A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A pI range from 3 to 10 was selected and second dimension was run using 9 -16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as pIs, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.