Analysis of an intact G-protein coupled receptor by MALDI-TOF mass spectrometry: molecular heterogeneity of the tachykinin NK-1 receptor.
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.
Alves
Isabel D
ID
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
Sachon
Emmanuelle
E
Bolbach
Gerard
G
Millstine
Lynda
L
Lavielle
Solange
S
Sagan
Sandrine
S
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008969:Molecular Sequence Data; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1