Fluorimetric screening assay for protein carbonyl evaluation in biological samples P. Stocker a,⇑ , E. Ricquebourg a , N. Vidal a,b , C. Villard c , D. Lafitte c , L. Sellami c,1 , S. Pietri a a Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France b SARL YELEN, 13820 Ensuès la Redonne, France c Centre de Recherches en Encologie Biologique et Onco-Pharmacologie (CRO2), Aix-Marseille Université, Institut National de la Santé et de la Recherche Médicale (INSERM), 13385 Marseille, France article info Article history: Received 5 February 2015 Received in revised form 20 April 2015 Accepted 22 April 2015 Available online 28 April 2015 Keywords: Protein carbonyls NBDH Oxidized BSA Fluorescence MALDI–TOF abstract Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitroben zo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments. Ó 2015 Elsevier Inc. All rights reserved. Oxidative stress can give rise to the formation of protein car- bonyl (PC) 2 derivatives due to cellular protein oxidation that occurs during pathological processes. Carbonylation of proteins is charac- terized by the introduction of carbonyl groups (C@O) in the protein structure, and the quantification of the carbonyl content of cell pro- teins is a useful indicator of oxidative protein damage and a fre- quently studied oxidation target product. Carbonylation is described as a nonreversible modification of proteins elicited by direct oxidation of amino acid side chains (Lys, Arg, Pro, and Thr) or by reaction of the nucleophilic side chains of Cys, His, and Lys residues with reactive carbonyl compounds produced during lipid peroxidation such as 4-hydroxy-2-nonenal, malondialdehyde, and acrolein [1]. Reactive carbonyl derivatives can also interact with the amino group of lysine residues of the protein by glycation and glycoxidation reactions. For example, the dicarbonyl methylglyoxal, which is mainly formed during glycolysis and sugar fragmentation reactions, irreversibly binds and modifies a number of proteins under physiological conditions [2], including bovine serum albumin (BSA) [3–6], ribonuclease A [7,8], lysozyme [9], and collagen [10]. High levels of protein carbonyls are observed in several human dis- eases such as diabetes, arthritis, Alzheimer’s disease, and inflamma- tory bowel disease [11–14]. Protein carbonyls are then described as a biomarker of protein oxidation and widely used in experimental and clinical studies [15]. Many biological assays, including fluorimetric, spectrophoto- metric, and immunological methods, are available for detection and quantification of protein carbonylation in human tissues and body fluids [16–18]. Interestingly, a recent study describes a new accurate PC detection method based on fluorescent hydrazides http://dx.doi.org/10.1016/j.ab.2015.04.021 0003-2697/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +33 4 91 28 87 92. E-mail address: p.stocker@univ-amu.fr (P. Stocker). 1 Current address: Shimadzu, Manchester M17 1GP, UK. 2 Abbreviations used: PC, protein carbonyl; BSA, bovine serum albumin; DNPH, 2,4-dinitrophenol hydrazine; NBDH, 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-flight; STZ, strepto- zotocin; BCA, bicinchoninic acid; PBS, phosphate-buffered saline; OxBSA, HOCl-oxidized BSA; TCA, trichloroacetic acid; ANOVA, analysis of variance; MS/MS, tandem mass spectrometry; LOD, limit of detection. Analytical Biochemistry 482 (2015) 55–61 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio