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 post translational modifications such as glycosylation in the diverse set of proteins expressed in a cellular system. High resolution separation systems, such as 2D-gel electrophoresis and more recently liquid chromatography (LC) have been used to fractionate these complex mixtures, however, subsequent mass analysis is hindered by the low level of the purified components. Off-line coupling of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) is an attractive technique for the analysis of such samples, but suffers from sensitivity to the degree of salt contamination that is unavoidable in the isolation of low level protein samples from biological extracts. In this publication we will report on a novel application of a commercially available system for the micro-purification of 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 analyses by protein sequencing and MALDI-TOF/MS. To demonstrate the applicability of these techniques we isolated and performed structural analysis of the following samples: a high-mannose glycopeptide isolated from a digest of the glycoprotein rt-PA, a poly-His tagged recombinant DNA-binding protein isolated by Ni 2+-chelating agarose and a polyclonal antibody sample.