Protocol development for the use of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) for detection of clostridium difficile toxin A and B from stool samples

Background: Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus. C. difficile is important in the medical community because it is the most common cause of antibiotic-associated diarrhea and infections can lead to serious complications such as toxic megacolon and pseudomembranous colitis. Infection is mediated by toxins A and B, both of which are proteins that undergo processing within the target cell to yield a 63 kDa active domain. Objective: This study investigates the use of MALDI-TOF-MS to detect the active domains of C. difficile toxins A and B from stool samples to explore the possibility of using MALDI as a diagnositc tool for C. difficile infections. Methods: Known toxin and antigen positive and negative stool samples sent from Royal Inland Hospital were diluted 10-fold in deionized water or phosphate buffered saline (PBS) and vortexed to create a relatively homogeneous suspension. Samples were then centrifuged and the pellet removed. Proteins in the supernatant were precipitated with acetonitrile or ammonium sulfate and the solution was centrifuged again. The pellet was resuspended in deionized water or TA30 and spotted on a MALDI plate with a sinnapinic acid (SA), SDHB (a mixture of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2-hydroxy-5-methoxybenzoic acid), or CHCA (α-Cyano-4-hydroxycinnamic acid) matrix co-crystalized. Results and Discussion: MALDI analysis showed no difference between samples diluted in deionized water and those diluted in PBS. Protein precipitation with acetonitrile produced higher quality spectra than protein precipitation with ammonium sulfate. Sample co-crystalization with a SA matrix provided higher quality spectra than sample co-crystalization with a SDHB or CHCA matrix. MALDI analysis showed no peaks in the 63 kDa range in any of the samples. Because stool is a complex combination of materials, MALDI mass spectra were expected to be complicated and show vast differences between samples. Surprisingly, all ten MALDI spectra acquired were relatively similar. Similar individual ion signals were seen between 20 and 60 kDa and above 70 kDa. No individual ion signals were seen in the 63 kDa range in any of the samples, regardless of their being toxin and antigen positive or negative. This suggests that there is an open mass window for unambiguous detection of the 63 kDa active domain. Conclusion: We were unable to use MALDI to detect the 63 kDa active domains of C. difficile toxins A and B from crude stool protein extracts. Further studies would be required to ascertain the possibility of using this technological tool to detect C. difficile toxins as an alternative method of diagnosis to the tests currently available. Although inconclusive, this study is a starting point for the investigation of MALDI as a diagnostic tool in a clinical setting.