COMPREHENSIVE 2-DIMENSIONAL HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY FOR THE ISOLATION OF OVEREXPRESSED PROTEINS AND PROTEOME MAPPING

A two-dimensional liquid chromatographic system is described here whic h uses size-exclusion liquid chromatography (SEC) followed by reversed -phase liquid chromatography (RPLC) to separate the mixture of protein s resulting from the lysis of Escherichia coli cells and to isolate th e proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to revers ed-phase chromatography to separate similarly sized proteins on the ba sis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet regi on at 215 nm regardless of the SEC mobile phase used. The two-dimensio nal (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y ax is and intensity represents quantity of analyte. Following chromatogra phic separation, the analytes are deposited into six 96-well (576 tota l) polypropylene microtiter plates via a fraction collector. Interesti ng fractions are analyzed by matrix-assisted laser desorption ionizati on time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mas s spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact pro teins without any additional sample preparation, electroblotting, dest aining, etc. The remaining 97% of a fraction can then be used for othe r analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC pr otein purification and identification system was used to isolate the s rc homology (SH2) domain of the nonreceptor tyrosine kinase pp60(c-src ) and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome. (C) 1998 Academic Press.