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 femtomole amounts of proteins in solution using a capillary immobilized protease column is presented. Dilute protein samples are passed through a “μ-digestion” column packed with Poroszyme^? immobilized trypsin 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 protein src kinase and 30 fmol of the protein lck kinase with a tryptic μ-digestion column, we obtained sufficient data from NanoES-MS and MS/MS to unambiguously identify both proteins using database searching. This approach was also used to locate a phosphorylation site on lck kinase starting with only 300 fmol of protein. The method was successfully used to identify an E. coli cold shock protein in a fraction collected from a two-dimensional HPLC separation of an E. coli cell lysate. The μ-digestion column was found to be less susceptible to adsorptive losses than solution digests, thus allowing for digestion and enhanced recovery of peptides from even low fmol amounts of protein in solution.