AbstractThe total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) where 500 μg was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in‐gel trypsin digestion, and fractionated by reversed phase‐high performance liquid chromatography (RP‐HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PDC1, ENO1 ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2. The chromatograms obtained from RP‐HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2‐D pattern of S. cerevisiae will be obtained in sequencable amounts.