RAPID MICROSCALE PROTEOLYSIS OF PROTEINS FOR MALDI-MS PEPTIDE-MAPPINGUSING IMMOBILIZED TRYPSIN

In this study we present a rapid method for tryptic digestion of prote ins using micro-columns with enzyme immobilized on perfusion chromatog raphy media. The performance of the method is exemplified with acyl-Co A-binding protein and reduced carbamidomethylated bovine serum albumin . The method proved to be significantly faster and yielded a better se quence 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 du e 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 s ample contains large amounts of salt or is too diluted for MALDI-MS an alysis. Furthermore, by step-wise elution from the reversed-phase colu mn, a complex digest can be fractionated, which reduces signal suppres sion and facilitates data interpretation in the subsequent MS-analysis . The method also proved useful for consecutive digestions with enzyme s of different cleavage specificity. This is exemplified with on-colum n tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion. (C) 1997 Elsevier Science B.V.