Greer Sylvester M, Parker W Ryan, Brodbelt Jennifer S
Department of Chemistry, University of Texas at Austin, 105 East 24th Street, Austin, Texas 78712, United States.
J Proteome Res. 2015 Jun 5;14(6):2626-32. doi: 10.1021/acs.jproteome.5b00165. Epub 2015 May 18.
Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of Halobacterium salinarum peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5-10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins.
最近的质谱研究报告称，通过使用多种蛋白酶或采用多种或替代激活方法，如电子转移解离与碰撞激活解离（CAD）相结合，蛋白质组覆盖率有所提高。在本研究中，与高能CAD（HCD）相比，评估了使用193nm紫外光解离分析由四种蛋白酶（胰蛋白酶、LysC、GluC和糜蛋白酶）产生的数千种盐生盐杆菌肽的情况。胰蛋白酶消化的蛋白质，HCD的序列覆盖率高于紫外光解离。LysC消化产生的紫外光解离和HCD的序列覆盖率相似；然而，对于GluC和糜蛋白酶消化的蛋白质，紫外光解离平均实现的序列覆盖率多5-10%。HCD对胰蛋白酶的肽鉴定更多（在1%的错误发现率下，HCD鉴定出4356个肽，而紫外光解离鉴定出3907个肽），而紫外光解离鉴定出LysC消化物（紫外光解离鉴定出1033个肽，HCD鉴定出844个肽）、糜蛋白酶消化物（紫外光解离鉴定出3219个肽，HCD鉴定出2921个肽）和GluC消化物（紫外光解离鉴定出2834个肽，HCD鉴定出2393个肽）中更多的肽，相应地鉴定出更多的蛋白质。
