Chemo-enzymatic derivatization of O-GlcNAcylated recombinant human tau: analysis during different reaction steps - RAW data files - LC-HRMS (6600, Sciex) aquisition

Chemo-enzymatic derivatization (GalNAzylation, biotinylation) of O-GlcNAcylated recombinant human tau, trypsinization, enrichment via coupling to streptavidin beads, and release of tagged peptides by β-elimination – Michael addition reaction. LC-HRMS (6600, Sciex) analysis during different steps of the sample preparation to monitor formation of reaction products and removal of non-modified tryptic peptides. Final extract contains purified sulfited peptides for the localization of tau O-GlcNAcylation sites.
"008": a digest of non-derivatized trypsinized O-GlcNAcylated recombinant human tau
And, from derivatized and trypsinized O-GlcNAcylated recombinant human tau, XICs of tryptic tau peptides in:
"031": the tryptic digest before addition of streptavidin beads
"041": the depleted fraction after incubation with streptavidin beads
"043": the final extract after release from streptavidin beads

对经O-连接N-乙酰葡糖胺（O-GlcNAc）修饰的重组人tau蛋白开展化学酶促衍生化（GalNAzylation、biotinylation），随后进行胰蛋白酶酶解；通过与链霉亲和素磁珠偶联实现肽段富集，再经β消除-迈克尔加成反应释放带标签的肽段。在样品前处理的不同阶段采用液相色谱-高分辨质谱（LC-HRMS，6600型，SCIEX公司）进行分析，以监测反应产物的生成与未修饰胰蛋白酶肽段的去除情况。最终提取物含有纯化的亚硫酸化肽段，可用于tau蛋白O-连接N-乙酰葡糖胺修饰位点的定位。
"008": 未经过衍生化的经胰蛋白酶酶解的O-连接N-乙酰葡糖胺修饰重组人tau蛋白的酶解产物
此外，针对经衍生化且经胰蛋白酶酶解的O-连接N-乙酰葡糖胺修饰重组人tau蛋白，其胰蛋白酶tau肽段的提取离子色谱（XIC）信息如下：
"031": 加入链霉亲和素磁珠前的胰蛋白酶酶解产物
"041": 与链霉亲和素磁珠孵育后的未结合组分
"043": 从链霉亲和素磁珠上洗脱得到的最终提取物