Protein Extraction and Digestion Protocol

On day 1,548, 15 ml of biomass was collected, pelleted by centrifugation at 7,942g and 4 °C for 20 min and stored at −80 °C. Biomass pellets were resuspended in 100 µl milliQ water and heated at 95 °C for 5 min with agitation before sonication for 10 min in a sonication bath (Branson 5800 Ultrasonic Cleaner). Samples were centrifuged at 14,000g for 20 min at 4 °C, and supernatant was transferred to a new Eppendorf tube as ‘soluble’ extracts and RapiGest SF (Waters) was added to a final concentration of 0.1% (v/v). Pellets were washed with 100 µl milliQ water before addition of 50 µl 2% (v/v) RapiGest SF solution and heated at 95 °C for 5 min. Samples were reduced with bond-breaker TCEP solution (Thermo Fisher Scientific) for 20 min before alkylation with 50 mM chloroacetamide for 20 min at room temperature in the dark. Enzymatic digestion was performed by incubating protein extracts overnight at 37 °C with 0.4 µg LysC (Wako Chemicals Europe) and 0.4 µg trypsin (Promega). RapiGest SF was removed by incubating tryptic digests in 0.5% trifluoroacetic acid at 37 °C for 40 min and subsequent centrifugation at 14,000g for 20 min at 4 °C. Samples were analysed both before and after salt-mediated organic solvent precipitation^{81 (link)}.