Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT

Abstract

One of the most effective methods for the direct identification of proteins from complex mixtures without first having to resolve them by polyacrylamide gel electrophoresis is to separate proteolytically generated peptides by microcapillary HPLC and then collect data directly on the eluent using a tandem mass spectrometer. Multidimensional HPLC separation techniques provide access to even more complex mixtures of proteins. A set of techniques for multidimensional analysis was developed in our lab; collectively they are known as multidimensional protein identification technology (MudPIT). These strategies employ a biphasic column with a section of reversed phase (RP) material flanked by strong cation exchange (SCX) resin and allow for multidimensional separation of peptides. A variation on MudPIT adds an additional section of RP material behind the SCX and RP. This 3-phase column can be used for “online” desalting of the sample. We compare the analysis of a complex mixture of proteins purified by their association with bovine brain microtubules using a single-dimension LC-MS/MS column, a 2-phase (standard) MudPIT column, and a 3-phase MudPIT column. We find that the 3-phase MudPIT column yields a greater number of protein identifications for this test sample and allows data to be collected on a set of hydrophilic peptides not sampled using the 2-phase MudPIT column.