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proteinidentify&protein表达谱构建
一,protein大规模identify (LC-MS/MS SHOTGUN)analyze:
1999年, Yatesresearch组提出“鸟枪法”(shotgun), 其基本技术路线is针对液体或SDS-PAGE条带of复杂混合物用enzyme (Trypsin) enzyme解成肽段混合物, 然后对肽混合物进行多维毛细管液相色谱分离and串联质谱analyzeas well as数据库检索, 从而确定proteinof种类, 可同时identify成百上千种protein. 他们把这种思路称for多维proteinidentify技术, 即Mud PIT(multidi-mensional protein identification technology). with传统of双向电泳技术相比具haveSensitivity更高, 动态Detection Range更广etc.特点. research对象is完整oftissue, 体液或其提取物, 其目ofin于identify出尽可能多of肽andprotein分子. 将溶液内protein分子或SDS-PAGE条带of复杂混合物enzyme解成肽段混合物, through液相色谱分离, such as2D-LC (阳离子柱SCXandC18反相柱串联) 或1D-LCofC18反相柱, 串联质谱测试, most后用相应of数据库进行检索匹配, 可同时identify成百上千种protein.
二,Experimental Procedure
典型数据结果
| 产品名称 | Sample Type | 送样量 | 预processandstore | 备注 |
| LC-MS/MSidentify | 胶条 | SDS-PAGE条带 | 将needidentifyofSDS-PAGE条带切下, 装进EP管, 常温或冰袋寄送, 颜色very浅of条带可取多pcs重复样本放一起, 常温或冰袋寄送 | |
| 胶点 | 肉眼可见 | 用移液枪头将2D点挖下来, 加入已装have超纯水ofEP管中, 做好标记, 常温或冰袋寄送 | ||
| protein干粉 | 100 μg | 冻干ofprotein粉末装入EP管, 常温或冰袋寄送 | ||
| protein溶液 | 1 μg/μL*20μL | 提好ofprotein溶液store于-80°C, 干冰寄送 | 溶液中generally不要haveSDS, CHAPS, Triton X-100, NP40及吐温20, 40etc.系列of去污剂, such as果have, 请in送样单上注明; 盐浓度小于50mM | |
| 外泌体-Cell Culture上清 | ≥30 mL | 10000g-20000gcentrifuge30-60min, (或using0.22um滤膜过滤) , 取上清, -80°Cstore, 干冰transport | ||
| 外泌体-Serum | ≥2 mL | 收集好of全血室温静置两hours, 3000gcentrifuge10min, 取上清, 液氮速冻, -80°Cstore, 干冰transport | ||
| 外泌体-血浆 | ≥2 mL | 收集好of全血加入抗凝剂, 室温静止30minutes, 1300g-2000gcentrifuge10min, 取上清, 液氮速冻, -80°Cstore, 干冰transport | ||
| 外泌体-尿液 | ≥30 mL | 5000×g 4°C centrifuge30-60min, 取上清, -80°Cstore, 干冰transport |
canthrough凝胶电泳 (such asSDS-PAGE) , efficient液相色谱 (HPLC) 或质谱analyze (MS) etc.方法来评估protein样本of纯度. 这些方法can帮助detect样本中is否存in其他杂质或protein.
完整性canthrough检查proteinof大小, 翻译后修饰 (such as糖基化, 磷酸化etc.) as well asis否存in降解产物来评估. 质谱analyzecanprovide这些信息, 并帮助确定proteinis否保持了其days然状态.
such as果protein样本中存in杂质或污染物, 它们可能会干扰identify过程. 因此, in某些情况下, 进一步纯化protein样本以提高其纯度可能is必要of.
选择哪种identify方法取决于research目of, 样本性质as well as实验室可用of技术and资源. 质谱analyzeis常用ofproteinidentify方法, 但也可能needusing其他技术, such as凝胶电泳, 免疫印迹 (Western blot) etc..
样本量取决于所usingofidentify方法as well asprotein本身of丰度. 某些方法可能need更多of样本才能获得reliableof结果.
样本processusuallyincludingprotein提取, 纯化, 浓度determineand可能ofSample Preparation步骤 (such as变性, 还原etc.) . 这些步骤应according to所usingofidentify方法进行optimize.
QEidentify结果shouldaccording to实验设计and所usingof方法进行仔细解读. usually, 这need综合考虑多种数据, such as质谱数据, 电泳图谱etc., 并with已知数据库进行relatively以进行identify.
Accuracyandreliable性canthroughusing内部control, 重复实验, with已知序列进行relativelyas well asusing统计方法来评估. 此外, 参with实验室间ofrelativelyandvalidate也can提高identify结果ofreliable性.
proteinQEidentify (Quality Evaluation) usually涉及对protein样本of质量, 纯度and完整性进行评估. inproteinQEidentify过程中, research者可能会遇到一些常见ofQuestionand疑问. 以下is一些可能of疑问及其解A:
1, such as何确定protein样本of纯度?
2, such as何validateprotein样本of完整性?
3, protein样本is否need进一步纯化以提高identifyAccuracy?
4, such as何选择合适ofproteinidentify方法?
5, proteinQEidentifyneed多少样本量?
6, such as何processprotein样本以进行QEidentify?
7, QEidentify结果such as何解读?
8, such as何评估QEidentifyofAccuracyandreliable性?
