Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation
Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation
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DOI：
10.1073/pnas.0405549101
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发表时间：
2004-09-14
影响因子：
11.1
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Olsen, JV;Mann, M
基于MS的蛋白质组学通常涉及胰蛋白酶多肽(串联MS或MS2)的裂解和通过搜索蛋白质序列数据库来鉴定它们。在离子陷阱仪器中，碎片可以进一步碎裂和分析，这一过程称为MS/MS/MS或MS3。在这里，我们报告了在线性离子陷阱中有效的离子捕获导致MS3的采集时间和光谱质量类似于使用传统的3D离子陷阱的MS2实验。N-末端或C-末端离子的碎裂导致信息量和低背景光谱，即使在肽的亚毫微米摩尔水平上也是如此。通常，C-末端离子被选择用于进一步的碎裂，而MS3光谱极大地限制了肽序列的C-末端氨基酸。MS3谱可以解决鉴定中的模糊性，这是蛋白质组学中的一个关键问题。由于线性离子陷阱的灵敏度和快速扫描速度，即使在对非常复杂的混合物进行测序时，也可以获得每个肽的几个MS3光谱。我们计算了实验中的MS3光谱来自于所考虑的多肽的给定N-端或C-端离子的碎裂的概率。该MS3识别分数可以与来自现有搜索引擎的前体多肽的MS2分数相结合。当MS3在线性离子陷阱-傅立叶变换质谱仪组合上进行时，准确的多肽质量进一步增加了多肽鉴定的信心。
MS-based proteomics usually involves the fragmentation of tryptic peptides (tandem MS or MS2) and their identification by searching protein sequence databases. In ion trap instruments fragments can be further fragmented and analyzed, a process termed MS/MS/MS or MS3. Here, we report that efficient ion capture in a linear ion trap leads to MS3 acquisition times and spectra quality similar to those for MS2 experiments with conventional 3D ion traps. Fragmentation of N- or C-terminal ions resulted in informative and low-background spectra, even at subfemtomol levels of peptide. Typically C-terminal ions are chosen for further fragmentation, and the MS3 spectrum greatly constrains the C-terminal amino acids of the peptide sequence. MS3 spectra allow resolution of ambiguities in identification, a crucial problem in proteomics. Because of the sensitivity and rapid scan rates of the linear ion trap, several MS3 spectra per peptide can be obtained even when sequencing very complex mixtures. We calculate the probability that an experimental MS3 spectrum originates from fragmentation of a given N- or C-terminal ion of a peptide under consideration. This MS3 identification score can be combined with the MS2 scores of the precursor peptide from existing search engines. When MS3 is performed on the linear ion trap-Fourier transform mass spectrometer combination, accurate peptide masses further increase confidence in peptide identification.