Steel maldi target plate

Sample extractions and analysis were performed as described by Yssouf et al. and Flaudrops et al.^{27 (link),60 (link)}. Two biological replicates cut from each truffle gleba were used for protein extraction. Each piece (8–10 mg) was mixed with 400 μL of 70% formic acid (Sigma, Lyon, France), 400 μL of 100% acetonitrile (VWR Prolabo) and ground with acid washed glass beads (Sigma Aldrich, Lyon France) in a polypropylene tube using the FAST Prep®−24 Instrument (MP Biomedicals, Illkirch-Graffenstaden, France). The homogenates were centrifuged for two minutes at 13,000 × g and, 1.5 μL of each supernatant was spotted onto a steel MALDI target plate (Bruker Daltonics, Wissembourg, France) in triplicate and then dried at room temperature for 15 minutes. 1.5 μL of a CHCA matrix suspension (saturated α-Cyano-4-hydroxy-cinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid and HPLC water) (Sigma) was then overlaid onto each spot to allow crystallisation. To control loading on the plate, matrix quality and the MALDI-TOF apparatus performance, 1.5 μL of the matrix solution was loaded in triplicate onto the plate with (positive control) or without (negative control) Bruker Protein Calibration Standard I. Finally, the plate was dried at room temperature for 15 minutes and was then immediately introduced into the Autoflex-Speed linear MALDI-TOF MS for analysis (Bruker Daltonics, Germany).

Sample preparation for MALDI-TOF mass spectrometry was performed as previously described with modifications [25 (link)]. The isolate was grown on solid medium until good sporulation was obtained. A mixture of spores and mycelium was harvested from the culture with the aid of a sterile cotton swab. The fungal material was transferred to a 1.5-mL microtube containing 300 μL of sterile distilled water, vortexed, and 900 μL 70% ethanol were added. After centrifugation for 10 min at 13,000 rpm, the pellet was resuspended in a mixture containing 50 μL of 70% formic acid and 50 μL of acetonitrile. After centrifugation for another 5 min at 13,000 rpm, 1.5 μL of the supernatant was spotted onto a steel MALDI target plate (Bruker Daltonik GmbH, Germany) and were allowed to dry at room temperature. Each spot was overlaid with 1.2 μL of alpha-cyano-hydroxycinnamic acid in 2.5% of trifluoroacetic acid and 50% of acetonitrile in water (Bruker Daltonik), and air dried at room temperature. Spectra were acquired using a Microflex LT mass spectrometer (Bruker Daltonik). Spectra were recorded in positive linear mode within a mass range from 2.000 to 20.000 Da and were analyzed using the software Flex Analysis (Bruker Daltonik).