Peptide-mass fingerprinting as a tool for the rapid identification and mapping of cellular proteins

摘要: For more than 25 years protein identification has largely depended on automated Edman chemistry (Hewick et al., 1981) or western blotting with an appropriate monoclonal antibody. Several limitations, however, have never been overcome. The procedure is inherently slow (generally one two peptide samples per day) and does not allow direct of many post-translational modifications. In addition, current detection limits are in the low-picomole to upper-femtomole range (Totty 1992). Protein by can be extremely rapid, but requires ready availability extensive library suitable antibody probes. Large-format 2D-electrophoresis systems now make it possible resolve several thousand cellular proteins from whole-cell lysates low- concentration (Patton 1990), presenting significant analytical challenges. recent introduction matrix-assisted laser-desorption (MALD) time-of-flight mass spectrometers (Karas Hillenkamp, 1988) led rapid analysis (at high sensitivity) mixtures. New strategies developed using a combination protease digestion, MALD spectrometry searching peptide-mass databases that promise acceleration (Henzel 1993; Pappin Mann James Yates 1993).