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

作者：Ho Jenny T C; White Jim F; Grisshammer Reinhard; Hess Sonja^*

来源：Analytical Biochemistry, 2008, 376(1): 13-24.

DOI：10.1016/j.ab.2007.12.025

• integral membrane protein
• intact protein
• detergents
• Electrospray ionization-mass spectrometry (ESI-MS)
• G protein-coupled receptor (GPCR)
• neurotensin receptor

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 NTSI by electrospray ionization (ESI)-MS with emphasis on methods to transfer intact NTSI 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 chromatographyESI-MS analysis. In a separate experiment, NTSI 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 NTSI protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.

• 出版日期2008-5-1