Mass spectrometric peptide fingerprinting of proteins after Western blotting on polyvinylidene fluoride and enhanced chemiluminescence detection.
The combined use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry has become a powerful and widely used tool in proteome studies. Following separation by electrophoresis, proteins can be transferred to an inert support such as polyvinylidene fluoride (PVDF) or nitrocellulose (NC) for the visualization of individual or specific classes of proteins by immunochemical detection methods. We developed a method that allows the mass spectrometric analysis of peptides derived from proteins detected by Western blotting on PVDF. Proteolysis buffer containing either dimethyl formamide (DMF) or Triton X-100 to recover peptides amenable to mass spectrometry was investigated. Although either one can be used, the buffer containing DMF required less sample handling prior to mass spectrometry. The approach was tested using commercially available proteins and serine-phosphorylated proteins from an HEK-293 nuclear extract.
Methogo
Ruth Menque
RM
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Dufresne-Martin
Geneviève
G
Leclerc
Patrice
P
Leduc
Richard
R
Klarskov
Klaus
K
D015153:Blotting, Western; D002244:Carbon / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003101:Collodion / Q000737:chemistry; D030562:Databases, Protein; D003902:Detergents / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007120:Immunochemistry; D049449:Luminescence; D008163:Luminescent Measurements; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D017830:Octoxynol / Q000494:pharmacology; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011145:Polyvinyls / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012694:Serine / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology; D005465:Fluorocarbon Polymers
D002244:Carbon; D003101:Collodion; D003902:Detergents; D004126:Dimethylformamide; D008567:Membranes, Artificial; D017830:Octoxynol; D010455:Peptides; D011145:Polyvinyls; D020543:Proteome; D012694:Serine; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; C028433:chinese ink; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers