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 干燥后的细胞球用200°L蛋白酶缓冲液(0.1MTris-HCl，pH 8.2，含0.01MCaCl2)溶解，将试管放入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。每个上清液都通过C18Sep Pak色谱柱。随后加入400%L的水，如前所述旋转和旋转，并将上清液装入各自的C189月白纸盒中。吸附的多肽和糖肽用9mL5%冰醋酸洗脱，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。洗脱液在氮气中干燥，其余溶液进行冷冻干燥。 干燥的洗脱液用30°L的水和20°L的0.1M磷酸二氢钠缓冲液(pH 7.5)重溶，用PNGase F处理，在370℃下孵育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-糖肽和多肽组分。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。 碳水化合物的Per-O-甲基化及C18Sep-Pak柱纯化 N-连接的多糖被全甲基化以用于寡糖图谱(Ciucanu和Kerek，1984)。将干燥的洗脱液溶解于二甲基亚砜中，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。 基质辅助激光解吸飞行时间质谱法分析低聚糖 最初使用MALDI/TOF-MS对N-连接的糖链进行分析。所使用的机器是4700蛋白质组学分析仪(应用生物系统)，它被设置在反射器正离子模式。以β-二羟基苯甲酸(DHBA，20 mg/mL溶液，50%甲醇：水)为基质，在MALDI板上结晶全甲基化葡聚糖。 线性离子陷阱-傅里叶变换离子回旋共振(FTICR)质谱仪(LTQ-FT，Thermo Science)分析低聚糖 MALDI-TOF MS检测到的N-连接低聚糖经LTQ-FT-MS确证，采用Atwood等人的方法进行质谱分析。(2007)。简而言之，将过甲基化的多糖溶解在1 mM氢氧化钠中50%的甲醇中，并直接注入仪器中。在与前一个窗口重叠2个质量单位的连续2.8个质量单位窗口中扫描总离子图谱、自动MS/MS分析(在29%碰撞能量下)、m/z范围从500到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.