Investigation of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) for Detection of Clostridium difficile Toxin A and B From Stools

Objective: Detection of Clostridium difficile toxins A and B from stools using matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI).
Methods: C. difficile toxin positive stool samples 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 SA (sinnapinic acid), SDHB (a mixture of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2-hydroxy-5-methoxybenzoic acid), or CHCA (α-Cyano-4-hydroxycinnamic acid) as matrices.
Results: MALDI analysis showed no difference between samples diluted in deionized water and those diluted in PBS. Protein precipitation with acetonitrile produced higher quality mass spectra than protein precipitation with ammonium sulfate. Sample co-crystalization with SA provided higher quality spectra than SDHB or CHCA. No peaks were seen in the 63 kDa range in any of the samples. Autocleavage of a commercially purchased toxin A also failed to show the expected peak at 63 kDa.
Discussion: Further processing of stool samples is necessary for MALDI to successfully detect the 63 kDa active domain. No individual ion signals were detected between 60-65 kDa. This suggests that a clear mass window is available for the detection of the 63 kDa active domain, had MALDI analysis been successful.