Immunodetection of small o-phenylenebismaleimide-labeled peptides through carrier protein display on polyvinylidene fluoride.
Protein modification and peptide analysis are important techniques for the elucidation of the structure and function of enzymes. We describe a new technique for the identification of peptides covalently modified with the maleimide cross-linker o-phenylenebismaleimide (OPBM). The method can identify labeled peptides without the use of sophisticated instrumentation or radioactive markers and takes advantage of the separating power of RPLC and of the sensitivity of immunoblotting. Chloroplast ATPase F1 was labeled at a single cysteine residue by OPBM and trypsinized. Fractions collected by RPLC were bound to polyvinylidene fluoride (PVDF). Despite the small size of the OPBM-labeled peptide (1.84 kDa) it was possible to immobilize it on PVDF by using glutaraldehyde to conjugate the peptide to a larger, unlabeled protein. Polyclonal antibodies raised against the cross-linker N,N',1,5-naphthalenebismaleimide (NBM) cross-react with OPBM. These antibodies detected the presence of OPBM displayed on the PVDF and correctly identified the RPLC fraction containing the OPBM-labeled peptide as verified by both mass spectroscopy and radiolabeling of OPBM. This method could be adapted to detect the presence of linear epitopes recognized by an antibody and is a broadly applicable technique for the immunodetection of peptides.
Evron
Yoav
Y
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Johnson
Eric A
EA
Pinaula
Simeon
S
McCarty
Richard E
RE
D025222:Chloroplast Proton-Translocating ATPases / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry*; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay / Q000379:methods; D008301:Maleimides / Q000737:chemistry* / Q000276:immunology; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods; D014357:Trypsin / Q000378:metabolism; D005465:Fluorocarbon Polymers
D025222:Chloroplast Proton-Translocating ATPases; D003432:Cross-Linking Reagents; D005976:Glutaral; D007106:Immune Sera; D008301:Maleimides; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C023860:N,N'-2-phenylenedimaleimide