MALDI-FTICR Imaging of Ascochyta Fungi

Ascochyta fabae isolate AF1 and A. viciae-villosae isolate AV1 (33 (link)) was grown on half-strength PDA in which a microscopic slide was embedded. After 2 weeks of growth, the embedded slide with the fungal colony growing on the thin layer of PDA (about 2 mm in diameter) was removed from the plate and dried for 1 h at 65°C for MALDI imaging analysis. The dried specimen on the slide was sprayed with 20 mg/ml of 2,5-dihydroxybenzoic acid (in a mixture that also contained 0.1% trifluoroacetic acid and 50% methanol) as a matrix, using a TM-sprayer system (HTX Technologies, Carrboro, NC, USA) with the following parameters: 12 passes; track spacing, 1.2 mm; nozzle temperature, 70°C; linear velocity, 110 cm/min; flow rate, 60 μl/min. A selected area of the specimen was analyzed on a 9.4T Fourier transform ion cyclotron resonance (FTICR) MS instrument (Bruker Daltonics, Billerica, MA, USA) in positive-ion mode. Data were acquired using ftmsControl software (Bruker Daltonics). Approximately 100 shots per spot were acquired with a Smartbeam II Nd:YAG laser using repetition rate of 1 kHz. Image acquisition was carried out using flexImaging 4.1 (Bruker Daltonics), and data were normalized to the total ion current. Ascochitine standard compound purified in our previous study (8 (link)) was spotted, and reference MS signals were obtained in a simultaneous run.