Comparative Proteomics in Primates

The following cell lines were used for this study: Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) derived from 5 human (Coriell YRI, NIGMS Human Genetic Cell Repository, GM18505, GM18507, GM18516, GM19193, GM19204), and 5 chimpanzee (Pan troglodytes) individuals (New Iberia Research Center: Min 18358, Min 18359; Coriell/IPBIR: NS03659, NS04973, Arizona State University, Pt91), and rhesus Herpesvirus papio transformed LCLs from 5 rhesus macaque (Macaca mulatta) individuals (Harvard Medical School, NEPRC: 150-99, R181-96, R249-97, 265-95, R290-96). Cells were maintained at identical conditions of 37° with 5% CO2 in RPMI media with 15% FBS, supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin. Internal standard LCL (Coriell YRI, NIGMS Human Genetic Cell Repository, GM19238) was grown in RPMI minus L-Lysine and L-Arginine, 15% dialyzed FBS, and L-13C615N4-arginine (Arg-10) and L-13C615N2-lysine (Lys-8) (Cambridge Isotopes, Andover, MA, USA) supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin under identical conditions as the unlabeled LCLs. The internal standard LCL was grown for 6 doublings to assure complete SILAC label incorporation. Complete label incorporation was verified by analyzing the protein lysate from the labeled LCL alone by high-resolution LC-MS/MS. LCLs were washed in PBS three times and then lysed using the UPX Universal Protein Extraction Kit (Expedeon Inc., San Diego, CA, USA). Protein quantitation was performed using the Qubit fluorometry assay (Invitrogen, Carlsbad California, USA) and the reducing agent-compatible (RAC) version of the BCA Protein Assay (Thermo Scientific, Waltham, Massachusetts, USA). 12ug of each sample was combined with 12ug of the SILAC labeled lysate from human LCL GM19238. Note that the SILAC lysate was prepared once and used as an internal standard through the quantification of the 15 cell lines. 24ug of each combined sample was then processed by SDS-PAGE using a 4-12% Bis Tris NuPage mini-gel (Invitrogen, Carlsbad California, USA). Calibration was with Thermo PageRuler broad range markers. Each of 40 gel segments were processed by in-gel digestion using a ProGest robot (DigiLab, Marlborough, MA, USA) with the following protocol: wash with 25mM ammonium bicarbonate followed by acetonitrile, reduce with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at room temperature, digest with trypsin (Promega, Madison, WI, USA) at 37°C for 4h, and quench with formic acid. The supernatant was analyzed directly without further processing. Each of gel digest was analyzed by nano-LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher LTQ-Orbitrap Velos Pro. Peptides were loaded on a trapping column and eluted over a 75um analytical column at 350nL/min using a 1-hour LC gradient. Both columns were packed with Jupiter Proteo resin (Phenomenex, Torrance, California, USA). The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. Low-level data analysis was performed using the open-source proteomics software tool PVIEW (Release December 23, 2012; http://compbio.cs.princeton.edu/pview). As input to PVIEW, we generated in silico translations of coding genes from the UCSC Genome Browser database based on gene models from build hg19 of the human genome. Each