Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics
Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics
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DOI：
10.1074/mcp.m113.035170
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发表时间：
2014-06-01
影响因子：
7
通讯作者：
Mirzaei, Hamid
中科院分区：
文献类型：
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作者：
Guo, Xiaofeng;Trudgian, David C.;Mirzaei, Hamid
自下而上的蛋白质组学在很大程度上依赖于用于蛋白质鉴定和定量的胰蛋白酶肽。由于肽长度、电离效率和翻译后修饰共定位等问题，胰蛋白酶消化通常提供有限的蛋白质序列覆盖。不幸的是，蛋白质中的感兴趣区域，例如，由于接近活性位点或存在重要的翻译后修饰，可能不被胰蛋白酶肽覆盖。检测限、定量准确度和同种型区分也可以通过更大的序列覆盖度来改善。选择性反应监测（SRM）也将极大地受益于能够鉴定另外的可靶向序列。在试图提高蛋白质序列的覆盖率和目标区域的蛋白质，不产生有用的胰蛋白酶肽，我们部署了一个多蛋白酶的HeLa蛋白质组的策略。首先，我们使用七种市售酶的单、双和三酶组合。共进行了48次手术。通过直接分析未分级的细胞裂解物消化物检测到5223种蛋白质;平均序列覆盖率为42%。另外的强阴离子交换分馏的最互补的pests允许识别超过3000个蛋白质，提高平均序列覆盖率。然后，我们构建了一个网络应用程序（https：//proteomics.swmed.edu/confetti），该应用程序允许社区检查靶蛋白或蛋白质同种型，以发现将产生跨越序列中某个感兴趣区域的肽的酶或酶组合。最后，我们研究了非胰蛋白酶消化的SRM的使用。从我们的强阴离子交换分馏数据，我们能够确定三个或更多的蛋白质型SRM候选人在一个单一的消化6056个基因。令人惊讶的是，在这些情况下，25%的消化产生最可观察到的蛋白质型肽既不是胰蛋白酶也不是Lys-C。对八种蛋白质的Asp-N与胰蛋白酶肽的SRM分析确定，Asp-N在八种情况中的五种中产生更高的信号。
Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins deter-mined that Asp-N yielded higher signal in five of eight cases.