PEPTIDE MASS FINGERPRINT SEQUENCE COVERAGE FROM DIFFERENTLY STAINED PROTEINS ON 2-DIMENSIONAL ELECTROPHORESIS PATTERNS BY MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROMETRY (MALDI-MS)/

Identification of proteins separated by two-dimensional electrophoresi s (2-DE) is a necessary task to overcome the purely descriptive charac ter of 2-DE and a prerequisite to the construction of 2-DE databases i n proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Un fortunately, proteins are modified by the silver staining procedure, l eading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still cl early below the sequence coverage starting with Coomassie Brilliant Bl ue (CBB) R-250-stained spots. Other factors additionally seem to modif y proteins during silver staining. By decreasing the protein amount, t he advantage of very sensitive detection on the gel is lost during ide ntification, because the resulting low sequence coverage is not suffic ient for secure identification. Low-quantity proteins can be identifie d better starting with CBB G-250 or Zn-imidazol-stained proteins. In c ontrast, for high-quantity CBB R-250-stained spots, a sequence coverag e of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of t ryptic digest with Asp-N- and Glu-C digest.