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方法简介
generally发生N-糖基化位点修饰ofprotein, 均have基本of氨基酸结构骨架. N-糖基化位点identifyusuallyusingPNGase Fenzyme释放糖链, days冬酰胺(N)发生脱氨基化, for了避免days然脱氨基化造成of假阳性, in 18 O水中进行enzyme切, 使去糖基化位点含have一pcs18 O, 形成+2.9890Da. through高精度LC-MS/MSdetect脱糖后肽段, confirm脱糖后分子量with理论分子量of变化as well as糖基化修饰肽段of序列, 从而确定proteinof糖基化位点.
Sample Requirements
关于样品:至少need20ugofprotein, 纯度>90% ; 含盐量: 挥发性无机盐<20mM, 不挥发性无机盐<5mM; protein溶液避免SDSetc.变性剂, 表面活性剂, 不含tris缓冲体系.
样品transport:请using足量of干冰transport, 并尽量选用较快of邮递方式, 以降低transport过程中样品降解of可能性.
N糖基化位点analyzeisproteinresearch中of一项important任务, 它涉及到对protein分子中特定氨基酸残基上连接ofN糖基化位点ofidentifyandanalyze. 这种analyze对于理解proteinof结构, 功能as well as其in生物体内of相互作用具haveimportant意义.
inN糖基化位点analyze中, usuallyneed对糖基化位点进行specificof质量标记, 使其with未修饰ofprotein质量之间have一pcs特定of差异. 然后, through质谱analyze来detect这种差异, 并进一步through串联质谱identifyisin哪pcs氨基酸残基上发生了糖基化.
常用of方法之一isN-糖苷enzymeF (PNGase F) enzyme解法. PNGase F作for脱氨基enzyme, can将原先糖基化ofdays冬酰胺脱氨基后转变fordays冬氨酸, 造成相对分子质量增加1. 随后, through质谱relatively含havedays冬氨酸of肽with含have非糖基化days冬酰胺of肽of相对分子质量, 即可确定发生N-糖基化of氨基酸位点.
此外, 质谱技术 (such asLC-MS/MS) inN糖基化位点analyze中扮演着关键角色. 这pcs过程usuallyincludingprotein提取and纯化, enzyme解, 质谱analyzeas well asData Analysisetc.步骤. 首先, 从cell或tissue样本中提取protein并进行纯化. 然后, using特定ofenzyme (such as胰proteinenzyme) 将protein分解成较小of肽段. 接下来, utilizing液相色谱-串联质谱技术对这些肽段进行质谱analyze, through电离anddetect肽段of质荷比来识别糖基化修饰of存in. most后, using专门of软件对质谱数据进行analyze, 以identifyN糖基化of特定位点.
N糖基化位点analyzeof结果对于生物药物of研发and生产具haveimportant意义. 糖基化位点of位置影响着proteinof生物学活性, 药效as well as药物代谢and免疫反应. 因此, accurate地定位andanalyzeN糖基化位点对于optimize药物设计, 提高药物stable性as well as预测药物of疗效and副作用至关important.
