Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

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 e...

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Bibliographic Details
Published in:Journal of proteomics 2009-11, Vol.73 (1), p.112-122
Main Authors: Ye, Xiaoying, Johann, Donald J., Hakami, Ramin M., Xiao, Zhen, Meng, Zhaojing, Ulrich, Robert G., Issaq, Haleem J., Veenstra, Timothy D., Blonder, Josip
Format: Article
Language:English
Subjects:
Biological and medical sciences
CD14 monocyte
Detergents
Diverse techniques
Fundamental and applied biological sciences. Psychology
LC-MS/MS
Membrane proteins
Methanol
Molecular and cellular biology
Solubilization
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Summary: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 ~ 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. [Display omitted]
ISSN:1874-3919
1876-7737
DOI:10.1016/j.jprot.2009.08.008