OLIGOSACCHARIDE PROFILING OF JURKAT CELL PELLETS

JURKAT 细胞沉淀的低聚糖分析

• 批准号: 7722682
• 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/7722682
• 关键词: Acetic Acid; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Buffers; Carbohydrates; Cells; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Endopeptidases; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Incubated; Institution; Ions; Isopropanol; Jurkat Cells; Lasers; Link; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylation; Methylene Chloride; Nitrogen; Oligosaccharides; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Planet Mars; Plant Resins; Polysaccharides; Procedures; Proteins; Proteomics; Range; Rate; Reaction; Research; Research Personnel; Resources; Sampling; Scanning; Sep-Pak C18; Series; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; capillary; chymotrypsin; instrument; ionization; methyl iodide; sodium borohydride; 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 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.

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25 μ L胰蛋白酶（2 mg/mL）和25 μ L胰凝乳蛋白酶（2 mg/mL）加入每个试管中，并在37 ℃下孵育18 h。 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，将上清液收集到各自的样品管中并冻干。 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。 通过冻干（N-聚糖）或在氮气流下（O-糖肽和肽）干燥两种级分。 通过消除释放O-连接聚糖 O-连接聚糖通过消除程序从糖肽上裂解。 简而言之，250？向每个样品中加入250 μ L 50 mM NaOH，然后检查pH值。确定pH值为碱性后，向样品中加入另外250 μ L含有19 mg硼氢化钠的50 mM NaOH，涡旋，并在45 ℃下孵育过夜。 然后将孵育的样品用10%乙酸中和，通过Dowex树脂的填充柱脱盐，然后冻干。 在氮气流下用甲醇：乙酸（9：1）清洗干燥样品的硼酸盐。 使样品通过C18反相柱以收集裂解的O-聚糖（用5%乙酸洗脱）。 在氮气流下干燥0-聚糖级分。 碳水化合物的全O-甲基化和C18 sep-pak柱的纯化 对N-和O-连接聚糖进行全甲基化，以进行寡糖分析（Ciucanu和Kerek，1984）。 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。 基质辅助激光解吸飞行时间质谱（MALDI-TOF）分析寡糖 最初使用MALDI/TOF-MS进行N-连接和O-连接聚糖的分析。所用的机器是4700蛋白质组学分析仪（Applied Biosystems），其设置为反射器正离子模式。在MALDI板上以？-二羟基苯甲酸（DHBA，20 mg/mL溶液，溶于50%甲醇：水）作为基质结晶全甲基化聚糖。 通过NanoSpray电离-线性离子阱质谱法（LTQ）分析寡糖 通过MALDI-TOF MS检测到的N-连接寡糖通过ESI-LTQ质谱法确认。 按照复杂碳水化合物研究中心开发的方法进行质谱分析（Aoki K，Perlman M，Lim JM，Cantu R，威尔斯L，Tiemeyer M.生物化学杂志2007年3月23日;282（12）：9127-42。Epub 2007年1月29日）。 通过使用NSI-LTQ/MSn确定质量分析。 简言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并以0.4 μ L/min的恒定流速直接注入仪器（LTQ，Thermo Finnigan）。毛细管温度设定为210 ℃，以正离子模式进行MS分析。MS/MS裂解的碰撞能量设定为28。 对于总离子图，自动MS/MS分析（在28个碰撞能量下），在连续的2.8质量单位窗口中扫描500至2000的m/z范围，该窗口与前一窗口重叠2个质量单位。