Mass Spectrometry, Supplemental

Protein Identification from SDS-PAGE:Protein in gel bands (self-made gel), have been reduced with DTT, alkylated with iodoacetomide, subjected to 3x alternating washes, and proteolysis with Trypsin (sequential grade, Promega) in 50mM TEAB buffer at 37C overnight. Peptides extracted and desalted on u-HLB Oasis plates, eluted with 60 acetonitrile/ 0.1%TFA, dried.
Dry peptides re-constituted in 20uL acetonitrile/0.1uL,and analyzed by LC/MS/MS on Orbitrap Lumos-ETD. Resolution set to 120K, 30K for the precursor and fragment ions, respectively. HCD energy at 31 with a isolation window set to 0.8Da without offset. Injected 10% for samples 3 and 4.

Data analysis:
MS/MS raw data were searched via PD2.3 with Mascot 6.1, against RefSeq2017_83_human database (containing common contaminants). Mascot .dat files were validated with PD2.3-Percolator.

基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）的蛋白质鉴定：凝胶条带中的蛋白质（自制凝胶）经二硫苏糖醇（DTT）还原、碘乙酰胺（iodoacetomide）烷基化后，进行3次交替洗涤，随后在50mM三乙胺碳酸氢盐（TEAB）缓冲液中，于37℃下用测序级胰蛋白酶（Trypsin，Promega公司）酶解过夜。肽段经Oasis u-HLB固相萃取板提取并脱盐，以60%乙腈/0.1%三氟乙酸（TFA）洗脱后冻干。冻干后的肽段复溶于20μL乙腈/0.1μL体系中，采用Orbitrap Lumos-ETD质谱仪进行液相色谱-串联质谱（LC/MS/MS）分析：前体离子与碎片离子的分辨率分别设为120K和30K；高能碰撞解离（HCD）能量为31，离子隔离窗口设为0.8道尔顿（Da）且无偏移。样品3和4的进样比例为10%。

数据分析：采用Proteome Discoverer 2.3（PD2.3）搭配Mascot 6.1搜索引擎，针对RefSeq 2017年第83版人类数据库（包含常见污染物序列）对MS/MS原始数据进行检索；通过PD2.3-Percolator对Mascot生成的.dat格式文件进行验证。