Training dataset: Generation of a spectral library from HEK-Ecoli Spike-in mass spectrometry data

The five raw files serve as a concise but meaningful training data set in the Galaxy training network (https://galaxyproject.github.io/training-material/). HEK and E.coli cell pellets were lysed with 5 % SDS, 50 mM triethylammonium bicarbonate (TEAB), pH 7.55. The obtained protein extracts were reduced by adding f.c. 5 mM TCEP and alkylated by the addition of f.c. 10 mM iodacetamide. Protein digestion and purification was performed on S-Trap columns. To ensure protein binding to the S-Trap columns, samples were acidified to a final concentration of 1.2 % phosphoric acid (~ pH 2). Six times the sample volume S-Trap buffer (90% aqueous methanol containing a final concentration of 100 mM TEAB, pH 7.1) was added to the samples which were then loaded on the columns and washed with S-Trap buffer. Protein digestion was performed with trypsin and LysC for one hour at 47 °C. Peptides were eluted in three steps with (1) 50 mM TEAB, (2) 0.2 % aqueous formic acid and (3) 50 % acetonitrile containing 0.2 % formic acid. Eluted peptides of HEK and E.coli were mixed in the following ratios (amount in µg): Sample HEK E.coli MS method<br> Sample1 2.5 0.00 DDA<br> Sample2 2.5 0.05 DDA<br> Sample3 2.5 0.15 DDA<br> Sample4 2.5 0.40 DDA<br> Sample5 2.5 0.80 DDA Additionally, iRT peptides were added and 1µg of each samples was measured with a Q-Exactive Plus mass spectrometer. Besides the five raw files, we uploaded two fasta files that serve as human and ecoli protein sequence databases, an transition list for the iRT peptides as well as an experimental design for the MaxQuant search.<br> Additionally, we uploaded the Galaxy MaxQuant training result files: protein groups, peptides, mqpar, msms, evidence and PTXQC.本数据集包含5个原始文件，作为Galaxy训练网络（对应链接：https://galaxyproject.github.io/training-material/）的简洁且具备实用价值的训练数据集。HEK细胞与大肠杆菌（Escherichia coli, E. coli）细胞沉淀采用含5%十二烷基硫酸钠（sodium dodecyl sulfate, SDS）、50 mM碳酸氢三乙胺（triethylammonium bicarbonate, TEAB，pH 7.55）的体系进行裂解。所得蛋白质提取物通过添加终浓度（final concentration, f.c.）为5 mM的三(2-羧乙基)膦（tris(2-carboxyethyl)phosphine, TCEP）进行还原，再通过添加终浓度为10 mM的碘乙酰胺（iodacetamide）完成烷基化反应。蛋白质酶解与纯化操作均在S-Trap层析柱（S-Trap columns）上完成。为确保蛋白质结合至S-Trap层析柱，需将样品酸化至终浓度为1.2%的磷酸（phosphoric acid）体系（约pH 2）。向样品中加入6倍样品体积的S-Trap缓冲液（含100 mM TEAB、pH 7.1的90%甲醇水溶液），随后将样品上样至层析柱，并用S-Trap缓冲液进行洗涤。采用胰蛋白酶与LysC蛋白酶在47 ℃下对结合在柱上的蛋白质进行1小时的酶解。肽段分三步洗脱：(1) 50 mM TEAB溶液；(2) 0.2%甲酸水溶液；(3) 含0.2%甲酸的50%乙腈（acetonitrile）溶液。HEK与大肠杆菌来源的洗脱肽段按照以下质量配比（单位：微克）混合：样本1：HEK肽段2.5 µg、大肠杆菌肽段0.00 µg，质谱方法为数据依赖采集（data-dependent acquisition, DDA）；样本2：HEK肽段2.5 µg、大肠杆菌肽段0.05 µg，质谱方法为DDA；样本3：HEK肽段2.5 µg、大肠杆菌肽段0.15 µg，质谱方法为DDA；样本4：HEK肽段2.5 µg、大肠杆菌肽段0.40 µg，质谱方法为DDA；样本5：HEK肽段2.5 µg、大肠杆菌肽段0.80 µg，质谱方法为DDA。此外，向所有混合样本中添加iRT肽段（iRT peptides），并取1 µg每份样本使用Q-Exactive Plus质谱仪（Q-Exactive Plus mass spectrometer）进行质谱检测。除上述5个原始文件外，本数据集还上传了2个分别对应人类与大肠杆菌蛋白质序列的FASTA格式文件（FASTA files）、iRT肽段的离子跃迁列表，以及用于MaxQuant搜库分析的实验设计文件。此外，本数据集还上传了Galaxy平台上MaxQuant训练分析的结果文件，包括蛋白质组文件、肽段文件、mqpar配置文件、msms二级质谱文件、证据文件以及PTXQC质量控制文件。