Abstract
BackgroundAcetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein–protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well.MethodsWistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase column and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOF-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data from mass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches.ResultsA set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis.ConclusionsThese data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.
Highlights
Diacetyl is a flavoring commonly used in foodstuffs, as it lends a buttery flavor to products such as popcorn, coffee blends, cakes, cookies, wines and other goods (McCoy et al, 2017; Shibamoto, 2014; Ryan et al, 2014; Papetti, Mascherpa & Gazzani, 2014; Park, Gilbert & Whittaker, 2018; More, Raza & Vince, 2012)
We reported that the reaction of peroxynitrite with α-dicarbonyls, namely diacetyl and methylglyoxal, in aerated phosphate buffer pH 7.4 results in the acetylation of free amino acids, peptides and proteins added to the reaction mixture (Alves et al, 2013; Massari et al, 2010; Massari et al, 2011; Tokikawa et al, 2014)
The lung extracts were analyzed by NanoLC-MS/MS, and significant differences were shown among the protein profiles in the control and diacetyl-treated groups
Summary
Diacetyl is a flavoring commonly used in foodstuffs, as it lends a buttery flavor to products such as popcorn, coffee blends, cakes, cookies, wines and other goods (McCoy et al, 2017; Shibamoto, 2014; Ryan et al, 2014; Papetti, Mascherpa & Gazzani, 2014; Park, Gilbert & Whittaker, 2018; More, Raza & Vince, 2012). Diacetyl/peroxynitrite-generated acetyl radicals have proven been proven to attack both the α- and ε-amino groups of free and blocked L-Lys, L-Lys-containing peptides and serum albumin (Alves et al, 2013) These findings have raised the hypothesis that radical acetylation of proteins contributes to transacetylase–promoted, post-translational protein modifications at sites where both methylglyoxal or diacetyl and peroxynitrite are present (Alves et al, 2013; Massari et al, 2010; Massari et al, 2011; Tokikawa et al, 2014). These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems
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