O-LINKED OLIGOSACCHARIDE PROFILING OF SIX SAMPLES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 N-连接葡聚糖的释放 干燥样品在胰酶缓冲液(0.1M Tris-HCl，含0.01M CaCl2的pH 8.2)中溶解。样品在100℃加热5分钟变性，冷却后用胰酶(37℃，过夜)消化。胰酶消化后，将样品在100℃加热5分钟以使胰酶失活。冷却至室温后，用PNGase F(New England BioLabs)处理N-聚糖以释放N-聚糖。消化后，样品通过C18反相柱。糖组分(N-糖链)首先用5%的冰醋酸洗脱，然后用20%的异丙醇-5%的冰醋酸、40%的异丙醇-5%的冰醋酸和100%的异丙醇依次洗脱到微离心管中。碳水化合物部分通过冷冻干燥，而其他丙醇部分在高速真空浓缩器中干燥，然后合并到一个试管中。 ？-除盐、除盐、除硼 O-连接的碳水化合物部分通过消除程序从糖蛋白中分离出来。简单地说，向每个样品中加入250°L的50 mM氢氧化钠，然后检查pH值。在确定pH为碱性后，再向样品中加入250°L的50 mM氢氧化钠，其中含有19 mg的硼氢化钠，旋转，并在450℃下孵育一夜。孵化后的样品用10%的醋酸中和，通过填充的DOWEXTM树脂柱(50W×8100，Sigma Aldrich)脱盐，然后冷冻干燥。干燥样品在全甲基化前用甲醇：冰醋酸(9：1)在氮气中清洗。 全O-甲基化碳水化合物的制备及C18的净化 碳水化合物部分溶解在二甲基亚砜中，然后用氢氧化钠和甲基碘甲基化(Ciucanu和Kerek，1984)。加水使反应熄灭，用二氯甲烷提取全氧甲基化碳水化合物。进一步清除了Per-O-甲基化多糖中的污染物。简而言之，将多聚糖装入C18Sep pak色谱盒，然后用纳米纯水和15%乙腈洗涤。然后用85%的乙腈洗脱多糖。纯化的多糖在氮气气流下干燥，并与甲醇溶解，然后进行质谱分析。 基质辅助激光解吸飞行时间质谱仪(MALDI-TOF) MALDI/TOF-MS色谱柱为反射镜正离子模式，以二羟基苯甲酸(DHBA，20 mg/mL甲醇：水)为基质。使用4700蛋白质组学分析仪(应用生物系统)获得所有光谱。 纳米喷雾电离-线性离子陷阱质谱(LTQ) 按照复合碳水化合物研究中心(青木K，珀尔曼M，LIM JM，坎图R，威尔斯L，蒂迈耶M.J生物化学)开发的方法进行质谱分析。2007年3月23日；282(12)：9127-42。EPub 2007年1月29日)。采用NSI-LTQ/MSN进行质量分析。简而言之，将过甲基化的葡聚糖溶于1 mM氢氧化钠中50%的甲醇中，并以0.4？L/分钟的恒定流速直接进入仪器(LTQ，Thermo Finnigan)。毛细管温度210℃，正离子模式进行MS分析。MS/MS裂解的碰撞能设为28。 对于总离子图谱、自动化的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从500到2000的m/z范围，这些窗口与前一个窗口重叠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. Release of N-linked glycans The dried samples were dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The samples then were denatured by heating for 5 minutes at 100¿C. After cooling, the samples were digested with the trypsin (37oC, overnight). After tryptic digestion, the samples were heated at 100¿ C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the N-glycans were treated with PNGase F (New England BioLabs) to release the N-glycans. After digestion, the samples were passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was first eluted 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 propanol fractions were dried in a speed vacuum concentrator and then combined into one tube. ¿- elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the glycoprotein 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 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 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 windows that overlapped the preceeding window by 2 mass units.