ABSTRACT
The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.
Subject(s)
Fluorescent Dyes/chemistry , HSP27 Heat-Shock Proteins/analysis , Immunoblotting/methods , Immunoconjugates/chemistry , Proteins/analysis , Proteomics/methods , Animals , Antibodies, Monoclonal/chemistry , Blotting, Western/methods , Carbocyanines/chemistry , Electrophoresis, Gel, Two-Dimensional/methods , Humans , Isoelectric Focusing/methods , WorkflowABSTRACT
The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.
Subject(s)
Fluorescent Dyes/chemistry , Immunoblotting/methods , Proteomics/methods , Animals , Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Polyacrylamide Gel/methods , HSP27 Heat-Shock Proteins/analysis , Humans , Immunoblotting/instrumentation , Myocardium/chemistryABSTRACT
Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label-free nanoscale LC-MS has been applied in this study to validate a Triton X-114-based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane-bound proteins with an enrichment of over 62% for this protein subset. LC-MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane-bound proteins. This approach should be applicable to a wider scale study of disease-associated changes in the cardiac membrane subproteome.
Subject(s)
Detergents , Heart Ventricles/metabolism , Membrane Proteins/analysis , Chromatography, Liquid/methods , Humans , Mass Spectrometry/methods , ProteomicsABSTRACT
The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.
