Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the detection of hemoglobins as the protein biomarkers for fecal occult blood

In this study, we discover that hemoglobins (Hb), highly water-soluble globular proteins that are the most predominant proteins detected by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry in blood, can be used as protein biomarkers for fecal occult blood (FOB). Hemoglobins were extracted from the feces with pure water and separated from the solids in feces through centrifugation. Singly charged molecular ions of Hb-related alpha chains (theoretical MW: 15 126) and beta chains (theoretical MW: 15 867) were detected by MALDI-TOF operated in linear mode using 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC) as the matrix (with a volumetric ratio of 1:1). The detection limit of FOB using this method is estimated to be lower than 0.1 microg blood per mg of feces, which is approximately 10 to 100 times lower than that of the conventional chemical approaches. The foods and dietary supplements that commonly interfere with the conventional chemical assays of FOB - such as animal blood food products and tablets containing iron and vitamin C - do not interfere with the detection of Hb biomarkers during MALDI-TOF analysis.