Pharmaceutical Sciences Faculty Publications

Title

Optimization of Protein Solubilization for the Analysis of the CD14 Human Monocyte Membrane Proteome Using LC-MS/MS

Document Type

Article

Publication Date

11-2-2009

Journal Title

Journal of Proteomics

ISSN

1876-7737

Volume

73

Issue

1

First Page

112

Last Page

122

DOI

10.1016/j.jprot.2009.08.008

PubMed ID

19709643

PubMed Central® ID

PMC3159575

Abstract

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol+PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was approximately 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.

Keywords

Bacteriorhodopsins, calibration, caseins, chromatography, liquid, membrane proteins, monocytes, proteome, proteomics, solubility, tandem mass spectrometry

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