SERUM NCAMP1 (COOMASSIE BLUE-STAINED GEL SLICES)

血清 NCAMP1（考马斯蓝染色凝胶切片）

• 批准号: 8170762
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170762
• 关键词: Acetonitriles; Algorithms; Blood capillaries; Charge; Color; Computer Retrieval of Information on Scientific Projects Database; Coomassie blue; Cysteine; Data; Databases; Digestion; Formic Acids; Funding; Gel; Grant; Ice; Incubated; Institution; Iodoacetamide; Ions; Mass Spectrum Analysis; Methods; Modification; Nitrogen; Parents; Peptides; Phase; Plant Resins; Procedures; Proteins; Proteome; Research; Research Personnel; Resolution; Resources; Sampling; Series; Serum; Site; Slice; Solutions; Source; Staining method; Stains; SwissProt; Trypsin; Tube; United States National Institutes of Health; Water; ammonium bicarbonate; base; capillary; ion source; mass spectrometer; pressure

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Methods: The coomassie blue-stained gel slices were cut into smaller pieces followed by destain, reduction, calboxyamidemethylation and in-gel tryptic digestion. The tryptic peptides were then extracted from the gel pieces and profiled by mass spectrometry. Detailed procedures for in-gel digestion and mass spec analysis are shown below. In-gel digestion Coomassie stained gel slices were cut into smaller pieces (~1 mm3) and destained alternately with 40mM Ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Destained gel was reswelled in 10 mM DTT in 40mM Ambic at 55¿ C for 1 hr. The DTT solution was exchanged with 55mM Iodoacetamide (IAM) and incubated in the dark for 45 min. Incubation was followed by washing alternately with 40mM AmBic and 100% acetonitrile twice. Dehydrated gel was reswelled with trypsin solution (trypsin in 40 mM Ambic) on ice for 45 min initially, and protein digestion was carried out at 37¿ C overnight. The supernatant was transferred into another tube. Peptides were extracted from the gels in series with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The sample solutions were dried and combined into one tube. Protein identification by LTQ orbitrap mass spectrometry The peptides were resuspended with mobile phase A (0.1% formic acid, FA, in water) and filtered with 0.2 ¿m filters prior to sampling. The sample was loaded by off-line onto a nanospray tapered capillary column/emitter self-packed with C18 reverse-phase (RP) resin in a Nitrogen pressure bomb for 5 min at 1000 psi and then separated via a 160 min linear gradient of increasing mobile phase B (80% acetonitrile, ACN, and 0.1% formic acid in water) at a flow rate of ~400 nL/min directly into the mass spectrometer. LC-MS/MS analysis was performed on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. A full FTMS spectrum at 30,000 resolution was collected at 4002000 m/z followed by 6 data dependent MS/MS spectra of ITMS in the most intense ion peaks from parent mass list following CID (36 % normalized collision energy). The resulting data was searched against the UniProtKB/Swiss-prot database using the TurboSequest algorithm (Proteome Discoverer 1.0, ThermoFisher) for protein identification. The SEQUEST parameters were set to allow 20.0 ppm of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Tryptic peptides were allowed with up to three missed internal cleavage sites and the differential modifications of 57.0215 Da for alkylated cysteine. The resulting peptides found by the sequest search were further filtered based on Xcorr value, 2.0/2.5/3.0 for +1/+2/+3 charged peptide respectively.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 方法： 将考马斯蓝染色的凝胶切片切成小块，然后进行脱色、还原、羧酰胺甲基化和凝胶内胰蛋白酶消化，然后从凝胶块中提取胰蛋白酶肽，并通过质谱分析进行分析。质谱分析如下所示。 凝胶内消化 将考马斯染色的凝胶切片切成小块 (~1 mm3)，并用 40mM 碳酸氢铵 (AmBic) 和 100% 乙腈交替脱色，直至脱色凝胶在 40mM Ambic 中的 10 mM DTT 中在 55° 下重新溶胀。 C 1 小时，将 DTT 溶液更换为 55mM 碘乙酰胺 (IAM)，并在黑暗中孵育 45 分钟，然后用 40mM AmBic 和 100% 乙腈交替洗涤两次。 40 mM Ambic) 最初在冰上 45 分钟，蛋白质消化在37°将上清液转移至另一管中，依次用 5% 甲酸中的 20% 乙腈、5% 甲酸中的 50% 乙腈、然后是 5% 甲酸中的 80% 乙腈提取肽。将溶液干燥并合并到一根管中。 通过 LTQ 轨道阱质谱法鉴定蛋白质 将肽用流动相 A（0.1% 甲酸，FA，水溶液）重悬，并用 0.2 ¿采样前，将样品离线加载到装有 C18 反相 (RP) 树脂的纳喷雾锥形毛细管柱/发射器上，在氮气压力弹中保持 5 分钟，压力为 1000 psi，然后通过过滤器分离。 160 分钟线性梯度增加流动相 B（80% 乙腈、乙腈和 0.1% 甲酸水溶液），流速约为 400 nL/min 直接进入质谱仪，在配备纳喷雾离子源的 LTQ Orbitrap XL 质谱仪 (ThermoFisher) 上进行，随后以 400 2000 m/z 收集分辨率为 30,000 的完整 FTMS 谱图。根据 CID 后来自母体质量列表的最强离子峰中的 6 个数据相关的 ITMS MS/MS 谱图（36% 归一化碰撞使用 TurboSequest 算法（Proteome Discoverer 1.0，ThermoFisher）在 UniProtKB/Swiss-prot 数据库中搜索所得数据以进行蛋白质鉴定。 SEQUEST 参数设置为允许 20.0 ppm 的前体离子质量容差和 0.8 Da 的片段。单同位素质量的离子耐受性 胰蛋白酶肽允许最多三个缺失的内部切割位点和不同的修饰。烷基化半胱氨酸为 57.0215 Da。根据 Xcorr 值进一步过滤通过隔离搜索找到的肽，+1/+2/+3 带电肽分别为 2.0/2.5/3.0。