Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS
Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS
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DOI：
10.1021/pr025557n
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发表时间：
2003-01-01
影响因子：
4.4
通讯作者：
Hancock, WS
中科院分区：
文献类型：
--
作者：
Choudhary, G;Wu, SL;Hancock, WS
本研究使用多种酶消化来增加通过鸟枪测序方法进行蛋白质组分析所鉴定的蛋白质的序列覆盖率。使用的酶是胰蛋白酶、Lys-C 和 Asp-N，它们分别在精氨酸和赖氨酸残基、赖氨酸和天冬氨酸残基处裂解。使用糖蛋白、组织纤溶酶原激活剂、t-PA 对该方法进行了评估，与单次酶消化相比，该方法提供了增强的序列覆盖率。然后用复杂的蛋白质组样本（即血浆）对该方法进行了评估。结果发现，胰蛋白酶和 Lys-C 能够检测重叠但不同的蛋白质组，并且数据的数字重组显着增加了蛋白质鉴定的数量以及每个蛋白质鉴定的肽的数量（这提高了分配的确定性）。
This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).