Multidimensional chromatography coupled to electrospray ionization time-of-flight mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins

The limitations of 2-D gels for global proteomics have encouraged the development of alternative approaches for identifying proteins in complicated mixtures, and determining their modification state. In this work, we describe the application of multidimensional liquid chromatography (SCX-RPLC) coupled with electrospray time-of-flight mass spectrometry and off-line fraction collection to analyze complex intact protein mixtures. Methods were developed using both standard proteins and an enriched yeast ribosomal fraction sample containing ∼100 proteins, which permitted assessment of the effectiveness of the individual separation dimensions, as well as investigation of the interplay between separation capacity and electrospray MS performance.