Tetraethylammonium bromide teab

Protein extraction and trypsin digestion were performed as described by Lu et al. (2022) with minor modifications. Briefly, frozen samples (n=3 for each season) were fully powdered under liquid nitrogen and then sonicated in lysis buffer (8 mol/L urea (Sigma-Aldrich, USA), 1% protease inhibitor (Merck Millipore, USA), and 1% phosphatase inhibitor (Millipore, USA); w:v=1:4), followed by centrifugation at 12 000 ×g for 10 min at 4 °C. Supernatants were collected to determine protein concentration using a BCA kit (Beyotime Biotechnology, China). Supernatants were added to 20% (w:v) trichloroacetic acid (TCA) (Sigma-Aldrich, USA), followed by incubation at 4 °C for 2 h. After centrifugation at 4 500 ×g for 5 min at 4 °C, the precipitates were collected and washed three times with cooled acetone, then dissolved in 200 mmol/L tetraethylammonium bromide (TEAB) (Sigma-Aldrich, USA). For digestion, trypsin (Promega, USA) (w:w=1:50) was added and left overnight. Samples were then reduced with 5 mmol/L dithiothreitol (Sigma-Aldrich, USA) for 60 min at 37 °C and alkylated with 11 mmol/L iodoacetamide (Sigma-Aldrich, USA) for 45 min at room temperature in darkness. Finally, the peptides were desalted using a Strata X SPE column (Dr. Maisch GmbH, GER) and vacuum-dried.

Urea, DL-dithiothreitol (DTT), iodoacetamide (IA), IPG buffer and formic acid (FA) were purchased from GE Healthcare. SDS, Tris-(hydroxymethyl)-amino-methane, trichloroacetic acid, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine were purchased from Amresco. Tetraethyl-ammonium bromide (TEAB) and Coomassie brilliant blue G-250 were obtained from Sigma-Aldrich (Merck KGaA). Trypsin was from Promega Corp. and acetonitrile (ACN; HPLC grade) and H₂O were purchased from Thermo Fisher Scientific, Inc.