Identification of gel-separated tumor marker proteins by mass spectrometry

Two-dimensional gel electrophoresis with subsequent analysis by mass spectr ometry was applied to study differences in protein expression between benig n and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were su bjected to isoelectric focusing with immobilized pH gradients followed by s econd-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacr ylamide gels. Polypeptides were identified using matrix-assisted laser deso rption/ionization and electrospray ionization mass spectrometry after in-ge l protein digestion. Some of the upregulated polypeptides in malignant cell s are of potential importance as markers of tumor proliferation. Twenty suc h proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins ide ntified span a wide range of functions, but several cases of protein trunca tion were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these overexpressed proteins support the presence of proteolytic processing steps in tumor material. The protei n processing and the difference between protein and mRNA abundancies in tum ors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.