COMPARISON OF IN-GEL AND ON-MEMBRANE DIGESTION METHODS AT LOW TO SUB-PMOL LEVEL FOR SUBSEQUENT PEPTIDE AND FRAGMENT-ION MASS ANALYSIS USINGMATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

The success of the mass spectrometric-based approaches for the identif ication of gel-separated proteins relies upon recovery of peptides, wi thout high levels of ionization-suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to det ermine whether in-gel or on-membrane digestion provided a significant advantage when low to sub-pmol quantities of gel-separated proteins we re analyzed by matrix-assisted laser-desorption/ionization mass spectr ometry (MALDI-MS) with respect to the number and size of released pept ides. Serial dilutions of five standard proteins of M-r 17000 to 97000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in-g el digestion (using a microcolumn clean-up protocol, Courchesne, P.L. and Patterson, S. D., BioTechniques, 1997, in press) or on-membrane di gestion following blotting to the PVDF-based membranes, Immobilon-P an d Immobilon-CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon-P and Immobilon-CD, 0.5 pmol; and in-gel, 125 fmol), and searches of Swiss-Prot or a nonre dundant database (>193000 entries) successfully identified all of the proteins, except beta-casein. Fragment-ion spectra using a curved-fiel d reflector MALDI-MS were obtained from more than one peptide per prot ein at loads down to 250 fmol (except p-casein). Using the uninterpret ed data, a search of the nonredundant database and a six-way translati on of GenBank dbEST (>2208000 entries total) was able to identify myog lobin, carbonic anhydrase II, and phosphorylase b.