Chen Emily I, Cociorva Daniel, Norris Jeremy L, Yates John R
Department of Cell Biology, 10550 North Torrey Pines Road, SR11, The Scripps Research Institute, La Jolla, California 92037, USA.
J Proteome Res. 2007 Jul;6(7):2529-38. doi: 10.1021/pr060682a. Epub 2007 May 27.
An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.
本研究对在混合有机-水体系和水体系中，使用或不使用与质谱兼容的去污剂，通过微液相色谱-串联质谱（microLC-MS/MS）进行肽段/蛋白质鉴定的胰蛋白酶消化策略进行了优化和比较。我们确定，在蛋白质组鸟枪法的蛋白水解消化方案中添加与质谱兼容的去污剂，可显著增加复杂蛋白质混合物中肽段和蛋白质的鉴定数量。在胰蛋白酶消化缓冲液中加入与质谱兼容的去污剂可提高蛋白质的溶解度和蛋白水解效率。当使用液相色谱-串联质谱时，结合了与质谱兼容去污剂的改良胰蛋白酶消化方案，能从5微克胰腺细胞裂解物中持续鉴定出300多种蛋白质，并且比用尿素进行胰蛋白酶消化产生更多的肽段鉴定结果。此外，通过合并使用三种不同与质谱兼容去污剂的胰蛋白酶消化所得到的蛋白质鉴定结果，鉴定出了700多种蛋白质。我们还观察到，混合水相和有机相溶剂体系的使用，与不同的与质谱兼容去污剂结合时，会影响蛋白质的鉴定。由不同的与质谱兼容去污剂和缓冲液组合产生的肽段混合物在疏水性上存在显著差异。我们的结果表明，包含与质谱兼容去污剂的蛋白质消化方案，在观察到的肽段鉴定中产生了定量和定性的变化，这导致总体上蛋白质鉴定数量增加，并可能增加低丰度蛋白质的鉴定。
