Early detection and rational therapy for gastric cancer are crucial. In this study we undertook comparative proteomics for identification of gastric carcinoma biomarkers using pooled laser capture microdissected GA cells and matched nonmalignant gastric mucosa epithelial cells. The method involved separation of total proteins by 1D SDS-PAGE, trypsin digestion, and postdigest (18)O/(16)O labeling followed by nano-HPLC-MS/MS for peptide identification and relative quantification. A total of 78 differentially expressed proteins were identified, among these proteins, 42 proteins were up-regulated in GA and 36 proteins were down-regulated. Some differentially expressed proteins were further validated by western blot analysis.