Alves Isabel D, Sachon Emmanuelle, Bolbach Gerard, Millstine Lynda, Lavielle Solange, Sagan Sandrine
Synthèse, Structure et Fonction de Molécules Bioactives, and Plateforme de Protéomique et de Spectrométrie de Masse, Université Pierre et Marie Curie-Paris 6, UMR 7613 CNRS, Paris, France. alves@ ccr.jussieu.fr
Anal Chem. 2007 Mar 15;79(6):2189-98. doi: 10.1021/ac062415u. Epub 2007 Feb 13.
Integral membrane proteins are among the most challenging targets for biomedical research as most important cellular functions are tied to these proteins. To analyze intrinsically their structure/function, their transduction mechanism, or both, these proteins are commonly expressed in cultured cells as recombinant proteins. However, it is not possible to check whether these recombinant proteins are homogeneously or heterogeneously expressed. Owing to difficulties in their purification, very few mass spectrometry studies have been performed with those proteins and even less with G-protein coupled receptors. Here we have set up a procedure that is highly compatible with MALDI-TOF mass spectrometry to analyze an intact histidine-tagged G-protein coupled, namely, the tachykinin NK-1 receptor expressed in CHO cells, solubilized and purified using cobalt or nickel chelating magnetic beads. The metal-chelating magnetic beads containing the receptor were directly spotted on the MALDI plate for analysis. SDS-PAGE, combined with in-gel digestion analyzed by mass spectrometry, Western blot ((His)6 and FLAG M2 tags), photoaffinity labeling with a radioactive agonist, and Edman sequencing, confirmed the identity of the purified protein as the human tachykinin NK-1 receptor. Mass spectrometry study of both the glycosylated and deglycosylated intact protein forms revealed the existence of several receptor species that is tempting to correlate with the unusual pharmacological behavior of the receptor.
整合膜蛋白是生物医学研究中最具挑战性的靶点之一，因为最重要的细胞功能都与这些蛋白相关。为了从本质上分析它们的结构/功能、转导机制或两者，这些蛋白通常作为重组蛋白在培养细胞中表达。然而，无法检查这些重组蛋白是均匀表达还是异源表达。由于其纯化困难，很少有针对这些蛋白的质谱研究，针对G蛋白偶联受体的研究更少。在这里，我们建立了一种与基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）高度兼容的方法，用于分析完整的组氨酸标签G蛋白偶联受体，即速激肽NK-1受体，该受体在CHO细胞中表达，使用钴或镍螯合磁珠溶解和纯化。将含有该受体的金属螯合磁珠直接点样到MALDI板上进行分析。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结合质谱分析的胶内消化、蛋白质免疫印迹（针对（His）6和FLAG M2标签）、放射性激动剂的光亲和标记以及埃德曼测序，证实纯化的蛋白为人速激肽NK-1受体。对糖基化和去糖基化完整蛋白形式的质谱研究揭示了几种受体种类的存在，这可能与该受体不寻常的药理行为相关。
