Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring
Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring
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DOI：
10.1016/j.jprot.2009.09.002
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发表时间：
2009-12-01
影响因子：
3.3
通讯作者：
Kuhn, Karsten
中科院分区：
文献类型：
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作者：
Byers, Helen L.;Campbell, James;Kuhn, Karsten
串联质量标签（TMT）适用于蛋白质和肽的全局和靶向定量方法。这些标签的不同形式允许产生具有相同共同结构的同量异位和同位素试剂组。这一功能允许将发现研究期间获得的数据直接转移到有针对性的调查中。在先前的发现研究中，使用这些试剂的同量异序组来鉴定在铁限制下表达的脑膜炎奈瑟菌蛋白。在这里，我们将这些试剂的同位素版本与单反应监测相结合，以验证在这些发现研究中发现的差异调节的选定候选物，这些候选物代表了众所周知的和新颖的铁调节蛋白，例如MtrCDE药物外排泵。在这种靶向方法（TMT-SRM）中，保持SRM的选择性，同时允许将内部参比标准品掺入实验中。通过监测184个转换，TMT-SRM导致代表12种蛋白质的33个肽的定量。所获得的数据证实了在发现阶段取得的结果。此外，通过基于MS的肽定量获得的这些数据通过蛋白质印迹结果（基于蛋白质水平定量的正交方法）独立确认。(C)2009 Elsevier B. V.保留所有权利。
Tandem Mass Tags (TMT) are suited to both global and targeted quantitation approaches of proteins and peptides. Different versions of these tags allow for the generation of both isobaric and isotopic sets of reagents sharing the same common structure. This feature allows for a straightforward transfer of data obtained during discovery studies into targeted investigations. In prior discovery studies, an isobaric set of these reagents was used to identify Neisseria meningitidis proteins expressed under iron-limitation. Here, we apply isotopic versions of those reagents in combination with single reaction monitoring to verify selected candidates found to be differentially regulated in these discovery studies, representing both well-known and novel iron-regulated proteins, such as the MtrCDE drug efflux pump. In this targeted approach (TMT-SRM), the selectivity of SRM is maintained while allowing the incorporation of an internal reference standard into the experiment. By monitoring 184 transitions, TMT-SRM resulted in the quantitation of 33 peptides representing 12 proteins. The acquired data corroborated the results obtained during the discovery phase. Furthermore, these data obtained by MS-based quantitation of peptides were independently confirmed by western blotting results, an orthogonal approach based on quantitation at the protein level. (C) 2009 Elsevier B.V. All rights reserved.