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 collectionAdult 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 digestionFrozen 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 12 000 ×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), 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). 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 phosphopeptidesPeptides 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 analysisAfter 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 searchThe 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 (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.

动物与样本采集
成年雄性Parki倭蛙（Nanorana parkeri，n=9/季）被捕获并采集其肝脏组织。简言之，解剖肝脏组织样本后立即液氮速冻，随后于实验室中保存在-80 ℃环境下。夏季样本（n=9）的受试蛙平均吻肛长（snout-vent length, SVL）为4.08±0.06 cm，平均体重（body mass, BM）为4.99±0.14 g；冬季样本的对应参数分别为4.06±0.06 cm与5.14±0.16 g。

蛋白质提取与胰蛋白酶消化
每个季节的冷冻样本（n=3）在液氮中充分研磨成粉末，随后于裂解缓冲液（8 mol/L尿素、1%蛋白酶抑制剂与1%磷酸酶抑制剂；w:v=1:4）中进行超声破碎，之后在4 ℃下以12000×g离心10 min。收集上清液，采用BCA蛋白定量试剂盒（碧云天生物技术，中国）测定总蛋白浓度。向上清液中加入终浓度20%（w:v）的三氯乙酸（trichloroacetic acid, TCA），4 ℃下静置孵育2 h。随后在4 ℃下以4500×g离心5 min，收集沉淀并以预冷丙酮洗涤三次，之后将沉淀溶于200 mmol/L四乙基溴化铵（tetraethylammonium bromide, TEAB）溶液中。消化步骤中，按酶底物质量比1:50加入胰蛋白酶，37 ℃下静置过夜。随后加入5 mmol/L二硫苏糖醇（dithiothreitol, DTT），37 ℃下还原反应60 min，再加入11 mmol/L碘乙酰胺，室温避光条件下烷基化反应45 min。最终肽段经Strata X固相萃取柱脱盐后，真空冷冻干燥。

串联质量标签（Tandem mass tag, TMT）标记、高效液相色谱（high-performance liquid chromatography, HPLC）分级与磷酸肽亲和富集
各样本的肽段经TMT试剂标记后，采用高pH反相HPLC进行分级分离。简言之，以10 mmol/L碳酸氢铵溶液（pH=9）为溶剂，采用8%~32%乙腈梯度在60 min内完成分离，共收集60个组分，随后合并为9个组分，经真空离心干燥后用于液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）分析。

LC-MS/MS分析
肽段经流动相A（0.1%甲酸、2%乙腈水溶液）溶解后，使用EASY-nLC 1200超高效液相色谱系统（赛默飞世尔科技，美国）进行分离，随后通过纳米喷雾电离（NSI）源电离，并采用Q Exactive™ HF-X质谱仪（赛默飞世尔科技，美国）完成分析。质谱扫描范围为350~1600 m/z，一级质谱分辨率为120000，二级质谱分辨率为30000。选取信号强度排名前20的肽段前体离子，以28%的归一化碰撞能量（normalized collision energy, NCE）进行高能碰撞解离（higher-energy collisional dissociation, HCD）。流动相B为含0.1%甲酸的90%乙腈水溶液。蛋白质组分析的洗脱梯度为：0~40 min，流动相B比例从6%升至22%；40~54 min，从22%升至32%；54~57 min，从32%升至80%；57~60 min，维持80% B，流速恒定为500 nL/min。磷酸蛋白质组分析的洗脱梯度为：0~38 min，流动相B比例从5%升至22%；38~53 min，从22%升至35%；53~57 min，从35%升至80%；57~60 min，维持80% B，0~53 min流速为350 nL/min，53~60 min流速为600 nL/min。

蛋白质组与磷酸蛋白质组数据库检索
原始MS/MS数据采用MaxQuant检索引擎（v.1.5.2.8）进行处理，将串联质谱谱图与结合了反向诱饵数据库的倭蛙（Nanorana parkeri）数据库（共37248条条目）进行比对检索。指定胰蛋白酶/P为酶切位点，允许最多2个漏切位点。前体离子质量误差在主检索中设置为5 ppm，首次检索中设置为20 ppm；碎片离子质量误差设置为0.02 Da。将半胱氨酸残基的氨基甲酰甲基化设为固定修饰，可变修饰包括蛋白质N端乙酰化、甲硫氨酸氧化、天冬酰胺/谷氨酰胺脱氨基化以及丝氨酸、苏氨酸、酪氨酸的磷酸化。采用TMT-6plex标记法进行定量分析。将错误发现率（false discovery rate, FDR）控制在1%以下，肽段最低得分设置为≥40。

蛋白质组和磷酸蛋白质组学生物信息学分析
采用UniProt-GOA数据库（http://www.ebi.ac.uk/GOA/）对蛋白质组与磷酸蛋白质组数据进行基因本体论（Gene Ontology, GO）注释，注释涵盖生物过程、细胞组分与分子功能三大类别。分别采用InterPro数据库（http://www.ebi.ac.uk/interpro/）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG，http://www.kegg.jp/）对蛋白质结构域与通路进行注释。采用WoLF PSORT软件预测蛋白质的亚细胞定位，使用Soft motif-x软件鉴定磷酸化修饰位点上下游各6个氨基酸残基范围内的序列基序。采用双侧Fisher精确检验对差异表达蛋白进行功能富集分析，校正后P值阈值设为0.05。采用STRING数据库（v11.0）对差异表达蛋白与磷酸化蛋白进行功能互作网络分析，并通过Cytoscape软件完成可视化。