AbstractThe success of the mass spectrometric‐based approaches for the identification of gel‐separated proteins relies upon recovery of peptides, without high levels of ionization‐suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in‐gel or on‐membrane digestion provided a significant advantage when low to sub‐pmol quantities of gel‐separated proteins were analyzed by matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI‐MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins ofMr17 000 to 97 000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in‐gel digestion (using a microcolumn clean‐up protocol, Courchesne, P.L. and Patterson, S. D.,BioTechniques, 1997, in press) or on‐membrane digestion following blotting to the PVDF‐based membranes, Immobilon‐P and 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 non‐redundant database (>193 000 entries) successfully identified all of the proteins, except β‐casein. Fragment‐ion spectra using a curved‐filed reflector MALDI‐MS were obtained from more than one peptide per protein at loads down to 250 fmol (except β‐casein). Using the uninterpreted data, a search of the nonredundant database and a six‐way translation of GenBank dbEST (>2 208 000 entries total) was able to identi