从复杂混合物中选择性富集含叠氮化物的肽段。
Selective enrichment of azide-containing peptides from complex mixtures.
作者信息
Nessen Merel A, Kramer Gertjan, Back JaapWillem, Baskin Jeremy M, Smeenk Linde E J, de Koning Leo J, van Maarseveen Jan H, de Jong Luitzen, Bertozzi Carolyn R, Hiemstra Henk, de Koster Chris G
机构信息
Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands.
出版信息
J Proteome Res. 2009 Jul;8(7):3702-11. doi: 10.1021/pr900257z.
Abstract
A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.
摘要
本文描述了一种从复杂肽混合物中分离含叠氮化物肽段的通用方法，旨在促进质谱分析以研究蛋白质组动力学的不同方面。该富集方法基于叠氮化物反应性环辛炔（ARCO）树脂对含叠氮化物肽段的共价捕获，并在两种不同应用中得到了验证。用含叠氮化物的交联剂双（琥珀酰亚胺基）-3-叠氮甲基戊二酸酯（BAMG）处理细胞色素c后得到的肽段富集显示出多个含交联的肽段。对用生物正交氨基酸叠氮高丙氨酸替代甲硫氨酸进行脉冲标记的大肠杆菌蛋白质组衍生的肽段进行分离，可鉴定出许多新合成的蛋白质。此外，该方法具有很高的特异性，因为富集后超过87%的所有肽段都含有（修饰的）叠氮高丙氨酸。
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