PROTEIN IDENTIFICATION AND N-LINKED OLIGOSACCHARIDE PROFILING

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 凝胶内消化 将考马斯亮蓝染色的凝胶切片切成更小的片（约1 mm 3），并用40 mM碳酸氢铵（AmBic）和100%乙腈交替脱色，直至颜色变澄清。将脱色的凝胶在55 ℃下在40 mM Ambic中的5 mM DTT中再溶胀1小时。将DTT溶液与15 mM碘乙酰胺（IAM）交换，并在黑暗中孵育45分钟。孵育后用40 mM AmBic和100%乙腈交替洗涤两次。脱水凝胶最初在冰上用胰蛋白酶溶液（140 mM Ambic中的胰蛋白酶）再溶胀45分钟，并在37 ℃下进行蛋白质消化过夜。将上清液转移至另一管中。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%甲酸溶液从凝胶中连续提取肽和糖肽。将样品溶液干燥并合并到一个试管中。 蛋白质鉴别（LC-MS/MS） 用200 μ L移动的A相（0.1%甲酸水溶液）重悬肽。然后将样品加载到纳米喷雾锥形毛细管柱/发射器上。（360 x75 x15英寸）m，PicoFrit，New Objective，Woburn，MA）用C18反相树脂自填充（10.5 cm，沃茨，米尔福德，MA）在氮气压力瓶中于1，000 psi（约5 uL上样量），然后通过160 min线性梯度递增的移动的相B（80%乙腈的0.1%甲酸溶液）以约500 nL/min的流速直接分离至质谱仪中。 在配备有纳米喷雾离子源的LTQ Orbitrap Discoverer质谱仪（Thermo Scientific）上进行LC-MS/MS分析。使用TurboSequest算法（Proteome Discoverer 1.1，Thermo Scientific）针对靶转运蛋白序列搜索所得数据。SEQUEST参数设定为允许单一同位素质量的30.0 ppm前体离子质量容限和0.8 Da碎片离子容限。允许消化的肽具有多达两个缺失的内部切割位点，并且允许分别用于烷基化半胱氨酸和甲硫氨酸氧化的57.02146 Da和15.9949 Da的差异修饰。 聚糖制剂 将提取的胰蛋白酶消化物通过C18 sep-pak柱，并用5%乙酸洗涤以除去污染物（盐、SDS等）。用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇连续洗脱肽和糖肽，并在高速真空浓缩器中干燥。合并干燥的样品，然后用50 mM磷酸钠缓冲液（pH 7.5）复溶，并在100 ℃下加热5 min至胰蛋白酶。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，使样品通过C18 sep-pak柱，用5%乙酸洗脱碳水化合物级分，并通过冻干干燥。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。 纳米喷雾电离-线性离子阱质谱 在配备有纳米喷雾离子源的LTQ Orbitrap Discoverer质谱仪（Thermo Scientific）上通过NSI-MS进行质量分析。简而言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并使用纳米喷雾源以0.5 L/min的注射器流速直接注入线性离子阱质谱仪中。毛细管温度设定为210 C，并在正离子模式下进行MS分析。在MS/MS模式中通过CID进行裂解，施加50%碰撞能量。使用Domon和Costello的命名法（Domon和Costello，1988）指导MS/MS谱图中碎片的描述。

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.