Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

详细信息    查看全文

• 作者：Gobom ; Johan ; Nordhoff ; Eckhard ; Ekman ; Rolf ; Roepstorff ; Peter
• 关键词：Immobilized proteases ; MALDI-MS ; Peptide mapping ; Protein identification ; Tryptic digestion
• 刊名：International Journal of Mass Spectrometry and Ion Processes
• 出版年：1997
• 出版时间：December, 1997
• 年：1997
• 卷：169-170
• 期：Complete
• 页码：153-163
• 全文大小：649 K

文摘

In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversed-phase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.