PROTEIN IDENTIFICATION AND N-LINKED OLIGOSACCHARIDE PROFILING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 凝胶内消化 考马斯蓝染色的凝胶切片切成小块(~1mm3)，用40 mM碳酸氢铵(AmBic)和100%乙腈交替脱色，直到颜色变得透明。去掉的凝胶在5 mM DTT中55℃下于40 mM Ambic中重新溶胀1小时。DTT溶液与15 mM碘乙酰胺(IAM)交换，在黑暗中孵育45min。孵育后用40 mM AmBic和100%乙腈交替洗涤2次。首先用胰酶溶液(140 mM Ambic中的胰酶)在冰上复温45min，然后在37℃下进行蛋白质消化过夜。将上清液转移到另一试管中。用20%乙腈-5%甲酸、50%乙腈-5%甲酸、80%乙腈-5%甲酸串联提取多肽和糖肽。将样品溶液干燥并合并到一个试管中。 LC-MS/MS法鉴定蛋白质 多肽用200°L流动相A(0.1%甲酸水)悬浮液。然后将样品加载到纳米锥形毛细管柱/发射器(360×75×15？m，PicoFrit，New Objective，Woburn，MA)上，用C18反相树脂(10.5 cm，Waters，Milford，MA)在氮气压力炸弹中以1,000 psi(~5 ul负载)自填充10分钟，然后通过160 min的线性梯度增加流动相B(80%乙腈在0.1%甲酸中)以~500 nL/min的流速直接进入质谱仪。 LC-MS/MS分析在装有纳米级离子源的LTQ Orbitrap Discoverer质谱仪(Thermo Science)上进行。使用TurboSequest算法(蛋白质组发现者1.1，Thermo Science)对照目标报告基因序列搜索得到的数据。SEQUEST参数被设置为允许30.0ppm的前体离子质量容差和0.8Da的碎片离子容差与单同位素质量。被消化的多肽最多可以有两个缺失的内部切割位点，对于半胱氨酸的烷基化和蛋氨酸的氧化，分别允许57.02146 Da和15.9949 Da的差异修饰。 葡聚糖制剂 提取的胰酶消化液通过C18Sep-pak小柱，并用5%的冰醋酸洗涤以去除污染物(盐、十二烷基硫酸钠等)。多肽和糖肽用20%异丙醇5%冰醋酸、40%异丙醇5%冰醋酸和100%异丙醇串联洗脱，在高速真空浓缩器中干燥。将干燥的样品合并，然后用50 mM的磷酸二氢钠缓冲液(pH 7.5)重置，在100℃加热5min以灭活胰酶。胰酶消化液与PNGase F在37℃下孵育一夜，以释放N-糖链。消化后，样品通过C18Sep-pak色谱柱，碳水化合物部分用5%的冰醋酸洗脱并冷冻干燥。根据Anuula和Taylor(Anuula and Taylor，1992)的方法对释放的N-连接低聚糖进行全甲基化，并用质谱仪进行分析。 纳米喷雾电离-线性离子陷阱质谱学 用NSI-MS在装有纳米喷雾离子源的LTQ Orbitrap Discoverer质谱仪(Thermo Science)上进行了质量分析。简单地说，将全甲基化的葡聚糖溶解在1 mM的氢氧化钠中50%的甲醇中，并使用纳米喷射源以0.5°L/分钟的注射器流速将其直接注入线性离子陷阱质谱仪。毛细管温度210℃，正离子模式进行MS分析。在MS/MS模式下，使用50%碰撞能量进行CID碎裂。Domon和Costello(Domon and Costello，1988)的命名被用来指导从MS/MS谱中获得的碎裂的描述。

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.