O-LINKED OLIGOSACCHARIDE PROFILING MASS SPECTROMETRY

O 联低聚糖分析质谱法

• 批准号: 7956032
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956032
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Buffers; Carbohydrates; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glass; Glycopeptides; Grant; Heating; Incubated; Institution; Iodoacetamide; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; New England; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Planet Mars; Plant Resins; Polysaccharides; Preparation; Procedures; Propanols; Proteomics; Reaction; Research; Research Personnel; Residual state; Resources; Sampling; Scanning; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; capillary; instrument; ionization; methyl iodide; sodium borohydride

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. The N-glycans were released and removed prior to b-elimination to avoid being mixed with the O-glycans in the latter's analysis. Briefly, glycoroteins were denatured by reduction and carboxyamidomethylation and then treated with Trypsin and PNGase F. Released N-linked glycans were separated from the residual peptides and O-linked glycopeptides through a C18 sep pak cartridge. Subsequently, the O-glycans were cleaved from the O-linked glycopeptides by ¿- elimination procedures. After ¿- elimination, released O-glycans were desalted and cleaned of borate, permethylated and analyzed by MALDI/TOF-MS and NSI-MSn. The procedures are shown in detail below. Release of N-linked glycans The dried sample was dissolved in Ambic buffer (50mM Ammonium Bicarbonate). The sample then was added with 25 mM DTT and incubated for 45 min at 50 for reduction, followed by carboxyamidomethylation with 90mM iodoacetamide, incubated at room temperature in the dark for 45 min. The sample then was digested with the trypsin (37oC, overnight). After digestion, trypsin was inactivated by heating at 100¿ C for 5 min. After cooling to room temperature, the tryptic digest was treated with PNGase F (New England BioLabs) to release the N-glycans. The tryptic/PNGase F digest then was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was eluted first with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the other iso-propanol fractions were dried in a speed vacuum concentrator and then combined into one glass tube. Release of O-linked glycans O-linked carbohydrates were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 500 ¿L of 50 mM Sodiumhydroxide (NaOH) containing 19 mg of sodium borohydride was added to the sample and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid, desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and lyophilized. The dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to separate the O-glycans from the peptides. O-linked glycans were eluted with 5% acetic acid and lyophilized, whereas the peptides were eluted with 100% iso-propanol and dried under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The dried carbohydrate fraction was dissolved in dimethylsulfoxide and methylated with NaOH and methyl iodide (Anumula and Taylor, 1992). The reaction was quenched by the 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 loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI/TOF-MS) MALDI/TOF-MS was performed in the reflector positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (NSI-LTQ/MSn) 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). 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 200 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 N-聚糖在b-消除之前被释放并去除，以避免在后者的分析中与O-聚糖混合。 简而言之，通过还原和羧酰胺甲基化使糖蛋白变性，然后用胰蛋白酶和 PNGase F 处理。通过 C18 sep pak 柱将释放的 N-连接聚糖与残留肽和 O-连接糖肽分离。 随后，通过 消除程序将 O-聚糖从 O-连接糖肽上裂解下来。 在 ¿- 消除后，释放的 O-聚糖被脱盐并清除硼酸盐，进行全甲基化并通过 MALDI/TOF-MS 和 NSI-MSn 进行分析。 详细步骤如下所示。 N-连接聚糖的释放 将干燥的样品溶解在 Ambic 缓冲液（50mM 碳酸氢铵）中。 然后向样品中加入25 mM DTT，并在50℃下孵育45分钟进行还原，然后用90mM碘乙酰胺进行羧酰胺甲基化，在室温下避光孵育45分钟。 然后用胰蛋白酶消化样品（37℃，过夜）。 消化后，100°C 加热 5 分钟，胰蛋白酶失活。 冷却至室温后，用 PNGase F（新英格兰生物实验室）处理胰蛋白酶消化物以释放 N-聚糖。 然后胰蛋白酶/PNGase F 消化物通过 C18 反相柱。 首先用 5% 乙酸洗脱碳水化合物部分（N-连接聚糖），然后用 5% 乙酸中的 20% 异丙醇、5% 乙酸中的 40% 异丙醇和 100% 异丙醇分别将 O-连接糖肽和肽依次洗脱到微量离心管中。 碳水化合物部分通过冻干干燥，而其他异丙醇部分在快速真空浓缩器中干燥，然后合并到一根玻璃管中。 O-连接聚糖的释放 通过β-消除程序将O-连接的碳水化合物从糖肽上裂解下来。 简而言之，将含有 19 mg 硼氢化钠的 500 µL 50 mM 氢氧化钠 (NaOH) 添加到样品中，并在 450°C 下孵育过夜。 然后用 10% 乙酸中和孵育的样品，通过 DOWEXTM 树脂填充柱（50W x 8 100，Sigma Aldrich）脱盐并冻干。 在氮气流下用甲醇:乙酸(9:1)清除干燥样品中的硼酸盐。 然后将样品通过 C18 反相柱，将 O-聚糖与肽分离。 O-连接聚糖用5%乙酸洗脱并冻干，而肽用100%异丙醇洗脱并在氮气流下干燥。 全O-甲基化碳水化合物的制备，用C18净化 将干燥的碳水化合物部分溶解在二甲亚砜中并用NaOH和碘甲烷甲基化（Anumula和Taylor，1992）。 通过添加水猝灭反应并用二氯甲烷萃取全-O-甲基化碳水化合物。 全O-甲基化聚糖被进一步清除污染物。 简而言之，将聚糖装入 C18 sep pak 柱中，然后用纳米纯水和 15% 乙腈洗涤。 然后用 85% 乙腈洗脱聚糖。 纯化的聚糖在氮气流下干燥并用甲醇溶解，然后进行质谱分析。 基质辅助激光解吸飞行时间质谱 (MALDI/TOF-MS) MALDI/TOF-MS 以反射器正离子模式进行，使用 ¿-二羟基苯甲酸（DHBA，20mg/mL 50%甲醇：水溶液）作为基质。 所有光谱均通过使用 4700 蛋白质组分析仪（Applied Biosystems）获得。 NanoSpray 电离-线性离子阱质谱 (NSI-LTQ/MSn) 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, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42）。使用 NSI-LTQ/MSn 进行质量分析。 简而言之，将全甲基化聚糖溶解在 50% 甲醇中的 1mM NaOH 中，并以 0.4 µL/min 的恒定流速直接注入仪器（LTQ，Thermo Finnigan）中。 毛细管温度设定为210℃，MS 分析在正离子模式下进行。 MS/MS 碎片的碰撞能量设置为 28。 对于总离子图谱，自动 MS/MS 分析（在 28 碰撞能量下），m/z 范围从 200 到 2000 在连续 2.8 质量单位窗口中扫描，该窗口与前面的窗口重叠 2 质量单位。