An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.