Protein carbonylation: 2,4-dinitrophenylhydrazine reacts with both aldehydes/ketones and sulfenic acids.
Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. beta-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.
Dalle-Donne
Isabella
I
Department of Biology, University of Milan, Italy. quack@unimi.it
Carini
Marina
M
Orioli
Marica
M
Vistoli
Giulio
G
Regazzoni
Luca
L
Colombo
Graziano
G
Rossi
Ranieri
R
Milzani
Aldo
A
Aldini
Giancarlo
G
D000447:Aldehydes / Q000737:chemistry*; D019295:Computational Biology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D007659:Ketones / Q000737:chemistry*; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D010659:Phenylhydrazines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D050050:Protein Carbonylation; D013434:Sulfenic Acids / Q000737:chemistry*
D000447:Aldehydes; D007659:Ketones; D010455:Peptides; D010659:Phenylhydrazines; D013434:Sulfenic Acids; C004787:2,4-dinitrophenylhydrazine; D003545:Cysteine