OLIGOSACCHARIDE PROFILING OF MEMBRANE PROTEIN FROM TETRAHYMENA THERMOPHILA

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。 N-连接葡聚糖的释放 富含蛋白质的粉末用200°L蛋白酶缓冲液(0.1MTris-HCl，pH 8.2，含0.01MCaCl2)溶解，将试管置于1000℃，5min的加热块中变性。冷却至室温后，向试管中加入L胰酶(2 mg/mL)和L胰凝乳酶(2 mg/mL)各50份，在370℃孵育约21h，在100oC加热5min使胰酶和胰凝乳酶失活，在室温下3000rpm旋转15min消化。上清液通过C18 Pep Pak色谱柱。随后将颗粒与400°L的H2O一起加入，如前所述旋转和旋转，并将上清液装入相同的C18 9月白纸盒中。吸附的多肽和糖肽用5%的冰醋酸洗脱，用20%的异丙醇-5%的冰醋酸洗脱，40%的异丙醇-5%的冰醋酸洗脱，100%的异丙醇洗脱。洗脱液在氮气中干燥，其余溶液冷冻干燥。 干燥洗脱液用30°L水和20°L 0.1M磷酸二氢钠缓冲液(pH 7.5)重溶，用PNGase F处理，在370℃孵育18h，释放N-连接的多糖。在第二次酶消化(PNGase F)后，通过C18Sep pak小柱分离碳水化合物部分和含有多肽的部分。N-糖链部分首先用5%的冰醋酸洗脱，然后用100%的异丙醇洗脱O-糖肽和多肽部分。这两个组分都通过冷冻干燥(N-多糖)或在氮气气流(O-糖肽和多肽)下干燥。 葡萄糖苷酶对释放的N-糖链的消化 冻干后释放的N-葡聚糖用200°L的磷酸二氢钠缓冲液(pH~6.8)溶解，然后用50°L的α-葡萄糖苷酶(嗜热脂肪芽孢杆菌；300单位/毫升在10 mM磷酸二氢钠缓冲液中)处理。在37oC下消化48h后，再加入50°L的α-葡萄糖苷酶(上同)，在相同条件下再孵育48h，终止酶消化，将混合物冷冻干燥。 碳水化合物的Per-O-甲基化及C18Sep-Pak柱纯化 酶消化液被全甲基化以进行N-连接的糖链分析(Ciucanu和Kerek，1984)。将干燥的消化液溶解在二甲基亚砜中，然后用氢氧化钠和碘甲烷甲基化。加水使反应熄灭，用二氯甲烷提取全氧甲基化碳水化合物。进一步清除了Per-O-甲基化多糖中的污染物。简而言之，将多糖溶解在甲醇：水(1：1)中，装入C18Sep pak色谱柱，然后用纳米纯水洗涤。对-O-甲基碳水化合物用15%的乙腈洗脱到螺帽管中，用85%的乙腈洗脱到另一个螺帽管中。用85%的乙腈洗脱后，在氮气中干燥，用甲醇溶解，进行质谱分析。 电喷雾电离-质谱法(ESI-MS)分析寡糖 本实验的目的是确定先前报告中提出的GlcNAc2Man6(m/z 1784)的糖链、6个己糖和2个HexNAcs在葡萄糖苷酶消化后的命运。将干燥的Per-O-甲基化多糖溶解在20°L甲醇中，并与30°L的1 mM氢氧化钠混合在50%的甲醇中。混合物由泵(哈佛仪器22)直接注入到LCQ Advantage(Thermo-Finnigan)仪器中。通过扫描2分钟获得m/z 200至2000的全质量谱，并对所选离子进行裂解(MS/MS)分析以确认所鉴定的多糖的存在。对于MS/MS分析，选择的离子被扫描，隔离宽度为2.2，碰撞能量为35%。

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.