RAPID MASS-SPECTROMETRIC IDENTIFICATION OF PROTEINS FROM 2-DIMENSIONAL POLYACRYLAMIDE GELS AFTER IN GEL PROTEOLYTIC DIGESTION

We report a rapid method for identifying proteins resolved by two-dime nsional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-ass isted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsi n solution to gel plug was quantitatively controlled to promote reprod ucible digestion and to maximize the digestion yield. To make the dige stion samples more compatible with MALDI-MS, the volatile salt ammoniu m bicarbonate in the digestion buffer was largely removed prior to pep tide extraction. Samples of mixed tryptic peptides from in-gel digesti on were used without purification to obtain molecular weights by MALDI -MS with a-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALD I-MS spectra were distinguished from those of impurities by using seve ral types of controls, and masses were corrected by using trypsin auto digestion fragments as internal calibration standards. Two different p eptide-matching computer programs were used to interrogate sequence da tabases and identify proteins. Identification was enhanced by generati on of orthogonal data sets (by using different proteases) and by inclu ding experimental values of isoelectric point (pI) and molecular weigh t to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine pr otein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abu ndance.