Analysis of Two-Dimensional Gel Proteins by Mass Spectrometry and Microsequencing

A method for in situ digestion that allows high recovery of peptides at the low picomole level from two-dimensional gel electroblotted proteins is described. Peptides obtained from in situ digests were analyzed by matrix-assisted laser desorption/ ionization and electrospray ionization mass spectrometry. Mass analysis of peptides from digestion of single 2-D gel spots provided information sufficient to identify proteins from a protein sequence database using a fragment ion searching algorithm. Separation of these peptides by capillary HPLC allowed high recovery for peptide sequence analysis. The use of a microreaction cartridge in a gas-phase sequencer with a 37-min fast cycle enabled peptide sequencing by Edman degradation at the subpicomole level.