Optimization of mass spectrometry-compatible surfactants for shotgun proteomics
Optimization of mass spectrometry-compatible surfactants for shotgun proteomics
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DOI：
10.1021/pr060682a
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发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Yates, John R., III
中科院分区：
文献类型：
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作者：
Chen, Emily I.;Cociorva, Daniel;Yates, John R., III
本研究采用MU LC-MS/MS联用技术，在有机-水和水的混合体系中，对胰酶消化多肽/蛋白质的方法进行了优化和比较。我们确定，通过鸟枪式蛋白质组学，在蛋白质分解消化方案中添加与MS兼容的洗涤剂可以显著提高复杂蛋白质混合物中的多肽和蛋白质的识别能力。通过在胰酶消化缓冲液中加入与MS兼容的洗涤剂，提高了蛋白质的溶解和蛋白质分解效率。当使用LC-MS/MS时，结合MS兼容洗涤剂的改进的胰酶消化方法一致地从5微克胰腺细胞裂解产物中鉴定出300多种蛋白质，并产生比尿素胰酶消化更多数量的多肽鉴定。此外，通过将来自胰酶消化的蛋白质鉴定与三种不同的MS兼容洗涤剂合并，超过700种蛋白质被鉴定。我们还观察到，混合水和有机溶剂系统的使用可以影响与不同MS兼容洗涤剂组合的蛋白质鉴定。不同MS相容洗涤剂和缓冲液组合产生的多肽混合物在疏水性上有显著差异。我们的结果表明，结合MS兼容洗涤剂的蛋白质消化方案会在观察到的多肽鉴定中产生定量和定性的变化，这导致整体蛋白质鉴定的增加，并可能增加对低丰度蛋白质的鉴定。
An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by mu LC-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 mu g 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.