title: Immunoaffinity purification and characterization of 4-hydroxy-2-nonenal- and malondialdehyde-modified peptides by electrospray ionization tandem mass spectrometry.
abstract: Two methods based on specific immunoaffinity enrichment followed by electrospray ionization (ESI) mass spectrometry (MS) have been developed for the specific analysis of 4-hydroxy-2-nonenal (HNE)- and malondialdehyde (MDA)-modified proteins (Michael and Schiff base adducts, respectively). Anti-HNE antibodies were immobilized on CNBr-activated sepharose, and the immunosorbent produced was used for the enrichment of HNE-adducted peptides originating from a model peptide modification and a tryptic digest of modified apomyoglobin. A further immunosorbent was produced by anti-dinitrophenyl immobilization and assayed for selective extraction of peptides modified with HNE and MDA that were initially converted to their respective hydrazones. Subsequent analysis and characterization of the different purified fractions by ESI-MS (MS/MS) revealed that the two immunosorbents enable efficient and specific enrichment of the carbonyl adducted proteins. This approach lowers substantially the detection limit of such modifications and, thus, enables better assessment and characterization of carbonyl modifications in biological and food systems.
authors.affiliation: Department of Quality and Safety Assurance, Nestle Research Center, Nestec Ltd., Vers-Chez-les-Blanc, 1000 Lausanne 26, Switzerland.
authors.forename: François
authors.initials: F
authors.lastname: Fenaille
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Tabet
authors.forename: Philippe A
authors.initials: PA
authors.lastname: Guy
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D007163:Immunosorbent Techniques; D008315:Malondialdehyde / Q000737:chemistry*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000447:Aldehydes; D010455:Peptides; D008315:Malondialdehyde; D014357:Trypsin; C027576:4-hydroxy-2-nonenal