Institute of Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Anal Chem. 2010 Sep 15;82(18):7588-95. doi: 10.1021/ac101306x.
This paper demonstrates the applications of a novel isobaric reagent, named deuterium ((2)H) isobaric amine-reactive tag (DiART), for quantitative proteomics. Peptides labeled with DiART were analyzed using an electrospray ionization (ESI)-based LTQ-Orbitrap mass spectrometer. Our data showed that (2)H-associated isotope effects, such as partial loss of (2)H labels during tandem mass spectrometry (MS/MS) and (2)H-related chromatographic shift, were either not observed or negligible. With the use of a hybrid collision-induced dissociation (CID)-higher energy C-trap dissociation (HCD) acquisition method, we were able to identify DiART-labeled peptides with high confidence and quantify them with high accuracy. Furthermore, we adopted a hybrid electron-transfer dissociation (ETD)-HCD acquisition protocol and developed a novel data analysis approach to measure phosphorylation of peptides. Our results showed DiART had excellent performance on LTQ-Orbitrap instruments and provided a cost-effective technique for large-scale quantitative proteomics measurements.
本文展示了一种新型等压试剂——氘代（2）H 等压胺反应标签（DiART）在定量蛋白质组学中的应用。使用电喷雾电离（ESI）-LTQ-Orbitrap 质谱仪分析用 DiART 标记的肽段。我们的数据表明，（2）H 相关的同位素效应，如串联质谱（MS/MS）过程中（2）H 标签的部分丢失和（2）H 相关的色谱位移，要么没有观察到，要么可以忽略不计。通过采用杂交碰撞诱导解离（CID）-高能 C 阱解离（HCD）采集方法，我们能够以高置信度鉴定 DiART 标记的肽段，并进行高准确度的定量分析。此外，我们采用了杂交电子转移解离（ETD）-HCD 采集方案，并开发了一种新的数据分析方法来测量肽段的磷酸化。我们的结果表明，DiART 在 LTQ-Orbitrap 仪器上具有优异的性能，为大规模定量蛋白质组学测量提供了一种具有成本效益的技术。
