Multiple separations facilitate identification of protein variants by mass spectrometry

Identification of variant proteins from complex biological samples promises to contribute much to our understanding the etiology of pathological states. Characterization of variants, either due to genetic mutations in protein sequences or to post-translational modifications, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. Identification of a few peptides by database retrieval is not adequate when the goal is to have a complete understanding of the modifications of the protein. Although one advantage of mass spectrometry is its ability to obtain specific responses to several components, the complexity of biological samples is often overwhelming, resulting in spectra lacking useful information. For complex mixtures, isolation procedures before mass spectrometric analysis may need to include a variety of chromatographic and electrophoretic separation techniques. In this report, we illustrate how several preparative steps were essential for obtaining information about modified human lens beta-crystallins. The preparative techniques prior to mass spectrometry included size exclusion chromatography, reversed phase chromatography, two-dimensional gel electrophoresis, in situ digestion of the proteins and peptide trapping and washing before a final reversed phase high performance liquid chromatographic separation on-line to the mass spectrometer. This approach for isolation and analysis, when customized for other proteins, should find application in many studies where protein variants of complex mixtures are to be identified.