2-DIMENSIONAL SEC RPLC COUPLED TO MASS-SPECTROMETRY FOR THE ANALYSIS OF PEPTIDES/

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC in terface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design p ermits the use of conventional analytical diameter HPLC columns, 7.8 m m for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 mu m diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimens ional chromatographic separation, an electrospray mass spectrometer de tects the peptide fragments. The mass spectrometer scans a 2000 m/z ra nge to identify the analytes from their molecular weights. The analyse s of tryptic digests of ovalbumin and serum albumin are each described .