Optibeam laser

Selected D. solani IPO2222 Tn5 transposon mutants were tested using a whole-cell MALDI-TOF MS spectral analysis as previously described (Vandroemme et al., 2013 (link)). Briefly, IPO2222 wild type and selected Tn5 mutants were grown on M9 medium supplemented with glucose to a final concentration of 0.4% at 28°C for 24 h prior to analysis. As a matrix ferulic acid (FA) (10 mg/ml) in 17% formic acid, 33% acetonitrile, 50% water was used. In each case, a 0,6 μl of matrix solution was used to overlay the sample spot, and the plate was then left to crystallize at room temperature. Directly after spot preparation (ca. 15 min), protein mass fingerprints were obtained using an 5800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Framingham, MA, United States), with detection in the linear middle mass (4000–20 000 Da), positive ion mode for a total of 1000 laser shots by an 1 kHz OptiBeam laser (YAG, 349 nm). Laser intensity remained fixed for all screened samples. Registered spectra were analyzed with Data Explorer software (AB Sciex). All MALDI-TOF MS spectra used in this study were averages of at least four replicated measurements per analyzed strain.

Morpholine (0.5 mL) was added to a stirred solution of Fmoc–Leu–Ala–Gly–Gly–DOX (50 mg, 47 µmol) in DMF (0.5 mL), and the stirring was carried on for a further 2 h. Afterward, the mixture was neutralized with trifluoroacetic acid (0.6 mL). The raw product was isolated on a semi-preparative HPLC System (Shimadzu, LC 20AD, Shimadzu, Canby, OR, USA) using a Gemini NX Column (5.0 μm, 10 × 150 mm) to give 24 mg in a 61% yield. The flow rate was set at 4.0 mL/min with the mobile phase consisting of solvent A (0.1% formic acid) and solvent B (100% ACN). The gradient was as follows: 0–15 min, 10–90% of solvent B; 15–20 min, 90% of solvent B. The chemical structure of the product was confirmed using MALDI TOF/TOF 5800 mass spectrometry (AB Sciex, Darmstadt, Germany). As a matrix, 2,5-dihydroxybenzoic acid was used. The measurement was done in reflector positive ion mode. Samples were prepared using the dried droplet preparation method by mixing 0.8 mL of an analyte solution with 0.8 mL of the matrix solution (directly on a plate). MS spectra were acquired from 789 to 961 m/z for a total of 1000 laser shots by a 1 kHz OptiBeam laser (AB Sciex, Darmstadt, Germany).