Lc ms grade

After tryptic digestion, peptides were separated in a first dimension based on hydrophobicity at high pH by using a reversed phase C18 column (X!Select, CSH, RP-C18, 2.1 x 150 mm, 3.5 μm, Waters) connected to a Waters Alliance e2695 HPLC bio-system and a Waters 996 PDA detector (Waters Corporation, Milford, MA, USA). Solvent A contains 200 mM ammonium formate at pH 10, while solvent C contains 100% water and solvent D 100% acetonitrile (ACN) (LC-MS grade, Biosolve, Valkenswaard, Netherlands). During the chromatographic run, an ACN gradient was performed, while continuously 10% of solvent A was added to become an overall pH of 10 during the entire run. The following gradient was used at a constant flow rate of 200 μL/min: 5% to 15% D over the first 5 min, 15% to 40% D over 80 min, 40% to 90% D over 8 min, 5 min 90% D, and 90% to 5% D over 2 min. In total, 30 fractions were collected starting from 10 to 100 min with an interval of 3 min/fraction. The peptide concentration of the different fractions was determined based on the area under the curve (AUC at 214 nm). Fractions were pooled in a concatenated way (e.g. fractions 1, 11 and 21) to obtain optimal orthogonality, yielding in total 10 fractions for further analysis. Collected fractions were lyophilized and re-suspended in RP mobile phase (97% water, 3% ACN, 0.1% FA).

Secondary metabolites were taken from the culture supernatants extracted using ethyl acetate (Rajan and Kannabiran, 2014 (link)), QuEChERS (Schenck and Hobbs, 2004 (link)), or solid phase extraction (Kamenik et al., 2010 (link)), which was found to be the most suitable and was carried out as follows. An Oasis HLB 3cc 60 mg cartridge (hydrophilic-lipophilic balanced sorbent, Waters, USA) was conditioned with 3 mL methanol (LC-MS grade, Biosolve, Netherlands), equilibrated with 3 mL water (prepared using Milli-Q water purifier, Millipore, USA) and then 3 mL culture supernatant (pH adjusted to 3 with formic acid, 98–100%, Merck, Germany) was loaded. Subsequently, the cartridge was washed with 3 mL water and absorbed substances were eluted with 1.5 mL methanol. The eluent was evaporated to dryness (Concentrator Plus, 2013 model, Eppendorf), reconstituted in 200 μL 50% methanol and centrifuged at 12,000 × g for 5 min.