Pandoravirus proteome - Pandoraviruses: amoeba viruses with genomes up to 2

Proteome analysis of a novel type of virus. The virus was grown in A. castellanii amobea before being purified. Viral particules were enriched after centrifugation and proteins solubilised by SDS before being stacked in the top of a SDS-PGE gel. After in-gel digestion, resulting peptides were injected for a 120min nanoLC-MS/MS analysis using an Ultimate U3000 system and a LTQ-Orbitrap Velos pro hybrid mass spectrometer (Top 20).Data processing and bioinformatics: Data were processed automatically using Mascot Daemon software (version 2.3.2, Matrix Science). Concomitant searches against Pandoravirus and A. castellanii protein sequence databanks as well as classical contaminants database and the corresponding reversed databases were performed using Mascot (version 2.4). ESI-TRAP was chosen as the instrument, trypsin/P as the enzyme and 2 missed cleavage allowed. Precursor and fragment mass error tolerances were set respectively at 10 ppm and 0.6 Da. Peptide modifications allowed during the search were: carbamidomethyl (C, fixes) acetyl (N-ter, variable), oxidation (M, variable) and deamidation (NQ, variable). The IRMa software (Dupierris et al., Bioinformatics, 2009, 25:1980-1, version 1.30.4) was used to filter the results: selection of rank 1 peptides, peptide identification FDR < 1% (as calculated by employing the reverse database strategy), and minimum of 1 specific peptide per identified protein group.

新型病毒的蛋白质组学分析。该病毒先在卡氏棘阿米巴（Acanthamoeba castellanii, A. castellanii）阿米巴原虫中增殖，随后进行纯化。通过离心富集病毒颗粒，经十二烷基硫酸钠（Sodium Dodecyl Sulfate, SDS）溶解蛋白后，将样品上样至十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE）凝胶顶部进行电泳。凝胶内酶解后，所得肽段经Ultimate U3000系统与LTQ-Orbitrap Velos Pro混合型质谱仪（Top 20模式）进行时长120分钟的纳升液相色谱-串联质谱（nanoLC-MS/MS）分析。数据处理与生物信息学分析：采用Mascot Daemon软件（版本2.3.2，Matrix Science公司）自动处理数据。使用Mascot（版本2.4）同时针对潘多拉病毒（Pandoravirus）、卡氏棘阿米巴（A. castellanii）的蛋白质序列数据库，以及经典污染数据库与对应的反向数据库进行检索。检索参数设置如下：以电喷雾电离-离子阱（ESI-TRAP）作为仪器类型，胰蛋白酶/P（trypsin/P）作为酶解酶，允许2个漏切位点；前体离子质量误差容忍度设为10 ppm，碎片离子质量误差容忍度设为0.6 Da。检索中允许的肽段修饰包括：半胱氨酸的氨基甲酰甲基化（carbamidomethyl，C，固定修饰）、N端乙酰化（acetyl，N-ter，可变修饰）、甲硫氨酸氧化（oxidation，M，可变修饰）以及天冬酰胺/谷氨酰胺脱酰胺（deamidation，NQ，可变修饰）。使用IRMa软件（Dupierris等，《生物信息学》，2009年，25卷：1980-1981，版本1.30.4）对结果进行过滤：筛选排名第一的肽段，肽段鉴定的错误发现率（False Discovery Rate, FDR）<1%（采用反向数据库策略计算），且每个鉴定到的蛋白质组至少包含1条特异性肽段。