181 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress ground dried biomass was extracted with 2 ml of 10 % sulphuric acid in methanol with 200 ppm sodium benzoate as internal standard (IS) and 2 ml chloroform. After retightening the caps, samples were heated at 100ºC for 20 hours. After cooling down for 3 hours, samples were vortexed for 1 min with addition of 1 ml mili-Q water and settled for 1 h. From the lower layer (chloroform layer), 1 ml was transferred to a 1.5 ml GC vial. GC-MS analysis were performed on an Agilent 7890A GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a HP-5MS column (30 m x 0.25 mm x 0.25 μm) using an 7693A automated sampler. The GC was connected to a JEOL JMS-T100 GCv mass spectrometer (JEOL Ltd., Akishima, Tokyo, Japan). For the analysis, 1 μl of each sample was injected (injector temperature 250°C) onto the GC column using a split ratio of 25:1 and the following temperature program: 70 °C for 3 min, ramp 10 °C/min to 320 °C, hold 5 min. A column flow with helium (5.0) of 1.0 ml/min was used. Electron Ionization Spectra were acquired at 3Hz (spectra per second) mass range 35-650, column bleeding (silanol 207) in the range 14.50-14.90 minutes was employed to do internal mass drift compensation for the Perfluorokerosene (PFK) calibrated MS resulting in a mass accuracy window smaller than 3 mmu for mass peaks with sufficient signal to noise. GC-MS peaks were manually integrated using MassCenter (JEOL Ltd., Akishima, Tokyo, Japan). Acquisition was performed using Mass Center System version 2.5.1a © 2001-2010 JEOL Ltd. Spectra were compared to the NIST/EPA/NIH Mass Spectral Library version NIST MS Search 2.3. build May 2017 with the NIST Mass Spectral Search Program. Identification and analysis of sialic acids (nonulosonic acids) by Mass Spectrometry and sialidase activity The identification and analysis of nonulosonic acids from freeze dried biomass was performed using acid hydrolysis, DMB labelling, followed by reversed phase chromatography coupled to high-resolution mass spectrometry PRM scanning using small mass channels as described before(Kleikamp et al., 2020). In short, lyophilized biomass was hydrolyzed by 2 M acetic acid for 2 hours at 80°C and dried with a Speed Vac concentrator. The released NulOs were labelled using DMB (1,2-diamino-4,5-methylenedioxybenzene dihydrochloride) for 2.5 hours at 55°C and analyzed by reverse phase chromatography Orbitrap mass spectrometry (QE 6
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