Identification of gel-separated tumor marker proteins by mass spectrometry Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clini- cal material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10±13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrom- etry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such pro- teins were identified, ten constituting novel identifications and ten sequence verifica- tions of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cyto- keratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over- expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abun- dancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes. Keywords: Two-dimensional gel electrophoresis / Mass spectrometry / Tumor markers EL 3773 Ann-Charlotte Bergman 1 Timothy Benjamin 2 Ayodele Alaiya 3 Mark Waltham 4 Kazuyazu Sakaguchi 5 Bo FranzØn 3 Stig Linder 3 Tomas Bergman 1 Gert Auer 3 Ettore Appella 5 Peter J. Wirth 2 Hans Jörnvall 1 1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 2 Laboratory of Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD, USA 3 Department of Oncology and Pathology, Karolinska Institutet and Hospital, Stockholm, Sweden 4 Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, MD, USA 5 Cell Biology, National Cancer Institute, NIH, Bethesda, MD, USA 1 Introduction Tumor progression can be evaluated via examination of the expression of proteins for which the quantity varies according to tumor type and development (marker pro- teins). In this respect two-dimensional (2-D) gel electro- phoresis is a powerful tool since the expression of a large number of proteins can be evaluated in single experi- ments. Identification at the subpicomole level of the great number of proteins that can be resolved (up to thousands) is the analytical challenge in this methodology. Over the years, identification has been carried out by comigration with known proteins, immunoblotting, and gel matching [1]. Today, mass spectrometry (MS) is the technique of choice [2]. We have employed 2-D analysis of human solid tumor cell extracts to examine protein expression patterns between fibroadenomas and invasive ductal carcinomas of the breast [3], adenocarcinomas and small-cell carcinomas of the lung, and benign, borderline and malignant tumors of the ovary [4]. In this manner, we have identified twenty proteins, ten of which were upregulated and previously not recognized as potential markers of breast, lung and ovarian tumors. In addition, we verified the identity of ten proteins that were assigned as markers in earlier studies but identified via gel matching and immunological tech- niques only. Of these twenty proteins, three were found to be truncated. 2 Materials and methods 2.1 Sample preparation All tissue samples were obtained shortly after resection (within 40 min), histopathologically characterized, and solubilized as described [5]. Briefly, tumor cells were col- lected from the cut surface of a nonnecrotic tumor tissue by scraping with a scalpel. Cells were collected in 2±5 mL ice-cold RPMI-1640 medium containing 5% fetal calf serum and 0.2 mM phenylmethylsulfonyl fluoride / Correspondence: Dr. Hans Jörnvall, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden E-mail: hans.jornvall@mbb.ki.se Fax: +46-8-337-462 Abbreviations: CD, cathepsin D; hnRNP, heterogeneous nucle- ar ribonucleoprotein Electrophoresis 2000, 21, 679±686 679  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/0303-0679 $17.50+.50/0 Proteomics and 2-DE