IN-GEL DIGESTION AND N-LINKED PROFILING (MASS SPECTROMETRY) OF THREE 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。 考马斯染色凝胶的还原、烷基化和脱色 将凝胶带转移到干净的试管中，并用镊子的尖端将其打碎。加入100mL0.01MDTT/0.1MTris，pH 8.5，在55℃加热1~2小时可使蛋白质还原。将试管冷却至室温后，取出液体，用100ml 0.015M碘乙酰胺/0.1M Tris，pH 8.5代替。这是允许反应30分钟。在黑暗中，液体被移走，凝胶被清洗，如下所述。 用200ml 0.05M Tris，pH 8.5/25%乙腈洗涤一次，用200ml 0.05M Tris，pH 8.5/50%乙腈洗涤两次，制备消化用凝胶。浑身发抖。去除洗涤剂后，将凝胶干燥30分钟。在极速真空集线器中。 凝胶内消化 在15ml 0.025M Tris，pH 8.5中加入0.08 mg胰酶(序列级，罗氏分子生物化学公司，印第安纳波利斯，IN)消化凝胶。如果需要的话，可以加入更多的缓冲液来完全膨胀凝胶。将试管放在32℃的加热块中，放置一夜. 多肽用2×50毫升50%乙腈/2%TFA提取。将组合提取物的体积减少到~15ml，并转移到注射瓶中。 由制造商(New England Biolabs，Beverly，MA)通过使用酶消化协议供应商的N-糖苷酶F(PNGase F，N-Glycanase)处理样品来从样品中释放N-连接的寡糖。样品在37℃下孵育过夜，消化后的样品应用于C18经典SEP-PAK。用5毫升5%的冰醋酸洗脱，以收集N-连接的糖。 样品组分用Ciukanu和Kerek(1984)Carbohyr的方法干燥和全甲基化。决议131：209-217(用氢氧化钠和甲基碘在干DMSO中处理)。全甲基化后，样品用二氯甲烷/水提取三次，以去除任何杂质。将得到的过甲基化样品重新悬浮在甲醇中，并用MALDI-TOF进行分析。

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. Reduction, alkylation, and destaining of Coomassie-stained gels The gel band is transferred to a clean tube and broken up with the tip of tweezers. The protein is reduced by adding 100ml 0.01M DTT/0.1M Tris, pH 8.5, and heating at 55¿ for 1-2h. After cooling the tube to room temperature, the liquid is removed and replaced with 100ml 0.015M iodoacetamide/0.1M Tris, pH 8.5. This is allowed to react for 30 min. in the dark after which the liquid is removed and the gel is washed as described below. The gel is prepared for digestion by washing once with 200ml 0.05M Tris, pH 8.5/ 25% acetonitrile and twice with 200 ml 0.05M Tris, pH 8.5/50% acetonitrile for 20 min. with shaking. After removing the washes, the gel is dried for 30 min. in a Speed-Vac concentrator. In-gel digestion The gel is digested by adding 0.08 mg trypsin(sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN) in 15ml 0.025M Tris, pH 8.5. More buffer is added if needed to completely swell the gel. The tube is placed in a heating block at 32¿ and left overnight. Peptides are extracted with 2X 50ml 50% acetonitrile/2% TFA. The combined extracts are reduced in volume to ~15ml and transferred to an injection vial. N-linked oligosaccaharides were released from the samples by treatment with peptide N glycosidase F (PNGase F, N-glycanase) using the enzyme digestion protocol supplier by the manufacturer (New England Biolabs, Beverly,MA). Samples were incubated overnight at 37¿C. The digested samples were applied to a C18 classic SEP-PAK. This was eluted with 5 ml of 5% acetic acid to collect the N-linked sugars. The sample fractions were dried and permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). After permethylation, the samples were extracted three times with methylene chloride/water in order to remove any impurities. The resulting permethylated samples were resuspended in methanol and analyzed by MALDI-TOF.