Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography–electrospray ionization–mass spectrometry

https://doi.org/10.1016/j.ab.2007.12.025Get rights and content

Abstract

The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)–MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI–MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography–ESI–MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC–ESI–tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.

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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