，嵌合体最显著地降低数据库搜索分数。在大规模数据集中，嵌合体MS/MS的识别率比非嵌合体光谱低2倍。重要的是，这以不取决于绝对前体离子强度而是取决于相对于中值前体强度分布的强度的方式发生。我们进一步表明，嵌合体通过增加假阴性而减少了可接受的肽鉴定的数量，而假阳性几乎没有增加。结果提供了一个框架，用于识别嵌合体和表征其对MS/MS假阴性类别的贡献。
A complicating factor for protein identification within complex mixtures by LC/MS/MS is the problem of "chimera" spectra, where two or more precursor ions with similar mass and retention time are co-sequenced by MS/MS. Chimera spectra show reduced scores due to unidentifiable fragment ions derived from contaminating parents. However, the extent of chimeras in LC/MS/MS data sets and their impact on protein identification workflows are incompletely understood. We report Chimera Counter, a software program which detects chimeras in data sets collected on an Orbitrap/LTQ instrument. Evaluation of synthetic chimeras created from pairs of well-defined peptide MS/MS spectra reveal that chimeras reduce database search scores most significantly when contaminating fragment ion intensities exceed 20% of the targeted fragment ion intensities. In large-scale data sets, the identification rate for chimera MS/MS is 2-fold lower compared to nonchimera spectra. Importantly, this occurs in a manner which depends not on absolute precursor ion intensity, but on intensity relative to the median precursor intensity distribution. We further show that chimeras reduce the number of accepted peptide identifications by increasing false negatives while showing little increase in false positives. The results provide a framework for identifying chimeras and characterizing their contribution to the poorly understood false negative class of MS/MS.