Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Abstract

To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n=2) and Cryptosporidium hominis (n=1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.