Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents
复制标题
DOI：
10.1074/mcp.m400129-mcp200
复制
发表时间：
2004-12-01
影响因子：
7
通讯作者：
Pappin, DJ
中科院分区：
文献类型：
--
作者：
Ross, PL;Huang, YLN;Pappin, DJ
我们在这里描述了一种多重蛋白质定量策略，提供了复杂混合物中蛋白质的相对和绝对测量。该方法的核心是一套多重等压试剂，可产生胺衍生肽。衍生肽在质谱中无法区分，但表现出强烈的低质量质谱/质谱特征离子，支持定量。在这项研究中，我们检测了野生型酵母菌株和等基因的upf1Delta和xrn1Delta突变株的全球蛋白表达，它们分别在无义介导的mRNA衰变和一般的5‘到3’衰变途径中存在缺陷。我们还演示了4倍复用的使用，使相对蛋白测量与使用合成等压肽标准测定靶蛋白的绝对水平同时进行。我们发现，Upf1p和Xrn1p的失活会对蛋白质表达产生共同和独特的影响。
We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.