Trypsin cleaves exclusively C-terminal to arginine and lysine residues
Trypsin cleaves exclusively C-terminal to arginine and lysine residues
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DOI：
10.1074/mcp.t400003-mcp200
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发表时间：
2004-06-01
影响因子：
7
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Olsen, JV;Ong, SE;Mann, M
基于质谱的蛋白质组学中的几乎所有大型项目都使用胰蛋白酶将蛋白质混合物转化为更容易分析的肽群。当针对序列数据库搜索肽片段化光谱时，可能需要匹配肽序列，以符合胰蛋白酶特异性，即仅C-末端对精氨酸或赖氨酸。但是，在许多已发表的报告中，大量蛋白质是通过非胰肽鉴定的。在这里，我们使用新的离子陷阱傅里叶转化质谱仪的每百万质量精度的子部分，与典型的离子陷阱实验相比，对肽鉴定的置信度提高了100倍以上，并表明胰蛋白酶仅裂解仅C-末端到精氨酸裂解和赖氨酸。我们发现，非特里肽肽仅作为蛋白质的C末端肽以及全胰岛素肽N末端的分裂产物出现。模拟较低的质量精度导致大量用非特殊肽命中率错误识别的蛋白质。我们的结果表明，在先前的研究中应重新检查此类肽命中率，并且肽鉴定应基于严格的胰蛋白酶特异性。
Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.