protein sequence entry retained the corresponding Ensembl gene identifier and gene symbol. Database searches were performed using +-4 p.p.m. MS1 tolerance and an MS2 window tolerance of +-0.5 Da. Up to 2 missed tryptic cleavages were allowed during search. Carboxyamidomethylation of cysteine was used as fixed modification. Up to two methionine oxidations were allowed as variable modifications of a tryptic peptide. Peptide spectrum matches were obtained at a stringent false discovery rate (FDR) of 1%. We used the median log2(sample/standard) ratio across all independent quantifications of a protein (distinct peptides including duplicate peptide measurements across fractions and for differing charge states). Note that use of the hg19 database was sufficient as SILAC pairs as the expected isotope shift used for quantification were only present for peptides that that had the same underlying sequence in the human internal standard line. Associated RNA-seq data have been deposited to GEO with accession number GSE49682.本研究使用的细胞系如下：源自5名人类个体的爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）转化淋巴母细胞样细胞系（lymphoblastoid cell lines, LCLs）（来自Coriell YRI、NIGMS人类遗传细胞库，编号GM18505、GM18507、GM18516、GM19193、GM19204），以及5只黑猩猩（Pan troglodytes）个体的同类LCLs（New Iberia研究中心：Min 18358、Min 18359；Coriell/IPBIR：NS03659、NS04973；亚利桑那州立大学：Pt91），另有5只恒河猴（Macaca mulatta）个体的狒狒疱疹病毒（Herpesvirus papio）转化的LCLs（哈佛医学院NEPRC：150-99、R181-96、R249-97、265-95、R290-96）。 所有细胞均在统一条件下培养：37℃、5% CO₂环境，使用添加15%胎牛血清（FBS）、2 mM L-谷氨酸、100 IU/ml青霉素及100 μg/ml链霉素的RPMI培养基。 内标LCL（Coriell YRI、NIGMS人类遗传细胞库，GM19238）的培养采用不含L-赖氨酸与L-精氨酸的RPMI培养基，添加15%透析型FBS、L-¹³C₆¹⁵N₄-精氨酸（Arg-10）与L-¹³C₆¹⁵N₂-赖氨酸（Lys-8）（剑桥同位素公司，安多弗，马萨诸塞州，美国），并补充2 mM L-谷氨酸、100 IU/ml青霉素及100 μg/ml链霉素，培养条件与未标记的LCLs完全一致。内标LCL经6次传代倍增，以确保细胞培养中氨基酸稳定同位素标记（Stable Isotope Labeling with Amino acids in Cell culture, SILAC）完全整合。通过单独分析标记LCL的蛋白质裂解液的高分辨LC-MS/MS结果，验证了标记完全整合。 收集LCLs后用PBS洗涤三次，使用UPX通用蛋白提取试剂盒（Expedeon公司，圣地亚哥，加利福尼亚州，美国）裂解细胞。蛋白定量采用Qubit荧光定量法（Invitrogen公司，卡尔斯巴德，加利福尼亚州，美国），以及兼容还原剂的BCA蛋白定量试剂盒（Thermo Scientific公司，沃尔瑟姆，马萨诸塞州，美国）。取每份样品12 μg，与12 μg来自人类LCL GM19238的SILAC标记裂解液混合。注：该SILAC裂解液仅制备一次，在15株细胞系的定量分析中均作为内标使用。 取每份混合后的24 μg样品，采用4-12% Bis-Tris NuPage迷你凝胶（Invitrogen公司，卡尔斯巴德，加利福尼亚州，美国）进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE），电泳校准采用Thermo PageRuler广谱分子量Marker。将凝胶切割为40个区段，使用ProGest机器人（DigiLab公司，马尔伯勒，马萨诸塞州，美国）进行胶内酶解，具体流程如下：先用25 mM碳酸氢铵洗涤，再用乙腈处理；以10 mM二硫苏糖醇于60℃还原，随后于室温下用50 mM碘乙酰胺烷基化；以胰蛋白酶（Promega公司，麦迪逊，威斯康星州，美国）于37℃酶解4小时，最后用甲酸终止反应。酶解后的上清液无需进一步处理，直接进行分析。 每份凝胶酶解产物均采用纳升液相色谱-串联质谱（nano-LC/MS/MS）进行分析，系统配置为Waters NanoAcquity HPLC系统连接ThermoFisher LTQ-Orbitrap Velos Pro质谱仪。肽段上样至捕集柱，以350 nL/min的流速在75 μm分析柱上洗脱，洗脱梯度时长为1小时。两根色谱柱均装填Jupiter Proteo树脂（Phenomenex公司，托伦斯，加利福尼亚州，美国）。质谱仪采用数据依赖型采集模式：MS1扫描在Orbitrap中完成，分辨率为60,000半高全宽（FWHM）；MS/MS扫描在LTQ中完成，选取丰度最高的15个离子进行碎裂分析。 基础数据分析使用开源蛋白质组学软件工具PVIEW（2012年12月23日发布；http://compbio.cs.princeton.edu/pview）完成。作为PVIEW的输入文件，我们基于人类基因组hg19版本的基因模型，从UCSC基因组浏览器数据库中获取编码基因的计算机翻译序列。每个蛋白质序列条目均保留对应的Ensembl基因标识符与基因符号。数据库搜索采用±4 ppm的MS1质量容差和±0.5 Da的MS2窗口容差，允许最多2次胰蛋白酶酶切漏切。半胱氨酸的羧酰胺甲基化作为固定修饰，甲硫氨酸的氧化作为最多2处的可变修饰。肽段谱匹配的筛选严格控制错误发现率（false discovery rate, FDR）为1%。我们采用某一蛋白质所有独立定量结果（涵盖不同组分、不同电荷状态下的重复肽段检测值的特异性肽段）的中位数log₂(样品/内标)比值。注：由于仅在与人类内标细胞系序列一致的肽段中存在用于定量的预期同位素位移，因此使用hg19数据库即可满足分析需求。 相关RNA-seq数据已提交至基因表达综合数据库（Gene Expression Omnibus, GEO），登录号为GSE49682。