IN-GEL DIGESTION AND O-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

两个样品的凝胶内消化和 O-连接寡糖分析

• 批准号: 7722677
• 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/7722677
• 关键词: Acetic Acid; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Carbohydrates; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Excision; Formic Acids; Funding; Gases; Gel; Glycopeptides; Grant; Ice; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Nitrogen; Oligosaccharides; Peptides; Planet Mars; Plant Resins; Polysaccharides; Preparation; Procedures; Proteins; Proteomics; Range; Rate; Reaction; Research; Research Personnel; Resources; Sampling; Scanning; Sep-Pak C18; Solutions; Source; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; capillary; formic acid; instrument; ionization; methyl iodide; reconstitution; 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. In-gel digestion, Peptide extraction from the gel The gel pieces first were cut into smaller pieces and then washed with 40mM AmBic. After 10 minutes, Ambic was removed and then the gel pieces were dehydrated with 100% acetonitrile for 10 minutes. The preceding washing steps were repeated until the gel turned white. After destaining, the gel pieces were reswelled in 10mM DTT in 40mM Ambic at 55¿ C for 1 hr. After cooling to room temperature, DTT solution was exchanged with 55mM IDA solution and then incubated in the dark for 45 minutes, and then gel pieces were washed with 40mM AmBic ( for 10 min) and exchanged with 100% acetonitrile (for 10 min). This second washing steps were repeated once. After drying with acetonitrile, the gel pieces were reswelled on ice with the trypsin solution (10ul of trypsin in 1ml of 40mM Ambic) for 45 minutes and then the proteins in the gel pieces were digested overnight at 37¿ C. The supernatant were transferred to a new tube and then the peptides and the glycopeptides were extracted from the gel pieces with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The sample solutions were dried and then reconstituted with nanopure water to composite them into one tube eventually. ¿- elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the O-linked glycopeptides 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 and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). 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 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 were dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) 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 (LTQ) 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 210oC 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 windows 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。 凝胶内消化，从凝胶中提取肽 首先将凝胶块切成较小的块，然后用40 mM AmBic洗涤。10分钟后，除去Ambic，然后用100%乙腈将凝胶片脱水10分钟。 重复前述洗涤步骤，直至凝胶变成白色。脱色后，将凝胶块在55 ° C下在40 mM Ambic中的IOmM DTT中再溶胀1小时。冷却至室温后，将DTT溶液与55 mM IDA溶液交换，然后在黑暗中孵育45分钟，然后将凝胶块用40 mM AmBic洗涤（10分钟），并用100%乙腈交换（10分钟）。 将该第二洗涤步骤重复一次。用乙腈干燥后，将凝胶块在冰上用胰蛋白酶溶液（10 μ l胰蛋白酶溶于1 ml 40 mM Ambic中）再溶胀45分钟，然后将凝胶块中的蛋白质在37 ℃消化过夜。将上清液转移到新管中，然后用20%乙腈/5%甲酸、50%乙腈/5%甲酸和80%乙腈/5%甲酸从凝胶片中提取肽和糖肽。将样品溶液干燥，然后用纳米纯水复溶，最终将它们复合到一个试管中。 除盐、脱盐、硼酸盐去除 O-连接的碳水化合物部分通过消除程序从O-连接的糖肽中裂解。 简而言之，250 º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。 然后将温育的样品用10%乙酸中和，并通过DOWEXTM树脂的填充柱（50 W × 8）脱盐  100，Sigma Aldrich），然后冻干。 在全甲基化之前，在氮气流下用甲醇：乙酸（9：1）清洗干燥的样品的硼酸盐。 制备全-O-甲基化的碳水化合物，通过C18净化 将冻干碳水化合物部分溶于二甲亚砜中，然后用NaOH和碘甲烷甲基化（Ciucanu和Kerek，1984）。 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，将聚糖加载到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。 在氮气流下干燥纯化的聚糖，并用甲醇溶解，然后通过质谱法进行分析。 基质辅助激光解吸飞行时间质谱 采用反射器正离子模式，以20 mg/mL的DHBA（50%甲醇：水）为基质，进行MALDI/TOF-MS。 通过使用4700蛋白质组学分析仪（Applied Biosystems）获得所有光谱。 NanoSpray电离-线性离子阱质谱（LTQ） 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。 对于总离子图谱，自动MS/MS分析（在28个碰撞能量下），在连续的2.8个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。