OLIGOSACCHARIDE PROFILING OF JURKAT CELLS AND LYSATES

Jurkat 细胞和裂解物的寡糖分析

• 批准号: 7722695
• 负责人: Parastoo Azadi
• 金额: $ 0.02万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-08 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722695
• 关键词: Acetic Acid; Acetic Acids; Acetonitriles; Buffers; Carbohydrates; Cells; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Dryness; Electrospray Ionization; Endopeptidases; Fourier Transform; Funding; Gases; Glycopeptides; Grant; Heating; Incubated; Institution; Ions; Isopropanol; Jurkat Cells; Link; Maps; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; Nitrogen; Oligosaccharides; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Polysaccharides; Proteins; Range; Reaction; Research; Research Personnel; Resources; Sampling; Scanning; Sep-Pak C18; Series; Source; Spectrometry, Mass, Electrospray Ionization; Stream; Temperature; Trypsin; Tube; United States National Institutes of Health; Water; chymotrypsin; instrument; methyl iodide; sodium phosphate

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 and lysates 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. Both tryptic and chymotryptic digests, supernatant (from cells) and lysates were 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 then lyophilized to dryness. 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 1:1 methanol:water 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-Linear Ion Trap-Fourier Transform Mass Spectrometry (ESI-LTQ-FT MS) The released N-linked oligosaccharides from the cells and lysates were profiled by ESI-LTQ-FT MS. 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 28 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.

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。子项目和 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 将干燥的细胞沉淀和裂解物用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，向每个试管中加入25 µ L（胰蛋白酶2 mg/mL）和25 µ L胰凝乳蛋白酶（2 mg/mL），并在370 C下孵育18小时。 胰蛋白酶和胰凝乳蛋白酶消化酶在3000 rpm、40 ℃下离心15 min，将上清液转移到另一个试管中。 随后向沉淀物中加入200 µ L H2O，如前所述涡旋并离心，将上清液收集至其各自的样品管中。 将胰蛋白酶和胰凝乳蛋白酶消化物、上清液（来自细胞）和裂解物冻干。 用200 μ L 5%乙酸溶解干燥的沉淀物，并使其通过C18 sep pak柱以除去污染物。 用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇连续洗脱糖肽。 洗脱液在N2流下干燥，用25 μ L H2O和20 μ L 0.1 M磷酸钠缓冲液（pH 7.5）重新溶解，用5 μ L PNGase F处理，并在37 ℃下孵育18 h。 在第二次酶消化（PNGase F）后，使混合物通过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，然后用100%异丙醇洗脱O-糖肽和肽级分。 然后将两个级分冻干至干。 碳水化合物的全O-甲基化和C18 sep-pak柱的纯化 将N-和O-连接聚糖全甲基化以进行低聚糖分析（Ciucanu和Kerek，1984年）。 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。 简而言之，将聚糖溶解于1：1甲醇：水中，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。 电喷雾电离-线性离子阱-傅里叶变换质谱法（ESI-LTQ-FT MS）分析寡糖 通过ESI-LTQ-FT MS分析从细胞和裂解物中释放的N-连接寡糖。简言之，将全甲基化聚糖溶解于含ImM NaOH的50%甲醇中，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28碰撞能量下）、m/z范围为500至2000。