Development of Structural Marker Peptides for Cystic Fibrosis Transmembrane Conductance Regulator in Cell Plasma Membrane by Reversed-Footprinting Mass Spectrometry
Development of Structural Marker Peptides for Cystic Fibrosis Transmembrane Conductance Regulator in Cell Plasma Membrane by Reversed-Footprinting Mass Spectrometry
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DOI：
10.1021/acs.analchem.5b01962
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发表时间：
2015-09-01
影响因子：
7.4
通讯作者：
Yao, Xudong
中科院分区：
文献类型：
--
作者：
Farrokhi, Vahid;Bajrami, Bekim;Yao, Xudong
提出了一种基于靶向质谱的方法，该方法采用蛋白质的快速光化学氧化（FPOP）在囊性纤维化跨膜传导调节因子（CFTR）膜转运蛋白的原始质膜位置处进行足迹分析。寻求两个分析必要条件：（1）数据采集和分析的总体简化和（2）较低的定量限，这使得能够直接分析固有的低丰度跨膜蛋白。这些目标是通过使用反向足迹技术实现的，该技术监测FPOP处理后剩余的未氧化肽。在寻找结构信息肽的过程中，设计了一种工作流程，用于准确和精确地定量由蛋白水解消化细胞质膜亚蛋白质组产生的CFTR肽。该样品制备策略减轻了对大量结构完整的CFTR的挑战性纯化的需要。在此基础上，提出了能够报告细胞CFTR结构和功能的结构标记肽的概念。反向足迹质谱扩展了FPOP技术，可以直接研究低丰度蛋白质在其内源性细胞位置的构象和相互作用变化。
A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment. In searching for structurally informative peptides, a workflow was designed for accurate and precise quantitation of CFTR peptides produced from proteolytically digesting the plasma membrane subproteome of cells. This sample preparation strategy mitigated the need for challenging purification of large quantities of structurally intact CFTR. On the basis of the interrogated peptides, it was proposed a concept of the structural marker peptide that could report CFTR structure and function in cells. The reversed-footprinting mass spectrometry extends the FPOP technology to study conformation and interaction changes of low-abundance proteins directly in their endogenous cellular locations.