Phosphoproteomics COMMSBIO-18-0285B

For Phosphoproteomics, Mtb strains (Mtb H37Rv Wild type and mutants MtbΔdosR, MtbΔdosS, and MtbΔdosT) were cultured in liquid media with shaking (aerobic, OD595=0.3) or left standing (hypoxic, day 1 to day 30) as described. The cultures were harvested by centrifugation followed by washing in 7H9 media and resuspension in lysis buffer. Bacterial cells were sonicated by bead beating, and after filtration through 0.22 ìm filters; clear lysates were used in downstream procedures. The lysates were reduced with 10 mM dithiothreitol followed by alkylation with 15 mM iodoacetamide and acetone precipitation. The protein pellets were washed in acetone/water (80/20), followed by evaporation of acetone and reconstitution in 200 μL of modified urea lysis buffer (5 M urea, 150 mM NaCl, 50 mM Tris-HCl pH 8.0). Protein concentration was estimated by the Bradford assay (Bio-Rad), and 1 mg of protein was digested with trypsin for 16 h at 37°C followed by enzyme inactivation with 10 μL trifluoroacetic acid (TFA). Next, Phosphopeptide enrichment using TiO2 chromatography was performed. The trypsin-digested peptides were enriched for phosphopeptides using Titansphere TiO2 tips (Thermo scientific USA). Phosphopeptides were serially eluted in 5 % NH4OH in water, 5 % pyrrolidine in acetonitrile, and 60 % acetonitrile in water. The three elutions were pooled together, neutralized with 50 % acetic acid, and dried. Samples were reconstituted in 50 μL 0.03 % TFA. Each enriched sample was desalted using a Stage Tip (ThermoFisher) per the vendor protocol. Peptides were dried and reconstituted in 70 μL of 0.03 % TFA prior to analysis. Next, Mass spectrometry was performed as following. The phosphopeptide-enriched samples were analyzed by mass spectroscopy in collaboration with MS Bioworks. The samples were analyzed by nanoLC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive using a 2 h reverse phase gradient. The phosphopeptides were loaded onto a trapping column and eluted over a 75 μm analytical column at 350 nL min-1; both columns were packed with Jupiter Proteome resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 full width at half maximum (FWHM) and 17,500 FWHM for MS and MS/MS, respectively. The fifteen most abundant ions were selected for MS/MS. The data analyses and phosphoproteins were detected as below. The proteins expressed during onset of hypoxia, i.e., at the early stage of transition during aerobic to hypoxic conditions, were identified by mass spectroscopy. Mascot DAT files were parsed into Scaffold software for validation, filtering and to create a non-redundant list per sample. Data were filtered using at 1 % protein and peptide FDR and requiring at least one unique peptide per protein. Scaffold results were imported into Scaffold PTM in order to assign site localization probabilities using A-score. A minimum localization probability filter of 50 % was applied for the analysis.针对磷酸化蛋白质组学（Phosphoproteomics）研究，本研究将结核分枝杆菌（Mycobacterium tuberculosis, Mtb）菌株（包括野生型Mtb H37Rv以及突变株MtbΔdosR、MtbΔdosS与MtbΔdosT）按照既定方法培养：于液体培养基中振荡培养（有氧条件，OD595=0.3），或静置培养（低氧条件，第1天至第30天）。培养物经离心收集，随后用7H9培养基洗涤并重悬于裂解缓冲液中。细菌细胞经珠磨超声破碎后，通过0.22 μm滤膜过滤，取澄清裂解液用于下游实验。裂解液先用10 mM二硫苏糖醇（dithiothreitol）还原，再经15 mM碘乙酰胺烷基化，随后进行丙酮沉淀。蛋白沉淀用丙酮/水（80/20，体积比）洗涤，蒸发去除丙酮后，用200 μL改性尿素裂解缓冲液（5 M尿素、150 mM氯化钠、50 mM Tris-HCl，pH 8.0）重溶。采用布拉德福德蛋白定量法（Bradford assay，Bio-Rad公司）测定蛋白浓度，取1 mg蛋白于37℃下用胰蛋白酶消化16 h，随后加入10 μL三氟乙酸（trifluoroacetic acid, TFA）灭活胰蛋白酶。随后采用二氧化钛色谱法（TiO₂ chromatography）进行磷酸肽富集：使用Titansphere TiO₂固相萃取柱（Thermo Scientific, 美国）对胰蛋白酶酶解后的肽段进行磷酸肽富集。磷酸肽依次用5%氨水水溶液、5%吡咯烷乙腈溶液及60%乙腈水溶液进行梯度洗脱。合并三次洗脱组分，用50%乙酸中和后冻干。样品用50 μL 0.03% TFA重溶，随后按照厂商方案采用Stage Tip（ThermoFisher）对富集后的样品进行脱盐处理。肽段经冻干后，用70 μL 0.03% TFA重溶，以待质谱分析。质谱分析流程如下：本研究与MS Bioworks合作，采用质谱技术对磷酸肽富集样品进行分析。使用纳流液相色谱-串联质谱（nanoLC-MS/MS），配备Waters NanoAcquity高效液相色谱系统并联用ThermoFisher Q Exactive质谱仪，以2小时反相梯度洗脱程序完成分析。将磷酸肽上样至捕集柱，以350 nL·min⁻¹的流速在75 μm分析柱上洗脱；两根色谱柱均装填有Jupiter Proteome树脂（Phenomenex公司）。质谱仪采用数据依赖型采集模式：Orbitrap分析仪在MS扫描时的分辨率为60000半高峰宽（full width at half maximum, FWHM），MS/MS扫描时分辨率为17500半高峰宽（FWHM）。选取丰度最高的15个离子进行MS/MS分析。数据分析与磷酸化蛋白质鉴定流程如下：通过质谱鉴定出低氧起始阶段（即有氧向低氧过渡的早期阶段）表达的蛋白质。将Mascot生成的DAT文件导入Scaffold软件进行验证、过滤，并为每个样品生成非冗余蛋白列表。数据过滤阈值设置为蛋白质和肽段错误发现率（false discovery rate, FDR）均不超过1%，且要求每个蛋白质至少包含1个独特肽段。将Scaffold分析结果导入Scaffold PTM软件，利用A-score赋值位点定位概率。本分析设置的最小位点定位概率阈值为50%。