PROTEIN IDENTIFICATION AND N-LINKED OLIGOSACCHARIDE PROFILING

蛋白质鉴定和 N 连接寡糖分析

• 批准号: 8363075
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363075
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Algorithms; Blood capillaries; Carbohydrates; Color; Coomassie blue; Cysteine; Data; Digestion; Formic Acids; Freeze Drying; Funding; Gel; Glycopeptides; Grant; Heating; Ice; Incubated; Iodoacetamide; Ions; Link; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methionine; Methods; Modification; National Center for Research Resources; Nitrogen; Nomenclature; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Plant Resins; Polysaccharides; Preparation; Principal Investigator; Propanols; Proteins; Proteome; Research; Research Infrastructure; Resources; Salts; Sampling; Sep-Pak C18; Series; Site; Slice; Solutions; Source; Speed; Staining method; Stains; Syringes; Technology; Temperature; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; base; capillary; cost; ion source; ionization; mass spectrometer; oxidation; pressure; reconstitution; sodium phosphate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: In-gel digestion Coomassie blue-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 5 mM DTT in 40mM Ambic at 55¿ C for 1 hr. The DTT solution was exchanged with 15 mM 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 140 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 and the glycopeptides were extracted from the gel 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 LC-MS/MS The peptides were resuspended with 200 ¿L of mobile phase A (0.1% formic acid in water). The sample was then loaded onto a nanospray tapered capillary column/emitter (360x75x15 ¿m, PicoFrit, New Objective, Woburn, MA) self-packed with C18 reverse-phase resin (10.5 cm, Waters, Milford, MA) in a Nitrogen pressure bomb for 10 min at 1,000 psi (~5 uL load) and then separated via a 160 min linear gradient of increasing mobile phase B (80% acetonitrile in 0.1% formic acid) at a flow rate of~500 nL/min directly into the mass spectrometer. LC-MS/MS analysis was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. The resulting data were searched against the target portein sequence using the TurboSequest algorithm (Proteome Discoverer 1.1, Thermo Scientific). The SEQUEST parameters were set to allow 30.0 ppm of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Digested peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da and 15.9949 Da were allowed for alkylated cysteine and oxidation of methionines, respectively. Glycan preparation Extracted tryptic digest was passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, SDS, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100¿ C for 5 min to inactivate trypsin. The tryptic digest was incubated with PNGase F at 37¿ C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Released N-linked oligosaccharides were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Nanospray Ionization-Linear Ion Trap Mass Spectrometry Mass analysis by NSI-MS was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer using a nanospray source at a syringe flow rate of 0.5 ¿l/min. The capillary temperature was set to 210 ¿C, and MS analysis was performed in positive ion mode. Fragmentation by CID in MS/MS modes 50% collision energy was applied. The nomenclature of Domon and Costello (Domon and Costello, 1988) was used to guide the depiction of fragmentation derived from MS/MS spectra.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： 凝胶内消化 将考马斯亮蓝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，直至颜色变清。脱色凝胶在 40mM Ambic 中的 5 mM DTT 中于 55°C 重新溶胀 1 小时。将 DTT 溶液更换为 15 mM 碘乙酰胺 (IAM)，并在黑暗中孵育 45 分钟。 孵育后用 40mM AmBic 和 100% 乙腈交替洗涤两次。脱水凝胶最初用胰蛋白酶溶液（140 mM Ambic 中的胰蛋白酶）在冰上重新溶胀 45 分钟，并在 37° C 过夜进行蛋白质消化。将上清液转移至另一个管中。依次用20%乙腈的5%甲酸溶液、50%乙腈的5%甲酸溶液、然后80%乙腈的5%甲酸溶液从凝胶中提取肽和糖肽。将样品溶液干燥并合并到一根管中。 通过 LC-MS/MS 鉴定蛋白质 用 200 µL 流动相 A（0.1% 甲酸水溶液）重悬肽。然后将样品加载到纳喷雾锥形毛细管柱/发射器（360x75x15 µm，PicoFrit，New Objective，Woburn，MA）上，在氮气压力弹中以 C18 反相树脂（10.5 cm，Waters，Milford，MA）自装填，在 1,000 psi（约 5 uL 负载）下保持 10 分钟，然后通过增加流动性的 160 分钟线性梯度进行分离 B 相（0.1% 甲酸中的 80% 乙腈）以约 500 nL/min 的流速直接进入质谱仪。 LC-MS/MS 分析在配备纳喷雾离子源的 LTQ Orbitrap Discoverer 质谱仪 (Thermo Scientific) 上进行。使用 TurboSequest 算法（Proteome Discoverer 1.1，Thermo Scientific）针对目标蛋白序列搜索所得数据。 SEQUEST 参数设置为允许 30.0 ppm 的前体离子质量容差和 0.8 Da 的单同位素质量碎片离子容差。消化的肽允许有最多两个缺失的内部切割位点，并且分别允许烷基化半胱氨酸和蛋氨酸氧化的 57.02146 Da 和 15.9949 Da 的差异修饰。 聚糖制备 提取的胰蛋白酶消化物通过 C18 sep-pak 柱，并用 5% 乙酸洗涤以去除污染物（盐、SDS 等）。将肽和糖肽依次用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇洗脱，并在快速真空浓缩器中干燥。将干燥的样品合并，然后用 50 mM 磷酸钠缓冲液（pH 7.5）重新配制，并在 100℃ 下加热 5 分钟以灭活胰蛋白酶。将胰蛋白酶消化物与 PNGase F 在 37℃ 下孵育过夜以释放 N-聚糖。消化后，将样品通过 C18 sep-pak 柱，用 5% 乙酸洗脱碳水化合物级分并冻干。根据 Anumula 和 Taylor 的方法（Anumula 和 Taylor，1992）对释放的 N-连接寡糖进行全甲基化，并通过质谱分析进行分析。 纳喷雾电离-线性离子阱质谱 NSI-MS 质量分析在配备纳喷雾离子源的 LTQ Orbitrap Discoverer 质谱仪 (Thermo Scientific) 上进行。简而言之，将全甲基化聚糖溶解在含 50% 甲醇的 1 mM NaOH 中，并使用纳喷雾源以 0.5 l/min 的注射器流速直接注入线性离子阱质谱仪。毛细管温度设置为210℃，MS分析在正离子模式下进行。在 MS/MS 模式下通过 CID 进行断裂，应用 50% 碰撞能量。 Domon 和 Costello 的命名法（Domon 和 Costello，1988）用于指导从 MS/MS 谱图衍生的碎片描述。