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方法简介
肽段覆盖率is指detect到of肽段氨基酸数量占该protein总氨基酸数量of比例. 质谱法对protein多肽类药物分子of肽段覆盖率进行analyzeof基本原理is将protein分子选用Specificityproteinenzyme进行切割, enzyme解后of供试品for复杂of肽段组成of混合物, 然后经过质谱进行detect, 上海拜谱生物for提高肽段覆盖率, inTrypsinenzyme切进行肽段覆盖率analyzeof同时, 也provideTrypsin, Chymotrypsin, Glu-C三种enzyme切以提高覆盖率.
Sample Requirements
关于样品:canprovideprotein冻干粉或protein溶液; 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.
样品制备: 对于protein多肽类药物, 样品制备is质谱analyzeof关键步骤. 合适of样品制备方法应be able to保持药物分子of完整性andstable性, 避免in预process过程中发生降解或修饰.
enzyme解策略: enzyme解is将protein转化for多肽片段of关键步骤. 选择合适ofenzyme (such as胰proteinenzyme) as well asoptimizeenzyme解条件, 以确保protein分子被充分且均一地切割成较小of多肽片段, 从而增加质谱detectof肽段覆盖率.
质谱条件: 质谱analyze时, need选择合适of质谱模式 (such as离子阱质谱, 轨道阱质谱etc.) as well asoptimize质谱参数 (such as离子源温度, 碰撞能量etc.) , 以获得高质量of质谱数据, 提高肽段ofdetectSensitivityand分辨率.
数据库比对: 获得of质谱数据needwith已知ofprotein数据库进行比对, 以识别肽段序列. 数据库of完整性andAccuracy对于肽段覆盖率of评估至关important. 此外, 合适of比对算法and参数设置也能提高比对结果ofAccuracyandreliable性.
软件analyze: utilizingprofessionalof质谱analyze软件对Raw Data进行processandanalyze, including峰detect, 噪声过滤, 肽段识别etc.步骤. 这些analyze过程对于提高肽段覆盖率至关important.
质谱indetermineprotein多肽类药物肽段覆盖率时of几pcs要点:
