The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues
The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues
复制标题
DOI：
10.1016/j.jasms.2007.04.021
复制
发表时间：
2007-08-01
影响因子：
3.2
通讯作者：
Whetton, Anthony D.
中科院分区：
文献类型：
--
作者：
Griffiths, John R.;Unwin, Richard D.;Whetton, Anthony D.
应用假设驱动的方法灵敏地测定酶消化蛋白质上的赖氨酸乙酰化位点。使用乙酰化牛血清白蛋白（AcBSA）消化物的连续稀释进行比较灵敏度测试，以评估与更传统的前体扫描（PS）方法相比，基于多反应监测（MRM）的方法的性能。这两种方法都能够选择性地检测乙酰化的肽在低飞摩尔水平时，加标到500 fmol的六蛋白胰蛋白酶消化的背景。MRM方法比前体扫描的灵敏度大约高10倍，其中检测到一个乙酰化肽，并在柱上以2 fmol的水平进行测序。该技术随后被应用到凝胶衍生的细胞角蛋白-8（CK 8）的样品中，通过Western印迹分析显示含有乙酰化赖氨酸残基。本文应用的策略，称为MRM启动的检测和测序（MIDAS），导致这种蛋白质上的乙酰化的新位点的容易识别。
The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)-based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein.