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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： 凝胶消化 切成小块（〜1 mm3），用40毫米碳酸氢铵（Ambic）和100％乙腈切成薄片，直到颜色变透明。在40毫米Ambic的55°C中，DeStained Gel在5 mm DTT中持续1小时。将DTT溶液与15毫米碘乙酰胺（IAM）交换，并在黑暗中孵育45分钟。孵育之后，用40mm的Ambic和100％乙腈洗涤两次。脱水的凝胶用胰蛋白酶溶液（140毫米Ambic中的胰蛋白酶溶液）在冰上持续45分钟，然后在37°C下进行蛋白质消化过夜。将上清液转移到另一个管中。从5％甲酸中的5％甲酸，5％甲酸中的50％乙腈中从凝胶中提取肽和糖肽，然后在5％的甲酸中提取50％的乙腈，然后在5％的甲酸中提取80％乙腈。将样品溶液干燥并组合成一个管。 通过LC-MS/MS鉴定蛋白质 将肽重悬于200卢斯A期A（水中0.1％的正式酸）。然后将样品加载到纳米喷雾的锥形毛细管/发射极（360x75x15€m，picofrit，picofrit，新目标，沃本，马萨诸塞州，马萨诸塞州）中，在nitrogen压力板中以10分钟为单位的PSI（〜5 ul 5 ul）在10分钟的班级中，并分离10分钟的A型倒数（10.5 cm，Waters，Milford，MA），然后〜5 ul load）（〜5 ul） B期（在0.1％甲酸中的80％乙腈中的80％乙腈）以〜500 nl/min的流速直接进入质谱仪。 LC-MS/MS分析是在LTQ Orbitrap发现器质谱仪（Thermo Scientific）等效的纳米喷离源上进行的。使用Turbosequest算法（Proteome Discoverer 1.1，Thermo Scientific）搜索所得数据针对目标港口序列。将隔离参数设置为允许30.0 ppm的前体离子质量耐受性和0.8 da的片段离子耐受性，具有单异位素质量。允许使用多达两个丢失的内部切割部位的消化宠物，并且允许57.02146 DA和15.9949 DA的差异修饰分别用于烷基化半胱氨酸和甲基氨酸的氧化。 聚糖准备 提取的胰蛋白酶消化物通过C18 sep-pak墨盒，并用5％乙酸洗涤以去除污染物（盐，SDS等）。在5％乙酸中以20％ISO丙醇为序列的肽和糖磷酸，在5％乙酸中40％ISO-丙醇和100％ISO丙醇，并在速度真空浓缩剂中干燥。将干燥的样品合并，然后用50 mM磷酸钠缓冲液（pH 7.5）重构，并在100°C加热5分钟以灭活胰蛋白酶。将胰蛋白酶消化物与37 c的PNGASE F一起孵育，以释放N-聚糖。消化后，样品通过C18 sep-pak弹药筒，用5％乙酸洗脱碳水化合物馏分，并通过冻干干燥。基于Anumula和Taylor的方法（Anumula and Taylor，1992）释放的N连接寡糖被氯化，并通过质谱法进行了介绍。 纳米喷雾电离线性离子陷阱质谱法 NSI-MS的质量分析是在LTQ Orbitrap发现器质谱仪（Thermo Scientific）等效的纳米喷离源上进行的。简而言之，将苄苄化聚糖溶解在1mM NaOH中，在50％的甲基戊二醇中，并使用纳米喷雾器直接以0.5 l/min的注射剂流速为单位源源质谱仪。毛细管温度设置为210€，MS分析以正离子模式进行。使用CID在MS/MS模式下通过50％碰撞能量进行碎片化。 Domon和Costello的命名法（Domon and Costello，1988）用于指导从MS/MS Spectra得出的片段化的描述。