Technical Abstract: A method has been developed to identify the alpha-subunit of shiga toxin 2 (alpha-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. E. coli O157:H7 (EDL933) was induced to express Stx2 by culturing in the presence of ciprofloxacin on solid agar. Bacterial cell lysates were incubated in the presence of furin (a human enzyme) that cleaves alpha-Stx2 into A1 (28 kDa) and A2 (5 kDa) protein fragments. After digestion with furin, the A1 and A2 fragments remain bound by a disulfide bond. A disulfide reduction step was performed to un-link A1 from A2. This sequential two-step process for the separation of the two protein fragments follows the model proposed for eukaryotic intracellular processing of alpha-Stx2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. The intensity of this peak increased with furin digestion time. In the absence of furin digestion and/or disulfide reduction, no peak was observed at m/z 5286, suggesting that both steps, performed sequentially, are necessary for release of the A2 fragment and its ionization by MALDI. The peak at m/z 5286 was analyzed by MALDI-TOF-TOF-MS/MS and top-down proteomic analysis which confirmed the ion at m/z 5286 is the A2 fragment of alpha-Stx2. No peak was observed that corresponded to the catalytically active A1 fragment although its presence was detected and confirmed by 1-D gel electrophoresis and bottom-up proteomic analysis.