Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 825, Issue 2, Pages 169-175Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2005.02.022
Keywords
rhodopsin; opsin; pigment; membrane protein; G protein-coupled receptor; mass spectrometry; HPLC; protein mappings protein identification; protein sequencing; LTQ
Funding
- NEI NIH HHS [EY-14793, EY-04939, EY-08239] Funding Source: Medline
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Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems. (c) 2005 Elsevier B.V. All rights reserved.
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