O-LINKED OLIGOSACCHARIDE PROFILING MASS SPECTROMETRY

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。 在b-消除之前释放并去除N-聚糖，以避免在后者的分析中与O-聚糖混合。 简言之，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，通过消除程序从O-连接的糖肽上切割O-聚糖。 消除后，将释放的O-聚糖脱盐并清除硼酸盐，全甲基化并通过MALDI/TOF-MS和NSI-MSn进行分析。 这些程序详细说明如下。 N-连接聚糖的释放 将干燥的样品溶解于Ambic缓冲液（50 mM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。 还原，然后用90 mM碘乙酰胺进行羧酰胺甲基化，在室温下避光孵育45 min。然后用胰蛋白酶消化样品（37 ℃，过夜）。 消化后，通过在100 ℃下加热5 min灭活胰蛋白酶。冷却至室温后，用PNGase F（新英格兰BioLabs）处理胰蛋白酶消化物以释放N-聚糖。 然后将胰蛋白酶/PNGase F消化物通过C18反相柱。 首先用5%乙酸洗脱碳水化合物级分（N-连接聚糖），然后用20%异丙醇/5%乙酸、40%异丙醇/5%乙酸和100%异丙醇将O-连接糖肽和肽依次洗脱至微量离心管中。 碳水化合物部分通过冻干干燥，而其它异丙醇部分在高速真空浓缩器中干燥，然后合并到一个玻璃管中。 O-连接聚糖的释放 O-连接的碳水化合物通过消除程序从糖肽中裂解。 简言之，向样品中加入500 μ L含有19 mg硼氢化钠的50 mM氢氧化钠（NaOH），并在45 ℃下孵育过夜。 然后将孵育的样品用10%乙酸中和，通过DOWEXTM树脂（50 W × 8）的填充柱脱盐  100，Sigma Aldrich）并冻干。 在氮气流下用甲醇：乙酸（9：1）清洗干燥样品的硼酸盐。 然后使样品通过C18反相柱，以将O-聚糖与肽分离。 用5%乙酸洗脱O-连接的聚糖并冻干，而用100%异丙醇洗脱肽并在氮气流下干燥。 制备全-O-甲基化的碳水化合物，通过C18净化 将干燥的碳水化合物部分溶于二甲亚砜中，并用NaOH和碘甲烷甲基化（Anumula和Taylor，1992）。 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（MALDI/TOF-MS） 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（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，威尔斯L，Tiemeyer M. 2007年3月23日;282（12）：9127-42）。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200至2000的m/z范围，该窗口与前一窗口重叠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. 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.