OLIGOSACCHARIDE PROFILING OF JURKAT CELL PELLETS

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，每管加入L胰酶2 mg/m L和L胰凝乳酶2 mg/m L，在370℃孵育18h，以3000转/分，40℃旋转15 m in，上清液转入另一管。随后将颗粒与200°L的水一起加入，如前所述旋转和旋转，上清液收集到各自的样品管中并冷冻干燥。 干燥后的消化液用200g/L的5%醋酸L溶解，通过C18Sep Pak色谱柱去除污染物。糖肽用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，用25°L水和20°L 0.1M磷酸二钠缓冲液(pH 7.5)重溶，用PNGase F的5°L处理，在370℃下孵育18h，经第二次酶切(PNGase F)后，通过C18Sep pak柱分离糖组分和含肽组份。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。 通过-消除释放O-连接的葡聚糖 通过消除程序将O-连接的糖链从糖肽上切割出来。简单地说，向每个样品中加入250°L的50 mM氢氧化钠，然后检查pH值。在确定pH为碱性后，再向样品中加入250°L的50 mM氢氧化钠，其中含有19 mg的硼氢化钠，旋转，并在450℃下孵育一夜。孵化后的样品用10%的冰醋酸中和，通过填充的Dowex树脂柱脱盐，然后冷冻干燥。干燥样品用甲醇：冰醋酸(9：1)在氮气中清洗。样品通过C18反相柱收集裂解的O-葡聚糖(用5%的冰醋酸洗脱)。O-葡聚糖组分在氮气中干燥。 碳水化合物的Per-O-甲基化及C18Sep-Pak柱纯化 N-和O-连接的多糖被全甲基化以进行寡糖图谱分析(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-连接和O-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。 低聚糖的纳米喷雾电离-线性离子俘获质谱学研究 用MALDI-TOF MS检测到N-连接的低聚糖，用ESI-LTQ质谱仪进行确证。按照复合碳水化合物研究中心(青木K，珀尔曼M，LIM JM，坎图R，威尔斯L，蒂迈耶M.J生物化学)开发的方法进行质谱分析。2007年3月23日；282(12)：9127-42。EPub 2007年1月29日)。采用NSI-LTQ/MSN进行质量分析。简而言之，将过甲基化的葡聚糖溶于1 mM氢氧化钠中50%的甲醇中，并以0.4？L/分钟的恒定流速直接进入仪器(LTQ，Thermo Finnigan)。毛细管温度210℃，正离子模式进行MS分析。MS/MS裂解的碰撞能设为28。对于总离子图谱，自动MS/MS分析(碰撞能量为28)，在连续的2.8质量单位窗口中扫描从500到2000的m/z范围，该窗口与前一个窗口重叠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. 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, 25 ¿L trypsin (2 mg/mL) and 25 ¿L chymotrypsin (2 mg/mL) were added to each tube and incubated at 370C for 18 h. The tryptic and chymotryptic digests were spun at 3000 rpm, 40C for 15 min and the supernatants were transferred into another tube. The pellets were added subsequently with 200 ¿L of H2O, vortexed and spun as described previously and the supernatants were collected into their respective sample tubes and lyophilized. The dried digests were dissolved with 200 ¿L of 5% acetic acid and passed through C18 sep pak cartridge to remove contaminants. The glycopeptides were eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The eluates were dried under a stream of N2, redissolved with 25 ¿L of H2O and 20 ¿L of 0.1 M sodium phosphate buffer (pH 7.5), treated with 5 ¿L of PNGase F, and incubated at 370C for 18 h. After the second enzymatic digestion (PNGase F), the mixture was 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). Release of O-linked glycans by ¿-elimination The O-linked glycans were cleaved from the glycopeptide by ¿-elimination procedures. Briefly, 250 ¿L of 50 mM NaOH were added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid, desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. The samples were passed through C18 reversed phase cartridge to collect the cleaved O-glycans (eluted with 5% acetic acid). The O-glycan fraction was dried under a stream of nitrogen. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The N-and O-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 and O-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 NanoSpray ionization-Linear Ion Trap Mass Spectrometry (LTQ) The N-linked oligosaccharides detected by MALDI-TOF MS was confirmed by ESI-LTQ mass spectrometry. 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. Epub 2007 Jan 29). 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 capillary temperature was set at 210o C and MS analysis was performed in the positive ion mode. The collision energy was set at 28 for MS/MS fragmentation. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit window that overlapped the preceeding window by 2 mass units.