N-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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10 mM DTT中55℃下于40 mM Ambic中重新溶胀1小时。DTT溶液与55 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(40 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。 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。 基质辅助激光解吸电离飞行时间质谱仪(MALDI/TOF-MS) MALDI/TOF-MS色谱柱为反射镜正离子模式，以二羟基苯甲酸(DHBA，20 mg/mL甲醇：水)为基质。使用4700蛋白质组学分析仪(应用生物系统)获得所有光谱。 纳米喷雾电离-线性离子陷阱质谱(LTQ) 按照复合碳水化合物研究中心(青木K，珀尔曼M，LIM JM，坎图R，威尔斯L，蒂迈耶M.J生物化学)开发的方法进行质谱分析。2007年3月23日；282(12)：9127-42。采用NSI-LTQ/MSN进行质量分析。简而言之，将过甲基化的葡聚糖溶于1 mM氢氧化钠中50%的甲醇中，并以0.4？L/分钟的恒定流速直接进入仪器(LTQ，Thermo Finnigan)。在正离子模式下进行MS分析。 对于总离子图谱、自动化的MS/MS分析(碰撞能量为35)，在连续的2.8个质量单位窗口中扫描从500到2000的m/z范围，这些窗口与前一个窗口重叠2个质量单位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 10 mM DTT in 40mM Ambic at 55¿ C for 1 hr. The DTT solution was exchanged with 55mM 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 40 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. 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. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) MALDI/TOF-MS was performed in the reflector positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol:water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (LTQ) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42.). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ,Thermo Finnigan) at a constant flow rate of 0.4 ¿L/min. The MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis (at 35 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.