N-LINKED GLYCOSYLATION SITE MAPPING

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： ~(180)O标记和LC-MS/MS定位N-糖基化位点 40微克NG用25 mM DTT在55℃下还原1h，并在黑暗中用90 mM碘乙酰胺甲基化45min。干燥的透析样品在50 mM碳酸氢铵(NH4HCO3)中重新悬浮，在25℃下用2g胰酶消化20h，然后在100℃下胰酶失活5min，然后用2g PNGaseF在36℃的18O水(H_218O)和2M的1M NH_4HCO_3中脱糖。 标记的多肽用200°L流动相A(0.1%甲酸水)重新悬浮。然后将样品以1,000磅/平方英寸(~5微升负载)的氮气压力炸弹自填充于纳米锥形毛细管柱/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)自填充5分钟，然后通过160 min的线性梯度增加流动相B，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)对照重组NG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。胰蛋白酶多肽最多可以有两个缺失的内部切割位点，烷基半胱氨酸、蛋氨酸氧化和18O标记的天冬氨酸分别允许57.02146 Da、15.9949 Da和2.98826 Da的差异修饰。

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: N-Glycosylation Site Mapping With 18O-labeling and LC-MS/MS Forty micrograms of NG was reduced with 25 mM DTT for 1 h at 55 ¿C and carboxyamidomethylated with 90mM iodoacetamide in the dark for 45 min. The dried dialyzed sample was resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 2 ¿g of trypsin at 25 ¿C for 20 h. Following deactivation of trypsin at 100 ¿C for 5 min, the sample was then deglycosylated with 2 ¿g of PNGaseF in 36 ¿L of 18O Water (H218O) and 2 ¿L of 1M NH4HCO3. The labeled 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 5 min at 1,000 psi (~5 uL load) and then separated via a 160 min linear gradient of increasing mobile phase B 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 recombinant NG 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. Tryptic peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da, 15.9949 Da and 2.98826 Da were allowed for alkylated cysteine, oxidation of methionines and 18O-labeled aspartic acid, respectively.