title: Improved gel electrophoresis matrix for hydrophobic protein separation and identification.
abstract: We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach.
authors.affiliation: Miniaturisation pour l'Analyse, la Synthèse & la Protéomique (MSAP), USR CNRS 3290, and Protéomique, Modifications Post-traductionnelles et Glycobiologie, IFR 147, Université de Lille 1 Sciences et Technologies, 59655 Villeneuve d'Ascq Cedex, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Tokarski
authors.forename: Marianne
authors.initials: M
authors.lastname: Fillet
authors.forename: Christian
authors.initials: C
authors.lastname: Rolando
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D058105:Polymerization; D012995:Solubility; D053719:Tandem Mass Spectrometry
chemical_list: D000178:Acrylamides; D008565:Membrane Proteins; C099046:N,N-dimethylacrylamide