185 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress peak areas out of the 34,330 features detected and was the highest intensity feature that changed with increased salinity (Fig. 2B). In silico structure prediction based on the tandem mass spectrometry (MS2) fragmentation of m/z 189.125 yielded as likely identity the known osmolyte N(ε)-acetyl-β-L-lysine. To validate this prediction, we grew the methanogen M. mazei under the same salinity (23.8g/l) that was previously reported to produce N(ε)-acetyl-β-L-lysine(Pflüger et al., 2003). The retention time (RT: 3.6 min) and MS2 fragmentation spectrum of N(ε)-acetyl-β-Llysine produced by M. mazei matched those of the “Ca. Methanoperedens” candidate osmolyte (Fig. 2C and Supplementary Fig. 8). To rule out the possibility that the accumulating metabolite was not N(ε)-acetyl-β-L-lysine but its common isomer (N(ε)-acetyl-L-lysine), we performed MS2 experiments of the isomer as “negative control” and confirmed that the MS2 spectra did not match (Supplementary Fig. 8). To conclude, we confirmed that “Ca. Methanoperedens” produced the compatible solute N(ε)-acetyl-β-L-lysine. 6
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