CONFIRMING GLYCOSYLATION SITES FOR A PROTIEN

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。 N-连接糖基化位点的18 O标记 将100微升样品（TEP 1 r）置于微量离心管中，加入100 μ L 50 mM碳酸氢铵缓冲液（pH 8.4），然后立即用25 mM二硫苏糖醇还原（50 ℃下45分钟），并用90 mM碘乙酰胺进行羧酰胺甲基化（室温下黑暗中45分钟）。将样品用测序级胰蛋白酶（Promega，麦迪逊，WI）消化，在37 ℃孵育过夜。胰蛋白酶消化后，向样品中加入1%甲酸以使酶失活，然后通过C18反相柱。将样品在快速真空中干燥，并加入碳酸氢铵缓冲液（50 mM，pH 8.4），然后在快速真空中干燥。将磷酸钠（100 mM，pH 7.5）加入干燥样品中。然后将样品干燥至完全，并在19 μ L H218 O中再水化。加入PNGase F，将混合物在37 ℃下孵育20小时，并在Speed Vac中干燥。在37 ℃下用测序级胰蛋白酶消化样品5小时。 通过使用NanoSep离心浓缩器（Pall Inc.）并通过液相色谱和质谱（LC-MS）分析。 液相色谱和质谱分析 将肽上样到填充有C18的毛细管柱上，并用0.1%甲酸（移动的相A）洗涤。随后，使用5 ~ 95%的线性梯度的移动的相B（80%乙腈+0.1%甲酸），以200 nl/min的流速洗脱肽160 min。使用LTQ-MS（Thermo Finnigan）通过纳米电喷雾电离质谱法（NSI-MS）分析洗脱的肽。

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. 18O-labeling of N-linked glycosylation sites One hundred micro liters of the sample (TEP1r) was placed in microcentrifuge tube and added 100 ¿L of 50 mM ammonium bicarbonate buffer (pH 8.4) and followed immediately by reduction with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylation with 90 mM iodoacetamide (45 min at room temperature in the dark). The sample was digested with sequencing grade trypsin (Promega, Madison, WI) overnight incubation at 37¿C. After trypsin digestion, the sample was added 1 % formic acid to deactivate the enzyme and then was passed through a C18 reversed phase cartridge. The sample was dried down in a speed vac and added with ammonium bicarbonate buffer (50mM, pH 8.4) and then dried down in a speed vac. Sodium phosphate (100mM, pH 7.5) was added to the dried sample. The sample then was dried to completeness and rehydrated in 19 ¿L of H218O. PNGase F was added and the mixture was incubated for 20 hours at 37¿C and dried in the Speed Vac. The sample was digested with sequencing grade trypsin for 5 hours at 37¿C. The sample was filtered by use of a NanoSep centrifugation concentrator (Pall Inc.) and analyzed by liquid chromatography and mass spectrometry (LC-MS). Liquid chromatography and mass spectrometric analysis The peptides were loaded onto a capillary column packed with C18 and washed with 0.1 % formic acid (mobile phase A). Subsequently, the peptide was eluted over 160 min with a flow of 200 nl/min using a linear gradient of 5 ~ 95 % of mobile phase B (80 % acetonitrile + 0.1 % formic acid). The eluted peptides were analyzed by nanoelectrospray ionization mass spectrometry (NSI-MS) using a LTQ-MS (Thermo Finnigan).