Biomarker Verification Using Selected Reaction Monitoring and Shotgun Proteomics
Biomarker Verification Using Selected Reaction Monitoring and Shotgun Proteomics
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DOI：
10.1007/978-1-4939-0685-7_20
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发表时间：
2014-01-01
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影响因子：
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通讯作者：
Rosa, Jose Cesar
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作者：
Castro-Gamero, Angel Mauricio;Izumi, Clarice;Rosa, Jose Cesar
鸟枪蛋白质组学液相色谱-电喷雾离子化-质谱（LC-ESI-MS/MS）已经用几种类型的方法主导了亚细胞、细胞、组织和整个生物体中的全局蛋白质表达的策略，如用于相对和绝对定量的同量异序标签（iTRAQ）、同位素编码的亲和标签（ICAT）、或在细胞培养物中使用氨基酸的稳定同位素标记（SILAC）和非标记（无标记）方法。鸟枪蛋白质组学几乎取代了经典的2D凝胶电泳。选择性反应监测（SRM），也称为多反应监测（MRM），是一种靶向定量技术，其使用胰蛋白酶肽的复杂混合物，其可以通过液相色谱与电喷雾三重四极杆质谱联用来选择性地检测;该系统可以在碰撞期间选择前体离子与它们相应的产物离子的组合，诱导解离以产生与特定蛋白质相关的特异性检测。在这里，我们描述了从复杂的生物基质和用于LC-SRM-MS和LC-ESI-MS/MS（标记或无标记）的伴随材料中有效产生完全酶促胰蛋白酶消化的方案。
Shotgun proteomics (liquid chromatography-electrospray ionization-mass spectrometry, LC-ESI-MS/MS) has dominated the strategies for global protein expression in subcells, cells, tissues, and whole organisms with several types of approaches, as isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and non-labeling (label free) methods. Shotgun proteomics practically replaced the classical 2D gel electrophoresis. Selected reaction monitoring (SRM), also denominated multiple reaction monitoring (MRM), is a targeted quantitative technology that uses a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray triple-quadrupole mass spectrometer; this system can select precursor ions in combination with their correspondent product ions during collision-induced dissociation to produce specific detection related to a particular protein. Here we describe protocols that are efficient to produce a complete enzymatic trypsin digestion from complex biological matrices and concomitant material to be used for LC-SRM-MS and LC-ESI-MS/MS (labeled or label free).