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Identification of transformation sensitive proteins recorded in human two‐dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing
Author(s) -
Rasmussen Hanne H.,
Mørtz Ejvind,
Mann Matthias,
Roepstorff Peter,
Celis Julio E.
Publication year - 1994
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150150159
Subject(s) - bottom up proteomics , mass spectrometry , peptide , peptide mass fingerprinting , protein mass spectrometry , chemistry , proteome , database , molecular mass , proteomics , computational biology , tandem mass spectrometry , chromatography , biology , biochemistry , gene , computer science , enzyme
A comprehensive human keratinocyte two‐dimensional (2‐D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte 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 date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up‐ or down‐regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis 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 allowing fast screening of proteins recorded in 2‐D gel databases. The mass spectrometric approach when combined with microse‐quencing strengthened identification, and added the possibility of full characterization of post‐translational modifications and sequence variations.