Journal
ANALYTICAL BIOCHEMISTRY
Volume 410, Issue 2, Pages 272-280Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.11.008
Keywords
Integral membrane protein; HPLC; Electrospray ionization mass spectrometry; Methanol; Detergent; Intact mass analysis; LC-MS; Isocratic elution
Funding
- Canadian Institutes for Health Research [1097737]
- Canadian Foundation for Innovation
- Genome Canada through the Ontario Genomics Institute
- GlaxoSmithKline
- Karolinska Institute
- Knut and Alice Wallenberg Foundation
- Ontario Innovation Trust
- Ontario Ministry for Research and Innovation
- Merck and Co., Inc.
- Novartis Research Foundation
- Swedish Agency for Innovation Systems
- Swedish Foundation for Strategic Research
- Wellcome Trust
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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 mu g 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. (C) 2010 Elsevier Inc. All rights reserved.
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