Castro-Gamero Angel Mauricio, Izumi Clarice, Rosa José César
Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
Methods Mol Biol. 2014;1156:295-306. doi: 10.1007/978-1-4939-0685-7_20.
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).
鸟枪法蛋白质组学（液相色谱 - 电喷雾电离 - 质谱联用，LC - ESI - MS/MS）凭借多种方法主导了亚细胞、细胞、组织和整个生物体中全局蛋白质表达的策略，例如用于相对和绝对定量的等压标签（iTRAQ）、同位素编码亲和标签（ICAT）、细胞培养中使用氨基酸的稳定同位素标记（SILAC）以及非标记（无标记）方法。鸟枪法蛋白质组学实际上取代了经典的二维凝胶电泳。选择反应监测（SRM），也称为多反应监测（MRM），是一种靶向定量技术，它使用胰蛋白酶肽的复杂混合物，可通过与电喷雾三重四极杆质谱仪联用的液相色谱进行选择性检测；该系统可以在碰撞诱导解离过程中选择前体离子及其相应的产物离子，以产生与特定蛋白质相关的特异性检测。在这里，我们描述了从复杂生物基质中高效产生完整的胰蛋白酶酶解以及用于LC - SRM - MS和LC - ESI - MS/MS（标记或无标记）的伴随材料的方案。
