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 将富含蛋白质的粉末用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，将50 μ L胰蛋白酶（2 mg/mL）和50 μ L胰凝乳蛋白酶（2 mg/mL）加入试管中，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旋转消化物15 min。使上清液通过C18 sep pak柱。 随后向沉淀物中加入400 μ L H2O，如前所述涡旋并离心，将上清液上样至相同的C18 sep pak柱中。 吸附的肽和糖肽用8 mL 5%乙酸洗涤，并用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。 最初在N2下干燥滤液，并冻干剩余的溶液。 用30 μ L H2O和20 μ L 0.1 M磷酸钠缓冲液（pH 7.5）重新溶解干燥的甘露聚糖，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，使消化物通过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-连接聚糖级分，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。 释放的N-聚糖的葡萄糖苷酶消化 将冻干释放的N-聚糖用200 μ L NaPO 4缓冲液（pH~6.8）溶解，并用50 μ L α-葡糖苷酶（B.嗜热脂肪菌; 300单位/mL，在10 mM NaPO 4缓冲液中）。 在37 ℃消化48小时后，再次向混合物中加入50 μ L α-葡糖苷酶（同上），并在相同条件下再孵育48小时。 随后终止酶消化并将混合物冻干。 碳水化合物的全O-甲基化和C18 sep-pak柱的纯化 对酶消化物进行全甲基化，以进行N-连接聚糖分析（Ciucanu和Kerek，1984）。 将干燥的消化物溶于二甲亚砜中，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。 通过电喷雾电离-质谱法（ESI-MS）分析寡糖 本实验的目的是确定葡糖苷酶消化后聚糖、6种己糖和2种HexNAc（在先前的报告中建议为GlcNAc 2 Man 6（m/z 1784））的去向。 将干燥的全-O-甲基化聚糖溶于20 μ L甲醇中，并与30 μ L 1 mM NaOH的50%甲醇溶液混合。 通过泵（哈佛装置22）将混合物直接注入LCQ Advantage（Thermo-Finnigan）仪器中。通过扫描2 min获得聚糖的完整质谱（m/z 200至2000），并对所选离子进行碎片化（MS/MS）分析，以确认是否存在已鉴别的聚糖。 对于MS/MS分析，以2.2的隔离宽度和35%的碰撞能量扫描所选择的离子。

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 protein-rich powder was dissolved with 200 ¿L protease buffer (0.1 M Tris-HCl pH 8.2 containing 0.01 M CaCl2) and the tube was 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 tube and incubated at 370C for about 21 h. Trypsin and chymotrypsin were deactivated by heating the tube at 100oC for 5 min and digest was spun at 3000 rpm under room temperature for 15 min. The supernatant was passed through a C18 sep pak cartridge. The pellet was added subsequently with 400 ¿L of H2O, vortexed and spun as described previously and the supernatant was loaded into the same C18 sep pak cartridge. The adsorbed peptides and glycopeptides were washed with 8 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 eluate was dried initially under N2 and the remaining solution was lyophilized. The dried eluate was 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 digest was passed through a C18 sep pak cartridge to separate the carbohydrate fraction and the peptide-containing fraction. The N-linked glycans fraction was 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). Glucosidase digestion of released N-glycans The lyophilized released N-glycan was dissolved with 200 ¿L of NaPO4 buffer (pH~6.8) and was treated with 50 ¿L of alpha-glucosidase (B. stearothermophilus; 300 units/mL in 10 mM NaPO4 buffer). After 48 h of digestion at 37oC, the mixture was added again with 50 ¿L of alpha-glucosidase (same as above) and incubated under the same condition for another 48 h. Enzymatic digestion was terminated subsequently and the mixture was lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The enzymatic digest was permethylated for N-linked glycan profiling (Ciucanu and Kerek, 1984). The dried digest was 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 ElectroSpray Ionization-Mass Spectrometry (ESI-MS) The objective of the current experiment was to determine the fate of the glycan, 6 hexoses and 2 HexNAcs proposed as GlcNAc2Man6 (m/z 1784) in the previous report after glucosidase digestion. The dried per-O-methylated glycans were dissolved in 20 ¿L methanol and mixed with 30 ¿L of 1 mM NaOH in 50% methanol. The mixture was infused directly by a pump (Harvard Apparatus 22) into an LCQ Advantage (Thermo-Finnigan) instrument. Full mass spectrum of glycans, m/z 200 to 2000 was obtained by scanning for 2 min and fragmentation (MS/MS) analysis of the selected ion was undertaken to confirm the presence of an identified glycan. For MS/MS analysis, the selected ion was scanned with an isolation width of 2.2 and at 35% collision energy.