IDENTIFICATION OF TRANSFORMATION SENSITIVE PROTEINS RECORDED IN HUMAN2-DIMENSIONAL GEL PROTEIN DATABASES BY MASS-SPECTROMETRIC PEPTIDE-MAPPING ALONE AND IN COMBINATION WITH MICROSEQUENCING

A comprehensive human keratinocyte two-dimensional (2-D) gel protein d atabase has been established to study the expression levels and proper ties of the thousands of proteins that orchestrate various keratinocyt e functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2 -D gel patterns as well as to reveal hitherto unknown proteins. To dat e, protein identification has been performed by one or a combination o f the following methods: (i) comigration with known proteins, (ii) Wes tern blotting using specific antibodies, (iii) microsequencing and (iv ) vaccinia virus expression of full length cDNAs. Recently, the system atic identification of proteins has gained a new dimension with the ad vent of computer programs for searching peptide molecular mass databas es with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up- or down-regula ted in simian virus SV40 transformed human keratinocytes (K14). Peptid e mass maps of several proteins, including keratins 7, 8, 18 and 19 we re obtained either by plasma desorption mass spectrometry (PDMS) analy sis of high performance liquid chromatography (HPLC) purified peptides or by matrix-assisted laser desorption/ionization mass spectrometry ( MALDI-MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification all owing fast screening of proteins recorded in 2-D gel databases. The ma ss spectrometric approach when combined with microsequencing strengthe ned identification, and added the possibility of full characterization of post-translational modifications and sequence variations.