Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations dur...

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Bibliographic Details
Published in:Journal of proteome research 2003-03, Vol.2 (2), p.153-161
Main Authors: Goshe, Michael B, Blonder, Josip, Smith, Richard D
Format: Article
Language:English
Subjects:
Affinity Labels
Bacterial Proteins - analysis
Biotinylation
Cysteine
Deinococcus - chemistry
Hydrophobic and Hydrophilic Interactions
Mass Spectrometry - methods
Membrane Proteins - analysis
Peptide Mapping
Proteomics - methods
Solvents
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Summary:The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses. Keywords: affinity labeling • biotinylation • membrane proteins • hydrophobic proteins • proteomics • mass spectrometry
ISSN:1535-3893
1535-3907
DOI:10.1021/pr0255607