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

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 Peptides 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 After 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 350–1 600 m/z with a resolution of 120 000 and a second-order mass scan resolution of 30 000. 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 The 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 (37 248 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 (ebi.ac.uk/GOA/) and included three categories: biological processes, cellular components, and molecular functions.

Protein domains and pathways were annotated using the InterPro (ebi.ac.uk/interpro/) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (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.