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 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.
Ye
Xiaoying
X
Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., NCI at Frederick, Frederick, Maryland 21702-1201, USA.
Johann
Donald J
DJ
Hakami
Ramin M
RM
Xiao
Zhen
Z
Meng
Zhaojing
Z
Ulrich
Robert G
RG
Issaq
Haleem J
HJ
Veenstra
Timothy D
TD
Blonder
Josip
J
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D002364:Caseins / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018950:Lipopolysaccharide Receptors / Q000032:analysis* / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism*; D011492:Protein Hydrolysates / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012995:Solubility; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002364:Caseins; D018950:Lipopolysaccharide Receptors; D008565:Membrane Proteins; D011492:Protein Hydrolysates; D020543:Proteome; C026375:trypticase-soy broth; D001436:Bacteriorhodopsins