Peptide-Mass Fingerprinting as a Tool for the Rapid Identification and Mapping of Cellular Proteins
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 Edman procedure is inherently slow (generally one or two peptide or protein samples per day) and does not allow direct identification of many post-translational modifications. In addition, current detection limits are in the low-picomole to upper-femtomole range (Totty et al., 1992). Protein identification by western blotting can be extremely rapid, but requires the ready availability of an extensive library of suitable antibody probes. Large-format 2D-electrophoresis systems now make it possible to resolve several thousand cellular proteins from whole-cell lysates in the low- to upper-femtomole concentration range (Patton et al., 1990), presenting significant analytical challenges. The recent introduction of matrix-assisted laser-desorption (MALD) time-of-flight mass spectrometers (Karas and Hillenkamp, 1988) has led to the rapid analysis (at high sensitivity) of peptide mixtures. New strategies have been developed using a combination of protease digestion, MALD mass spectrometry and searching of peptide-mass databases that promise rapid acceleration in the identification of proteins (Henzel et al., 1993; Pappin et al., 1993; Mann et al., 1993; James et al., 1993; Yates et al., 1993).
KeywordsDigestion Procedure Octyl Glucoside Imperial Cancer Research Fund Potential Cleavage Site Change Of200
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