Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: An analytical challenge for studying complex protein mixtures

The large-gel two-dimensional electrophoresis (2-DE) technique, developed b y Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MAL DI-TOF-MS) provides the sample throughput necessary to identify thousands o f different protein species in an adequate time period. Spot excision, in s itu proteolysis, and extraction of the cleavage products from the gel matri x, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumen tation determine how many protein digests can be analyzed per day per instr ument. The present paper focuses on this analytical interface and reports o n an integrated protocol and technology developed in our laboratory. Automa ted identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of th e information contained in the experimental data, and scores the search res ults accordingly. This challenge is heading a second part of the paper.