Gastric cancer mortality is second only to lung cancer, and its prognosis is dismal. Using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry, we previously identified a single best mass, which could separate gastric cancer from patients without cancer, with a sensitivity of 89.9% and a specificity of 90%. Using protein liquid chromatography systems with various chromatography media and MS/MS analysis, we were able to identify thrombin light chain A, a proteolytic fragment of prothrombin, as the single best mass for early detection of gastric cancer patients. These findings indicate that disturbances in the coagulation-system are early events in gastric cancer biology and that a decrease or loss of thrombin light chain A, which we termed negative serum protein profiling, may contribute to the diagnosis of cancer patients.