Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography–electrospray ionization–mass spectrometry
Section snippets
Materials
Water, acetonitrile, CNBr (5 M in acetonitrile), formic acid, ammonium bicarbonate, trifluoroacetic acid (TFA), iodoacetamide (IAM), dithiothreitol (DTT), chloroform, and methanol were purchased from Sigma–Aldrich (St. Louis, MO, USA). RapiGest SF was purchased from Waters (Milford, MA, USA). The model TM peptide (IYSKVLVTAIYLALFVVGTVGNSVTAFTLARKKSLQSLQSTVHYHLGSLALSDLLILLLAMPVELY) was synthesized at the Center for Biologics Evaluation and Research (Food and Drug Administration, Bethesda, MD,
Results and discussion
A combination of the detergents CHAPS and LM in the presence CHS and glycerol is essential to maintain NTS1 in a soluble and functional form during solubilization and purification [26]. Although critical for purification, these reagents have adverse effects on proteolytic digestion and LC–ESI–MS analyses. Therefore, the NTS1 sample was treated in a manner to substitute the detergents with LC–ESI–MS-compatible solvents while maintaining receptor solubility throughout. The amino acid sequence of
Conclusion
A mixture of detergents and salts is essential to maintain solubility and functionality of GPCRs, whether they originate from natural sources or are expressed in a recombinant system. To perform comprehensive analysis of GPCRs by MS, experimental methodology is required to minimize detergents and salts in the receptor sample. With an appropriate MS-compatible sample preparation, we were able to perform MS analyses both with the intact NTS1 and on its peptidess derived from digestion. To
Acknowledgments
We thank G. Abdoulaeva and N. Y. Nguyen from the Center for Biologics Evaluation and Research (Food and Drug Administration, Bethesda, MD, USA) for the synthesis of the model TM peptide. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Neurological Disorders and Stroke, and Betty and Gordon Moore Foundation. Experiments by J.T.C.H. and S.H. were
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