178 Chapter 6 0.1% formic acid, and solvent B consisted of 80% ACN in H2O and 0.1% formic acid. The mass spectrometer was operated in data-dependent acquisition mode, where the top 10 most intense precursor ions were selected for fragmentation using higher-energy collisional dissociation (HCD) using a normalized collision energy (NCE) of 28. MS1 spectra were acquired from 385-1250 m/z at 70K resolution, and an AGC target of 3e6, and a max IT of 75 ms. Fragmentation spectra were measured at 17K resolution, with an isolation window of 2 m/z, an AGC target of 2e5, and a max IT of 75 ms. Unassigned, singly and >5x charged ions were excluded from fragmentation. Mass spectrometric raw data were database searched using a protein reference database containing ORFs identified from the contigs of the metagenomics sequencing data obtained from the same community, using PEAKS Studio X (Bioinformatics Solutions Inc., Waterloo, Canada). Database searching was performed allowing 20 ppm parent ion and 0.02 m/z fragment ion mass error, 3 missed cleavages, carbamidomethylation as fixed and methionine oxidation and N/Q deamidation as variable modifications. Peptide spectrum matches were filtered for 1% false discovery rates (FDR) and identifications with ≥2 unique peptides were considered as significant. Quantitative analysis of the changes between conditions, was performed using the PEAKSQ module (Bioinformatics Solutions Inc., Canada). Normalization was based on the total ion current (TIC), and only proteins with at least 2 unique peptides and identified in at least 2 out of 3 biological replicates were considered. Peptide spectrum matches were filtered with a 1% false discovery rate (FDR). ANOVA was used to determine the statistical significance of the changes between the conditions. Proteomics raw data, reference sequence database and database search files have been deposited in the ProteomeXchange consortium database with the dataset identifier PXD048239. Non-targeted metabolomics Biomass from 5 mL samples (n=4 per salinity) was collected in Nylon membrane filters (0.45 µm, 47 mm (WHA7404004) (Whatman, Maidstone, UK) using a vacuum system and were washed using about 5-10 ml of ice-cooled nitrate-free freshwater bioreactor medium. From here onwards, samples were processed as described before (Lawson et al., 2021), with some modifications. Washed biomass was vacuum-filtered using the same filter with acetonitrile:methanol:deionized water (40:40:20) (extraction solvent). Immediately after, filters were placed upside
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