Training dataset: Statistical analysis of a HEK/Ecoli Spike-in DIA dataset using MSstats

The uploaded 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 two different ratios and four replicates of each Spike/in ratio were measured and analysed using OpenSwathWorkflow in Galaxy. Results were exported using PyProphet and can be used for the statistical analysis and detection of the two different Spike-in Ratios. The Spike-in ratios were the following: Sample HEK E.coli <br> Spike_in_1 2.5 0.15<br> Spike_in_2 2.5 0.80 Besides the two PyProphet export files, we uploaded a sample annotation file as well as a comparison matrix file.<br> Additionally, we uploaded the Galaxy MSstats training result files: MSstats_ComparisonResult_export_tabular and MSstats_ComparisonResult_msstats_input.本次上传的文件为Galaxy训练网络（Galaxy training network，https://galaxyproject.github.io/training-material/）配套的简洁且兼具实用价值的训练数据集。将HEK细胞与大肠杆菌（E. coli）细胞沉淀采用含5%十二烷基硫酸钠（SDS）、50 mM三乙胺碳酸氢盐（TEAB）、pH 7.55的裂解体系进行处理。所得蛋白提取物通过添加终浓度5 mM的三(2-羧乙基)膦（TCEP）完成还原，再以终浓度10 mM的碘乙酰胺（iodacetamide）进行烷基化修饰。蛋白的酶解与纯化均在S-Trap柱（S-Trap columns）上进行。为保障蛋白结合至S-Trap柱，将样品酸化至终浓度1.2%的磷酸（phosphoric acid，pH约为2）。随后向样品中加入6倍样品体积的S-Trap缓冲液（含100 mM TEAB的90%甲醇水溶液，pH 7.1），将混合液上样至层析柱后，使用S-Trap缓冲液进行洗涤。以胰蛋白酶（trypsin）与赖氨酸C内切酶（LysC）在47℃下酶解1小时。肽段分三步洗脱：(1) 50 mM TEAB溶液；(2) 0.2%甲酸（formic acid）水溶液；(3) 含0.2%甲酸的50%乙腈（acetonitrile）水溶液。将HEK与大肠杆菌来源的洗脱肽段以两种不同比例混合，每种混合比例设置4次重复检测，通过Galaxy平台中的OpenSwathWorkflow工具完成检测与分析。分析结果通过PyProphet导出，可用于两种不同混入比例（Spike-in ratio）的统计分析与靶标检出。两种混入比例具体如下： | 混入组别 | HEK占比 | 大肠杆菌占比 | | ------- | ------ | ---------- | | Spike_in_1 | 2.5 | 0.15 | | Spike_in_2 | 2.5 | 0.80 | 除两份PyProphet导出文件外，本次上传还包含样本注释文件与比对矩阵文件。此外，还上传了Galaxy平台的MSstats训练结果文件：MSstats_ComparisonResult_export_tabular与MSstats_ComparisonResult_msstats_input。