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质谱分析仪

质谱分析仪. 扬州大学生物科学与技术学院. 质谱分析原理.

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质谱分析仪

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  1. 质谱分析仪 扬州大学生物科学与技术学院

  2. 质谱分析原理 • 质谱方法(Mass Spectroscope,MS)是通过正确测定蛋白质分子的质量而进行蛋白质分子鉴定、蛋白质分子的修饰和蛋白质分子相互作用的研究。质谱仪通过测定离子化生物分子的质荷比便可得到相关分子的质量。但长期以来，质谱方法仅限于小分子和中等分子的研究，因为要将质谱应用于生物大分子需要将之制备成气相带电分子，然后在真空中物理分解成离子。但如何使蛋白分子经受住离子化过程转成气相带电的离子而又不丧失其结构形状是个难题。20世纪70年代，解吸技术的出现成功地将蛋白分子转化成气相离子。尔后快原子轰击与其紧密相关的溶液基质二次离子质谱法使得具有极性的、热不稳定的蛋白分子可经受住电离过程。但这些方法仅限于10kD以下蛋白分子的研究。80年代电喷雾电离(ESI)和软激光解吸(SLD)电离技术的发展则使得质谱方法应用于高分子量蛋白分子的研究。

  3. 质谱分析 Ion source Ion separator detector 离子资源: 底物变成离子气 质量分析: 根据质量/电荷(m/z) 检测: femtomole –attomole (10-15– 10-18 mole)

  4. 质谱分析仪类型 MALDI-TOF Matrix Assisted Laser Desorption Ionization Time Of Flight 基质辅助激光解吸/电离飞行时间质谱 ESItandem MS (with HPLC, LC tandem MS or LC MS/MS) • Electro Spray Ionization MS Quadrupole • 电子喷雾离子化质谱

  5. 商品化的MALDI-Tof Microflex ™, Bruker Voyager DE-PRO™, ABI MALDI micro™, Micromass

  6. MALDI-TOF 质谱仪原理

  7. MALDI-TOF 质谱仪原理

  8. 两种主要类型的MALDI-TOF

  9. 图像分辨率的增强

  10. MALDI-Tof的典型结果

  11. MALDI-TOF检测的多肽指纹 Gel Database Protein ? 1 2 3 tryptic stored data or theoretical peptides digestion mass spectrometry compare: ?? is identical to ??

  12. ESI四极质谱 • Nano electrospray: >30 min spray time for 1 mL sample • Highly charged molecules are selected by ac modulation of transverse fields

  13. 四极质谱过滤

  14. ESI Quadrupole MS 的典型结果 From Eckerskorn in “Bioanalytik”, Lottspeich and Zorbas (Eds)

  15. 3倍 四极质谱分析仪 CID: Collision Induced Dissociation for acquiringMolecular weight and Structural information

  16. Q-TOF 质谱分析仪 Collision Cell

  17. CID 片段的命名法

  18. CID 质谱

  19. 商品化的LC/MS/MS API 4000, API Q-Tof ultima API, Micromass HCT plus, Bruker

  20. LC/MS/MS解释的结构信息 • Peptide sequence • Post translational modifications • Proteolytic processing, truncation • Trypsin, Endoproteinase mapping • Acylation • Missing of N-terminal peptide • Phosphorylation • Differential mapping /phosphatase treatment • Fe 3+ -loaded IMAC column • Glycosylation • Neuraminidase treatment

  21. 鉴定和注释蛋白质的路线 • 通过肽质谱指纹图（peptide mass fingerprinting，PMF）和数据库搜寻匹配 • 通过测出样品中部分肽段二级质谱信息或氨基酸序列标签和数据库搜寻匹配

  22. 质谱分析仪 (I) MALDI-TOF: 仪器类型: TOF 分析仪 样品状态: 固态. 优点: (1) easy (2) fast (3) high-through (4) sensitive. 缺点: (1) only fingerprint of protein, no sequence information (2) results is highly dependent on sample quality.

  23. 质谱分析仪 (II) (LC) -tandem MS: 仪器类型: (1) Triple quadrupole (2) Ion trap (3) Q-TOF 样品状态: 液态 . 优点: (1) de novo sequencing data available. (2) high sensitivity 缺点: (1) Lower through put (2) pricey

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