OLIGOSACCHARIDE PROFILING OF MEMBRANE PROTEIN FROM TETRAHYMENA THERMOPHILA

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。子项目及 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。 N-连接聚糖的释放 将干燥的细胞沉淀用200 μ L蛋白酶缓冲液（含有0.01M CaCl 2的0.1M Tris-HCl pH 8.2）溶解，并将管置于加热块中（100 ℃，5分钟）以使蛋白质变性。 冷却至室温后，向试管中加入50 μ L胰蛋白酶（2 mg/mL）和50 μ L胰凝乳蛋白酶（2 mg/mL），并在37 ℃下孵育约21小时。 通过在100 ℃下加热试管5 min使胰蛋白酶和胰凝乳蛋白酶失活，并在室温下以3000 rpm旋转离心15 min。使各上清液通过C18 sep pak柱。 随后向沉淀中加入400 µ L H2O，如前所述涡旋并离心，将上清液上样至相应的C18 sep pak柱中。 用9 mL 5%乙酸洗涤吸附的肽和糖肽，并用5%乙酸中的20%异丙醇、5%乙酸中的40%异丙醇和100%异丙醇连续洗脱。 洗脱液最初在N2下干燥，剩余溶液冻干。 用30 μ L H2O和20 μ L 0.1 M磷酸钠缓冲液（pH 7.5）重新溶解干燥的洗脱液，用PNGase F处理，并在370 ℃下孵育18 h以释放N-连接聚糖。 在第二次酶消化（PNGase F）后，使酶通过C18 sep pak柱以分离碳水化合物级分和含肽级分。 首先用5%乙酸洗脱N-连接聚糖，然后用100%异丙醇洗脱O-糖肽和肽级分。 通过冻干（N-聚糖）或在氮气流下（O-糖肽和肽）干燥两种级分。 碳水化合物的全O-甲基化和C18 sep-pak柱的纯化 对N-连接聚糖进行全甲基化，以进行寡糖分析（Ciucanu和Kerek，1984）。 将干燥的洗脱液溶于二甲亚砜中，然后用NaOH和碘甲烷甲基化。 通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。 进一步清除全-0-甲基化聚糖的污染物。 简言之，将聚糖溶解于甲醇：水（1：1）中并加载到C18 sep pak柱中，然后用纳米纯水洗涤。 用15%乙腈将全-O-甲基碳水化合物洗脱到螺旋盖管中，并用85%乙腈洗脱到另一个螺旋盖管中。 将用85%乙腈洗脱的聚糖在氮气流下干燥，并用甲醇溶解，用于质谱分析。 基质辅助激光解吸飞行时间质谱（MALDI-TOF）分析寡糖 最初使用MALDI/TOF-MS进行N-连接聚糖的分析。使用的机器是4700蛋白质组学分析仪（Applied Biosystems），其设置在反射器正离子模式。在MALDI板上以？-二羟基苯甲酸（DHBA，20 mg/mL溶液，溶于50%甲醇：水）作为基质结晶全甲基化聚糖。 通过线性离子阱-傅里叶变换离子回旋共振（FTICR）质谱仪（LTQ-FT，Thermo Scientific）分析寡糖 通过MALDI-TOF MS检测到的N-连接寡糖通过LTQ-FT MS确认。 按照Atwood等人（2007）开发的方法进行质谱分析。 简言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并直接注入仪器中。 在与前一窗口重叠2个质量单位的连续2.8质量单位窗口中扫描总离子图、自动MS/MS分析（在29%碰撞能量下）、m/z范围为500至2000。

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. Release of N-linked glycans The dried cell pellets were dissolved with 200 ¿L protease buffer (0.1 M Tris-HCl pH 8.2 containing 0.01 M CaCl2) and the tubes were placed in a heating block (1000C, 5 min) to denature the protein. After cooling to room temperature, 50 ¿L trypsin (2 mg/mL) and 50 ¿L chymotrypsin (2 mg/mL) were added to the tubes and incubated at 370C for about 21 h. Trypsin and chymotrypsin were deactivated by heating the tubes at 100oC for 5 min and digests were spun at 3000 rpm at room temperature for 15 min. Each of the supernatants was passed through a C18 sep pak cartridge. The pellets were added subsequently with 400 ¿L of H2O, vortexed and spun as described previously and the supernatants were loaded into the respective C18 sep pak cartridges. The adsorbed peptides and glycopeptides were washed with 9 mL of 5% acetic acid and eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The eluates were dried initially under N2 and the remaining solutions were lyophilized. The dried eluates were redissolved with 30 ¿L of H2O and 20 ¿L of 0.1 M sodium phosphate buffer (pH 7.5), treated with PNGase F, and incubated at 370C for 18 h to release the N-linked glycans. After the second enzymatic digestion (PNGase F), the digests were passed through a C18 sep pak cartridge to separate the carbohydrate fraction and the peptide-containing fraction. The N-linked glycans were eluted first with 5% acetic acid followed by the elution of O-glycopeptide and peptide fraction with 100% isopropanol. Both fractions were dried either by lyophilization (N-glycan) or under a stream of nitrogen gas (O-glycopeptide and peptide). Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The N-linked glycans were permethylated for oligosaccharide profiling (Ciucanu and Kerek, 1984). The dried eluates were dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were dissolved in methanol:water (1:1) and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. Oligosaccharide Profiling by Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) Profiling of N-linked glycans was performed initially using MALDI/TOF-MS. The machine used was a 4700 Proteomics analyzer (Applied Biosystems), which was set in the reflector positive ion mode. Permethylated glycans were crystallized on a MALDI plate with ¿-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol:water) as a matrix. Oligosaccharide Profiling by Linear Ion Trap-Fourier transform Ion Cyclotron Resonance (FTICR) Mass Spectrometer (LTQ-FT, Thermo Scientific) The N-linked oligosaccharides detected by MALDI-TOF MS were confirmed by LTQ-FT MS. Mass spectrometric analysis was performed following the method developed by Atwood et al. (2007). Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into the instrument. Total ion mapping, automated MS/MS analysis (at 29% collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit window that overlapped the preceding window by 2 mass units.