Failure in regulation of genes involved in growth and division of cells may result in pathological conditions, particularly cancer. Regulation is exerted at various levels of the transcriptional and post-transcriptional processes involving mainly nucleic acid-binding proteins. Here, we systematically explored the proteome of ten different cell lines in search for proteins potentially serving as molecular markers and/or targets for monitoring prognostic outcome and clinical therapies. High-throughput analysis, two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry, identified 72 nucleotide-binding proteins and their interacting partners, which were differentially expressed in the cell lines investigated. Out of the 72 identified proteins, 33 of them were specifically expressed in a single cell line (for e.g., replication protein A 32 kDa, transcription intermediary factor 1, heterogeneous ribonucleoproteins). Moreover, tumor-related proteins including breast carcinoma amplified sequence 2, zinc finger proteins, chromobox protein homologs were identified in individual cell lines. The present findings demonstrate that rich protein information can be obtained by means of proteomic analysis for better understanding of oncogenesis and pathogenesis in a global way, which in turn represents the basis for the rational designs of diagnostic and therapeutic methods.