Elsevier

Analytical Biochemistry

Volume 410, Issue 2, 15 March 2011, Pages 272-280
Analytical Biochemistry

High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

https://doi.org/10.1016/j.ab.2010.11.008Get rights and content
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Abstract

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography–mass spectrometry (LC–MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2–5 μg of protein. The method is also compatible with our standard LC–MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.

Keywords

Integral membrane protein
HPLC
Electrospray ionization mass spectrometry
Methanol
Detergent
Intact mass analysis
LC–MS
Isocratic elution

Cited by (0)

1

Present address: Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.

2

Present address: School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland.

3

Present address: Institute of Molecular Biology and Biophysics, ETH Zürich, HPK D14.3, 8093 Zürich, Switzerland.

4

Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.