CHARACTERIZATION OF FEMTOMOLE LEVELS OF PROTEINS IN SOLUTION USING RAPID PROTEOLYSIS AND NANOELECTROSPRAY IONIZATION MASS-SPECTROMETRY

A method for the rapid proteolytic digestion of low picomole to low fe mtomole amounts of proteins in solution using a capillary immobilized protease column is presented. Dilute protein samples are passed throug h a ''mu-digestion'' column packed with Poroszyme(TM) immobilized tryp sin for generation of proteolytic fragments in less than 10 min. After digestion, nanoelectrospray ionization mass spectrometry (NanoES) is used to generate a peptide map, and peptides of interest are subjected to MS/MS from the same sample. By digesting only 100 fmol of the prot ein src kinase and 30 fmol of the protein lck kinase with a tryptic mu -digestion column, we obtained sufficient data from NanoES-MS and MS/M S to unambiguously identify both proteins using database searching. Th is approach was also used to locate a phosphorylation site on lck kina se starting with only 300 fmol of protein. The method was successfully used to identify an E. coli cold shock protein in a fraction collecte d from a two-dimensional HPLC separation of an E. coli cell lysate. Th e mu-digestion column was found to be less susceptible to adsorptive l osses than solution digests, thus allowing for digestion and enhanced recovery of peptides from even low fmol amounts of protein in solution . (C) 1997 American Society for Mass Spectrometry.