Msp 96 target

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).

The assay was performed as previously described with slight modifications (Hrabak et al. 2012 (link)). A bacterial cell suspension was prepared in the suspension buffer (20 mmol/L Tris–HCl; 20 mmol/L NaCl, pH 7.0) with an addition of 50 mmol/L NH₄HCO₃. The reaction was performed in Tris–HCl buffer supplemented with 50 mmol/L NH₄HCO₃ (pH adjusted by HCl to 7.0). The incubation time was as previously described (2 h) (Hrabak et al. 2012 (link)). For the measurement, 1 μl of the sample was applied on a target (Bruker Daltonics GmbH, Bremen, Germany; MSP 96 Target, Catalog Nr. 224989) and allowed to dry. The spot was then covered by the matrix solution [10 mg/mL of 2,5-dihydroxybenzoic acid and 0.1 μmol/L reserpine in 50 % ethanol] and measured after drying in a range between 350 and 700 mass to charge ratio (m/z). All chemicals except meropenem (Astra Zeneca UK) were obtained from Sigma-Aldrich Co., Prague, Czech Republic. Similar measurement was performed using ceftazidime (GlaxoSmithKline, Prague, Czech Republic) as an indicator β-lactam.

For direct spotting on the MALDI target (MSP 96 Target, Catalog No. 224989, Bruker Daltonics GmbH, Bremen, Germany), bacterial culture was transferred by a toothpick forming a thin film on the spot. After drying, the spot was covered by 1 microliter of a matrix solution [10 mg/ml of alfa-cyano-4-hydrocinnamic acid (CHCA) (Bruker Daltonik, Bremen, Germany) in 50% acetonitrile (Sigma-Aldrich, Prague Czech Republic) and 2.5% trifluoroacetic acid (Sigma-Aldrich, Prague Czech Republic)].
On-plate extraction (semi-extraction) was performed by spotting of bacteria as described above. After drying, the spot was covered by 1 microliter of 70% formic acid and allow to dry. Then, 1 microliter of matrix was applied.
In the wet deposition, 1 microliter of 70% formic acid was pipetted on the spot. Instantly, bacteria were collected manually with a platinum inoculation loop and resuspended, in the formic acid droplet, on the spot. After the spot was dried, 1 microliter of the matrix was applied.

A matrix assisted laser desorption ionization-time of flight mass analysis (MALDI-TOF-MS) was performed for 26 pure cultures isolated from tomato and pepper plants. In brief, a single colony from an overnight culture was deposited on a polished steel MSP 96 target (Bruker Daltonics, Billerica, MA, USA) and overlaid with 1 μL of a saturated-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonics). Unidentified strains were resubmitted using the extended protocol. Prior to the matrix, 1 μL of 70% formic acid was added to the bacterial spot and left dry out. Mass spectra were acquired using the microflex LT mass spectrometer (Bruker Daltonics) and analyzed with the research-use-only (RUO) software workflow and reference library MBT v. 4.1.100. All of the isolates were successfully identified with a score >1.7.