A rapid and sensitive procedure for the micro-purification and subsequent characterization of peptides and protein samples by N-terminal sequencing and matrix assisted laser desorption ionization time of flight mass spectrometry

The characterization of the proteome, a key activity in the post-genome era , is made extremely challenging by the microheterogeneity introduced by pos t translational modifications such as glycosylation in the diverse set of p roteins expressed in a cellular system. High resolution separation systems, such as :2D-gel electrophoresis and more recently liquid chromatography (L C) have been used to fractionate these complex mixtures, however, subsequen t mass analysis is hindered by the low level of the purified components. Of f-line coupling of matrix assisted laser desorption ionization time of flig ht mass spectrometry (MALDI-TOF/MS) is an attractive technique for the anal ysis of such samples, but suffers from sensitivity to the degree of salt co ntamination that is unavoidable in the isolation of low level protein sampl es from biological extracts. In this publication we will report on a novel application of a commercially available system for the micro-purification o f peptides and proteins. In this procedure micro-columns (normally used for sequencing of electroblotted samples) were used to rapidly purify protein digests or crude extracts of proteins in sufficient amounts for further ana lyses by protein sequencing and MALDI-TOF/MS. To demonstrate the applicabil ity of these techniques we isolated and performed structural analysis of th e following samples: a high-mannose glycopeptide isolated from a digest of the glycoprotein rt-PA, a poly-His tagged recombinant DNA-binding protein i solated by Ni2+-chelating agarose and a polyclonal antibody sample. (C) 199 8 Elsevier Science B.V. All rights reserved.