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。 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，并用镊子尖端将其弄碎。通过加入100 ml 0.01 M DTT/0.1 M Tris（pH 8.5）并在55 ℃下加热1- 2 h来还原蛋白质。 将管冷却至室温后，除去液体，并用100 ml 0.015 M碘乙酰胺/0.1 M Tris（pH 8.5）替换。 使其在黑暗中反应30分钟，之后除去液体并如下所述洗涤凝胶。 通过用200 ml 0.05 M Tris（pH 8.5）/ 25%乙腈洗涤一次，并用200 ml 0.05 M Tris（pH 8.5）/50%乙腈洗涤两次，振荡20 min，制备用于消化的凝胶。 除去洗液后，将凝胶在Speed-Vac浓缩器中干燥30分钟。 凝胶内消化 通过将0.08 mg胰蛋白酶（测序级，Roche Molecular Biochemicals，Indianapolis，IN）加入15 ml 0.025 M Tris（pH 8.5）中消化凝胶。如果需要，加入更多的缓冲液以使凝胶完全溶胀。将管置于32 ℃的加热块中并放置过夜。 用2X 50 ml 50%乙腈/2% TFA提取肽。将合并的浸提液体积减少至约15 ml，并转移至注射小瓶中。 通过用肽N处理从样品中释放N-连接的寡糖 使用由制造商（新英格兰Biolabs，Beverly，MA）供应的酶消化方案，将所述酶消化至糖苷酶F（PNGase F，N-聚糖酶）。 将样品在37 ℃下孵育过夜。 将消化的样品应用于C18经典SEP-PAK。用5 ml 5%乙酸洗脱，收集N-连接糖。 将样品级分干燥并通过Ciukanu和Kerek（1984）Carbohydr. Res. 131：209-217（用氢氧化钠和碘甲烷在无水DMSO中处理）。全甲基化后，用二氯甲烷/水提取样品三次，以去除任何杂质。将所得全甲基化样品重悬于甲醇中，并通过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.