Identification of harmless and pathogenic algae of the genus Prototheca by MALDI-MS

The only plants infectious for mammals, green algae from the genus Prototheca, are often overseen or mistaken for yeast in clinical diagnosis. To improve this diagnostical gap, a method was developed for fast and reliable identification of Prototheca. A collection of all currently recognized Prototheca species, most represented by several strains, were submitted to a simple extraction by 70% formic acid and ACN; the extracts were analyzed by means of MALDI-MS. Most of the peaks were found in the range from 4 to 20 kDa and showed a high reproducibility, not in absolute intensities, but in their peak pattern. The selection of measured peaks is mostly due to the technique of ionization in MALDI-MS, because proteins in the range up to 200 kDa were detected using gel electrophoresis. Some of the proteins were identified by peptide mass fingerprinting and MS(2) analysis and turned out to be ribosomal proteins or other highly abundant proteins such as ubiquitin. For the preparation of a heatmap, the intensities of the peaks were plotted and a cluster analysis was performed. From the peak-lists, a principal component analysis was conducted and a dendrogram was built. This dendrogram, based on MALDI spectra, was in fairly good agreement with a dendrogram based on sequence information from 18S DNA. As a result, pathogenic and nonpathogenic species from the genus Prototheca can be identified, with possible consequences for clinical diagnostics by MALDI-typing.