Proteomic Analysis of Thiol Modifications and Assessment of Structural Changes in Hemoglobin Induced by the Aniline Metabolites N-Phenylhydroxylamine and Nitrosobenzene

Abstract

MS-based proteomic analysis was combined with in silico quantum mechanical calculations to improve understanding of protein adduction by N-phenylhydroxylamine (PhNHOH) and nitrosobenzene (NOB), metabolic products of aniline. In vitro adduction of model peptides containing nucleophilic sidechains (Cys, His, and Lys) and selected proteins (bovine and human hemoglobin and β-lactoglobulin-A) were characterized. Peptide studies identified the Cys thiolate as the most reactive nucleophile for these metabolites, a result consistent with in silico calculations of reactivity parameters. For PhNHOH, sulfinamides were identified as the primary adduction products, which were stable following tryptic digestion. Conversely, reactions with NOB yielded an additional oxidized adduct, the sulfonamide. In vitro exposure of human whole blood to PhNHOH and NOB demonstrated that only sulfinamides were formed. In addition to previously reported adduction of β93Cys of human Hb, two novel sites of adduction were found; α104Cys and β112Cys. We also report CD and UV-Vis spectroscopy studies of adducted human Hb that revealed loss of α-helical content and deoxygenation. The results provide additional understanding of the covalent interaction of aromatic amine metabolites with protein nucleophiles.

Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate

“…After overnight in-gel digestion with Trypsin, the peptides were extracted and analyzed with HRMS (Thermo, Q Exactive HF-X). The dHNN modifications, determined by Protein Discoverer software (ThermoFisher), were defined as an increase of molecular weight by 370.153 Da, 354.158 Da, 402.143 Da, or 386.148 Da on the cysteine residues characterized in the MS 2 fragments ( Callan et al, 2009 ; Möller et al, 2017 ). The abundance of each peptide was determined in the MS 1 .…”

“…After simplyBlue TM SafeStain (Thermo fisher), the dtSARM1-dN band was sliced, dehydrated with 100% ACN, and the proteins were alkylated by 22.5 mM IAA for 30 min in dark after the reduction by 10 mM DTT at 55℃ for 30 min. After overnight in-gel digestion with Trypsin, the peptides were extracted and analyzed with HRMS (Thermo, Q Exactive (Callan et al, 2009b, Moller et al, 2017. The abundance of each peptide was determined in the MS 1 .…”

Quo vadis blood protein adductomics?

“…n N -hydroxy-aniline, (2:1) sulfinamide adduct, as in the whole blood experiments, only Cys sulfinamide modifications were observed for Hb in the presence of either GSH or a mixture of GSH and GSSG (Moller et al 2017 );…”

“…Molar ratios of compound to hemoglobin are placed in brackets. a) MDI (1:1) (Mhike et al 2013 ) ä MDI (10:1) (Mhike et al 2013 ) b 24TDI (1:1) (Mhike et al 2016 ) ß 24TDI (10:1) (Mhike et al 2016 ) c styrene oxide (SO) (5:1) (Basile et al 2002 ), α-His-45/His-50 and ß-Cys-93/His-97,respectively, are found alternatively alkylated, ç) SO (10 –4 :1) (Basile et al 2002 ) d diepoxybutane (10:1), * Cys-104 or His-103 (Basile et al 2002 , 2001 ) e formaldehyde (3:1 and 100:1) (Ospina et al 2011 ) f methylbromide (1:1) (Ferranti et al 1996 ), g sulfur mustard (50:1) (Hallez et al 2021 ) h GSSG (100:1) glutathionylation (Chen et al 2014 ) i peroxynitrite (150:1), nitration (Chen and Chen 2012 ; Kojima et al 2016 ) j H 2 O 2 (1:1), oxidation (Kojima et al 2016 ; Xiang et al 2013 ) k 1-chloro-2,4-dinitrobenzene (5:1) (Ndreu et al 2020 ) l 1,2-epoxy-3-phenoxypropane (5:1) (Ndreu et al 2020 ) m 12-mesyloxy-nevirapine (100:1) (Antunes et al 2010 ) a surrogate of the metabolite 12-sulfoxy-nevirapine, the valine adducts were analyzed by the Edman degradation procedure using phenylisothiocyanate n N -hydroxy-aniline, (2:1) sulfinamide adduct, as in the whole blood experiments, only Cys sulfinamide modifications were observed for Hb in the presence of either GSH or a mixture of GSH and GSSG (Moller et al 2017 ); o N -hydroxy-4-aminobiphenyl CAS:6810-26-0 (3:1) (Pathak et al 2016 ) p 2-hydroxyamino-9 H -pyrido[2,3-b]indole (HONH-AαC) CAS:176853-90-0 (3:1) (Pathak et al 2016 ) q acetaldehyde (4300:1 to 430,000:1) (Birt et al 1998 ) r 16α-hydroxyestrone (16αOHE1) (1:1), CAS:566-76-7, ketoamine type and ketohydroxyamine type adduct found, only ketoamine adduct at α-Ser-63 (Charneira et al 2020 ) s reaction with 4-methylene-2,5-cyclohexadien-1-one (5:1) yielding 4-hydroxybenzyl-adducts (Rajczewski et al 2021 ), the valine adducts were analyzed separately (Degner et al 2018 ) ...…”

The life of Hans-Günter Neumann and his contributions to chemical carcinogenesis

“…Also adducts of reactive compounds or metabolites with serum albumin are valuable biomarkers of exposure to important carcinogenic food contaminants or drugs (Sabbioni and Turesky 2017). Nowadays, progress in analytical methodologies allows for untargeted screening of albumin and hemoglobin adducts in human blood (Carlsson et al 2019;Möller et al 2017). Such 'protein adductomics' will expand our knowledge on human exposure to toxic chemicals, including carcinogenic agents from exogenous and endogenous sources.…”