MALDI-TOF Analysis of Fungal Spores

First, we tested the detection limit of MALDI-TOF using Scutellospora ovalis with 1, 2, 4, 5 and 10 spores for the analyses to determine the optimal signal-to-noise ratio. The number of spores per sample for the other isolates was calculated using the estimated spore volume of each isolate (Supplementary Table S1). The final protocol was designed considering the protocols developed for pathogenic yeasts^{64 (link)} and cyanobacteria^{32 (link)}. Spore proteins were extracted using formic acid (FA) as follows: fungal spore samples were transferred to a sterile 1.5-mL tube with 20 µL of ultrapure water. After centrifugation for 3 minutes at 13,000 × g, the supernatant was discarded and the spore pellet was incubated for 5 minutes in 10 µL of (70:30 [vol/vol]) formic acid/acetonitrile (Sigma-Aldrich, Lyon, France). Spores were crushed using a pestle for 30 seconds and incubated for 5 minutes at room temperature before use. Each sample was checked under a stereomicroscope to ensure that spores were completely crushed. Successive aliquots of 1.5 μL of the supernatant were transferred to a polished steel MSP 96 target (Bruker) until the sample was consumed and was allowed to dry at room temperature before being overlaid with 1 μL of a saturated a-cyano-4-hydroxycinnamic acid (HCCA) matrix solution in 50% acetonitrile-2.5% trifluoroacetic acid (Bruker).