Mass spec raw data.xlsx

Agilent 1260 Infinity nHPLC stack and Thermo Orbitrap Velos Pro hybrid mass spectrometer were used for analysis of 8-µl samples with a C-18 column (75 μm x 15 cm; 300 Å; 5 μm; Phenomenex). All data were acquired in collision-induced dissociation mode. The phase A was 0.1% FA in ddH2O and phase B was 0.1% formic acid (FA) in 15% ddH2O/85% acetonitrile). The mobile phase gradient was: 10 min at 2% phase B, 90 min at 5-40% phase B, 5 min at 70% phase B and 10 min at 0% phase B. The MS detection included a full scan (m/z 300 -1200) with resolution at 60k and data-dependent MS2 scans on the top abundant ions (15 ions). The MS data files were converted to MzXML using ReAdW (v. 3.5.1). MzXML2 Search was used to create a Mascot generic format file. Data were analyzed using the SEQUEST engine and searches were performed using the Uniref100 database. The peptide ID lists were then further analyzed by Scaffold viewer. The mass spectrometry peptide identifications were filtered by Scaffold. In short, protein probabilities were set to ≥0.99 with false discovery rate <1% cutoff, minimum two peptides assigned per protein, and individual peptide probabilities were ≥0.8. Quantification across experiments was based on normalized spectral counting between samples for MS spectra assigned to each protein.

本研究采用安捷伦1260 Infinity型纳升高效液相色谱（Agilent 1260 Infinity nHPLC）联用系统与赛默飞Orbitrap Velos Pro混合型质谱仪，对8 μL样品进行分析，所用色谱柱为菲罗门（Phenomenex）品牌C18色谱柱（规格：75 μm × 15 cm，孔径300 Å，粒径5 μm）。所有数据均采用碰撞诱导解离（collision-induced dissociation, CID）模式采集。流动相A为含0.1%甲酸（FA, formic acid）的双蒸水（ddH₂O）溶液，流动相B为含0.1%甲酸（FA）的15%双蒸水/85%乙腈混合溶液。流动相洗脱梯度设置为：以2%流动相B平衡10 min，经5%~40%流动相B洗脱90 min，再以70%流动相B冲洗5 min，最后以0%流动相B平衡10 min。质谱检测采用全扫描模式，扫描范围为m/z 300~1200，分辨率60k，并对丰度最高的15个离子进行数据依赖型二级质谱（MS²）扫描。质谱原始数据文件通过ReAdW（v3.5.1）转换为MzXML格式，随后经MzXML2 Search工具生成Mascot通用格式文件。数据分析采用SEQUEST引擎，检索数据库为Uniref100数据库。后续通过Scaffold Viewer对肽段鉴定结果列表进行进一步分析，并由Scaffold软件对质谱肽段鉴定结果进行过滤：设置蛋白置信度≥0.99，假发现率（false discovery rate, FDR）<1%，每个蛋白至少匹配2条肽段，单条肽段置信度≥0.8。实验间定量分析基于各蛋白匹配质谱光谱的样本间归一化光谱计数法完成。