Training dataset: DIA data analysis of a HEK/Ecoli Spike-in dataset using OpenSwathWorkflow

The eight 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 two different ratios and four replicates of each Spike/in ratio were measured: Sample HEK E.coli MS method<br> Sample1 2.5 0.15 DIA<br> Sample2 2.5 0.15 DIA<br> Sample3 2.5 0.15 DIA<br> Sample4 2.5 0.15 DIA<br> Sample5 2.5 0.80 DIA<br> Sample6 2.5 0.80 DIA<br> Sample7 2.5 0.80 DIA<br> Sample8 2.5 0.80 DIA Additionally, iRT peptides were added and 1µg of each samples was measured using data independent acquisition with a Q-Exactive Plus mass spectrometer. Briefly, a scan range from 400-1000 m/Z was first covered by an MS1 scan followed by 25 consecutive MS2 scans (each 24 m/z broad). In the next cycle another MS1 scan was acquired followd by 26 MS2 scans (also 24m/z broad) in which the window centers were shifted by 50% compared to the previous cycle of MS2 scans. The resulting raw files contain overlapping MS2 scans. Besides the eight raw files, we uploaded a spectral library, a transition list for the iRT peptides as well as an sample annotation file.<br> Additionally, we uploaded the Galaxy PyProphet score training result files: PyProphet score report and PyProphet score.本数据集包含8个原始文件，可作为Galaxy训练网络（https://galaxyproject.github.io/training-material/）中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5%十二烷基硫酸钠（SDS）、50 mM 碳酸氢三乙胺（triethylammonium bicarbonate, TEAB）、pH 7.55的体系进行裂解。所得蛋白提取物通过添加终浓度5 mM的三(2-羧乙基)膦（TCEP）进行还原，并通过添加终浓度10 mM的碘乙酰胺进行烷基化。蛋白酶解与纯化均在S-Trap色谱柱上完成：为确保蛋白结合至S-Trap色谱柱，将样品酸化至终浓度1.2%的磷酸（约pH 2）；向样品中加入6倍样品体积的S-Trap缓冲液（含终浓度100 mM TEAB的90%含水甲醇溶液，pH 7.1），随后将样品上样至色谱柱，并用S-Trap缓冲液洗涤。使用胰蛋白酶（trypsin）与LysC蛋白酶在47℃下酶解1小时。肽段通过三步进行洗脱：(1) 50 mM TEAB；(2) 0.2%含水甲酸；(3) 含0.2%甲酸的50%乙腈溶液。 将洗脱得到的HEK与大肠杆菌肽段按两种不同的加标比例混合，每种加标比例设置4个重复样本并进行检测，具体样本信息如下： | 样本编号 | HEK投料量 | E. coli投料量 | 质谱方法 | |----------|----------|-------------|---------| | Sample1 | 2.5 | 0.15 | DIA | | Sample2 | 2.5 | 0.15 | DIA | | Sample3 | 2.5 | 0.15 | DIA | | Sample4 | 2.5 | 0.15 | DIA | | Sample5 | 2.5 | 0.80 | DIA | | Sample6 | 2.5 | 0.80 | DIA | | Sample7 | 2.5 | 0.80 | DIA | | Sample8 | 2.5 | 0.80 | DIA | 此外，向所有样品中添加iRT内参肽段，并取1μg样品采用数据非依赖性采集（data independent acquisition, DIA）模式，通过Q-Exactive Plus质谱仪进行检测。具体采集参数如下：首先采集扫描范围为400-1000 m/z的一级质谱（MS1）扫描，随后进行25次连续的二级质谱（MS2）扫描（每个扫描窗口宽度为24 m/z）；在下一个循环中，再次采集一级质谱扫描，随后进行26次二级质谱扫描（同样为24 m/z宽度），且本次二级扫描的窗口中心相较于上一轮二级扫描循环偏移50%，所得原始文件包含重叠的二级质谱扫描数据。 除上述8个原始文件外，本数据集还上传了谱图库、iRT肽段的跃迁列表以及样本注释文件。此外，我们还上传了Galaxy平台上的PyProphet评分训练结果文件：PyProphet评分报告与PyProphet评分文件。