Surviving winter on the Qinghai-Tibetan Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri

Animals and sample collection\tAdult male N. parkeri frogs (n=9 in each season) were captured and their liver tissues were collected. In brief, liver tissue samples were dissected and snap-frozen in liquid nitrogen, followed by storage at −80 °C in the laboratory. The mean snout-vent length (SVL) of frogs was 4.08±0.06 cm and mean body mass (BM) was 4.99±0.14 g in summer (n=9); comparable data were 4.06±0.06 cm and 5.14±0.16 g in winter.Protein extraction and trypsin digestion\tFrozen samples (n=3 for each season) were fully powdered under liquid nitrogen and then sonicated in lysis buffer (8 mol/L urea, 1% protease inhibitor, and 1% phosphatase inhibitor; w:v=1:4), followed by centrifugation at 12\u202f000 ×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), followed by incubation at 4 °C for 2 h. After centrifugation at 4\u202f500 ×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). For digestion, trypsin (w:w=1:50) was added and left overnight. Samples were then reduced with 5 mmol/L dithiothreitol for 60 min at 37 °C and alkylated with 11 mmol/L iodoacetamide for 45 min at room temperature in darkness. Finally, the peptides were desalted using a Strata X SPE column and vacuum-dried.Tandem mass tag (TMT) labeling, high-performance liquid chromatography (HPLC) fractionation, and affinity enrichment of phosphopeptides\tPeptides from each sample were labeled with TMT reagent and fractionated by high-pH reverse-phase HPLC. Briefly, the peptides were separated with a gradient of 8%–32% acetonitrile in 10 mmol/L ammonium bicarbonate (pH=9) over 60 min into 60 fractions, then combined into nine fractions and dried by vacuum centrifugation for liquid chromatography-tandem mass spectrometry (LC-MS-MS).LC-MS/MS analysis\tAfter dissolution in mobile phase A (0.1% formic acid, 2% acetonitrile in water), the peptides were separated using the EASY-nLC 1200 ultra-high-performance liquid system (Thermo Fisher Scientific, USA), followed by ionization in an NSI source and analysis using the Q ExactiveTM HF-X system (Thermo Fisher Scientific, USA). The mass spectrometry scan range was 350–1\u202f600 m/z with a resolution of 120\u202f000 and a second-order mass scan resolution of 30\u202f000. The top 20 peptide parent ions with the highest signal intensity were used for higher-energy collisional dissociation (HCD) fragmentation at a normalized collision energy (NCE) of 28%. Mobile phase B contained 0.1% formic acid and 90% acetonitrile. For proteomic analysis, the elution settings were: 40 min, 6%–22% B; 14 min, 22%–32% B; 3 min, 32%–80% B; 3 min, 80% B, with a constant flow rate of 500 nL/min. For phosphoproteomic analysis, the gradient was as follows: 38 min, 5%–22% B; 15 min 22%–35% B; 4 min 35%–80% B; 3 min, 80% B, with a flow rate of 350 nL/min at 0–53 min and 600 nL/min at 53–60 min.Proteomic and phosphoproteomic database search\tThe raw MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8), and tandem mass spectra were searched against the Nanorana_parkeri database (37\u202f248 entries) concatenated with the reverse decoy database. Trypsin/P was designated as a cleavage enzyme and missing cleavages of less than 2 were permitted. The mass error for precursor ions was set at 5 ppm in the main search and 20 ppm in the first search, and a value of 0.02 Da was used for mass error of fragment ions. Carbamidomethyl on Cys was specified as a fixed modification, and variable modifications included acetylation on protein N-terminal, oxidation on Met, deamidation (NQ), and phosphorylation on Ser, Thr, and Tyr. TMT-6plex was performed for quantification. The false discovery rate (FDR) was adjusted to <1% and the minimum score for peptides was set to >40.Bioinformatics analysis for proteomics and phosphoproteomicsGene Ontology (GO) annotation of proteomic and phosphoproteomic data was conducted using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) and included three categories: biological processes, cellular components, and molecular functions. Protein domains and pathways were annotated using the InterPro (http://www.ebi.ac.uk/interpro/) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/), respectively. Subcellular localization was predicted using WoLF PSORT software, and sequence patterns around the phosphorylation modification site (spanning six amino acids both upstream and downstream) were identified using Soft motif-x. Two-tailed Fisher’s exact test was used to test functional enrichment of differentially expressed proteins with a critical corrected P-value of 0.05. Functional interaction network analysis of differentially expressed proteins and phosphorylated proteins was performed using STRING (v11.0) and visualized using Cytoscape.