2 5 dihydroxybenzoic acid matrix

Compounds were tentatively identified by accurate mass match of obtained high resolution FT-ICR m/z values to the Metlin database [41 ]. Identifications were confirmed using MALDI-TOF MS/MS on matrix coated barley tissue sections or on methanolic extracts. For extracts, 2 mg to 40 mg of freeze-dried, milled tissue were extracted with 1 ml water by ribolysing (3 x 45 s, 15 min pause, 6.5 m/s) using 500 mg ribobeads (0.5 mm diameter, Roth) for tissue disruption. Samples were centrifuged 15 min at 14,000 rpm. 1 μl to 2 μl of the supernatant were spotted on a ground steel target (Bruker Daltonics) with 1 μl 2,5-dihydroxybenzoic acid matrix (30 mg/ml in 50% methanol, 0.2% TFA). Dextrin 20 (Serva) with a concentration of 1 mg/ml was used as a reference sample for oligosaccharide analysis. The MALDI-TOF was calibrated on peptide standards. MS/MS was performed on selected precursor ions with individual laser settings using collision induced dissociation (CID) with argon as collision gas at a pressure of 2 x 10⁶ mbar and a collision energy of 8 keV. The software functions Mass frontier (Thermo scientific) and IsotopePattern (Bruker Daltonics) served for structure and formula elucidation of metabolites.