Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry

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• 作者：Wenjing Li ; Jianshi Yu ; Maureen A. Kane
• 关键词：Native mass spectrometry ; Quantification ; Noncovalent protein complex ; Cellular retinol ; binding protein ; Vitamin A
• 刊名：Journal of The American Society for Mass Spectrometry
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：28
• 期：1
• 页码：29-37
• 全文大小：
• 刊物主题：Analytical Chemistry; Biotechnology; Organic Chemistry; Proteomics; Bioinformatics;
• 出版者：Springer US
• ISSN：1879-1123
• 卷排序：28

文摘

Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein–ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1–10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system.