Speciation analysis of fungi by liquid atmospheric pressure MALDI mass spectrometry

Fungal pathogens pose a growing threat to global health, necessitating rapid and accurate identification methods. Here, liquid atmospheric pressure matrix-assisted laser desorption/ionisation (LAP-MALDI) mass spectrometry (MS) is applied to fast lipid and protein profiling of Candida albicans and Saccharomyces cerevisiae from cultured colonies. Species-specific lipid profiles were observed in the m/z 600-1100 range, dominated by phospholipids as confirmed by tandem mass spectrometry (MS/MS). Following simple solid phase extraction clean-up, LAP-MALDI mass spectra revealed multiply charged protein ions suitable for MS/MS analysis. For C. albicans, the fully mature, species-specific WHS11 protein (~ 7 kDa; P43074) was detected intact and confidently identified by top-down MS/MS proteoform sequencing, including the cleavage of the N-terminal methionine initiator and the associated N-terminal acetylation. For S. cerevisiae, a set of proteoforms were sequenced by MS/MS analysis, which led to the identification of two species-specific proteins within the 'UniProtKB reference proteomes + Swiss-Prot' target database. One of these was also detected intact, and sequenced and identified as the fully mature HSP12 protein (~ 11.5 kDa; P22943). This work demonstrates the potential of LAP-MALDI MS and MS/MS biotyping as a powerful, label-free platform for rapid fungal classification and proteoform characterisation, offering substantial improvements over conventional MALDI biotyping.