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HPLC and Mass Spectrometry of Integral Membrane Proteins

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The Protein Protocols Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

HPLC separations of membrane proteins can be conveniently divided into two categories. Firstly, there are many methods available for isolation of functional membrane proteins. Typically the proteins are maintained in configurations as close to their native state as possible through the use of mild detergents that provide solubility without denaturation, allowing convenient ion-exchange or size-exclusion chromatography, for example. Proteins or complexes isolated in this way are subsequently used for functional analysis or crystallization, and so on. These isolation techniques have been well reviewed and readers are referred to literature specific to the protein or membrane of interest. The second category of separations are those used to separate membrane proteins from detergents and salts for the purpose of protein chemistry; while tempting to call these methods “denaturing” there is substantial evidence that this is not always the case. The focus of this chapter is to review the latter category of HPLC techniques with specific reference to those methods that provide conditions compatible with mass spectrometric analysis, especially on-line electrospray ionization.

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Acknowledgments

The techniques described here are evolved from huge amounts of groundwork by many others, especially Gobind Khorana and John Findlay. My career in HPLC and mass spectrometry would not have been possible without the generous mentorship of Kym Faull and my colleagues at UCLA. I have learnt hugely from my peers and I acknowledge invaluable contributions from Sir John E. Walker, Ian Fearnley, H. Ron Kaback, and Bill Cramer amongst many others to whom I apologize for not mentioning. I specifically thank John R. Bowyer and Patrick Camilleri who understood and shared my interest in techniques compatible with mass spectrometry and Chris Gerrish for his skillful help with HPLCs.

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Authors and Affiliations

  1. The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Julian P. Whitelegge

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  1. Julian P. Whitelegge
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Editors and Affiliations

  1. School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK

    John M. Walker

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Whitelegge, J.P. (2009). HPLC and Mass Spectrometry of Integral Membrane Proteins. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_123

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  • DOI: https://doi.org/10.1007/978-1-59745-198-7_123

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-474-6

  • Online ISBN: 978-1-59745-198-7

  • eBook Packages: Springer Protocols

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