Characterization of Complex Protein Gel Bands by Offline LC-MALDI QIT TOF MS

Introduction

Traditionally, identification of complex protein mixtures has required a series of time-consuming protocols. Protein mixtures are separated using 1D or 2D gel electrophoresis, protein bands or spots excised and digested producing a peptide mass fingerprint (PMF) to identify the protein using MALDI mass spectrometry and database searching. Limitations can arise, including co-migration of more than one protein, suppression effects during ionization, or the PMF not correlating with any protein in the databases. In many cases, it is simply the complexity of the sample which may contain hundreds or thousands of tryptic peptides from a number of different proteins that proves to be the restrictive factor when attempting to assign putative protein identification. It is clear that complicated samples require an added dimension of separation prior to MALDI analysis and the use of reverse phase liquid chromatography followed by deposition of the separated peptides directly onto a MALDI target has been recently reported. In this instance, the separated peptides are analyzed initially by MS techniques to provide a “candidate list” and subsequently by MS/MS which can provide sufficient information to generate useful sequence data and protein identification via database searching. Here, in an attempt to circumnavigate many of these issues we have analysed digested 1D gel samples using offline LC-MALDI QIT TOF MS.

December 6, 2013 GMT