N-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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10 mM DTT中再溶胀1小时。将DTT溶液与55 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（40 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。 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。 基质辅助激光解吸电离飞行时间质谱 采用反射器正离子模式，以20 mg/mL的DHBA（50%甲醇：水）为基质，进行MALDI/TOF-MS。 通过使用4700蛋白质组学分析仪（Applied Biosystems）获得所有光谱。 NanoSpray电离-线性离子阱质谱（LTQ） 按照复杂碳水化合物研究中心开发的方法进行质谱分析（Aoki K，Perlman M，Lim JM，Cantu R，威尔斯L，Tiemeyer M. 2007年3月23日;282（12）：9127-42.）。 通过使用NSI-LTQ/MSn确定质量分析。 简而言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并以0.4 μ L/min的恒定流速直接注入仪器（LTQ，Thermo Finnigan）中。以正离子模式进行MS分析。 对于总离子图，自动MS/MS分析（在35碰撞能量下），在连续的2.8质量单位窗口中扫描500至2000的m/z范围，该窗口与前一窗口重叠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.