Bottom-up proteomics analysis for adduction of the broad-spectrum herbicide atrazine to histone
Shaogang Chu1, Robert J Letcher2
1Ecotoxicology and Wildlife Health Division, Wildlife and Landscape Science Directorate, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1A 0H3, Canada.
Abstract:
Histones are the major proteinaceous components of chromatin in eukaryotic cells and an important part of the epigenome. The broad-spectrum herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1, 3, 5-triazine) and its metabolites are known to form protein adducts, but the formation of atrazine-histone adducts has not been studied. In this study, a bottom-up proteomics analysis method was optimized and applied to identify histone adduction by atrazine in vitro. Whole histones of calf thymus or human histone H3.3 were incubated with atrazine. After solvent-based protein precipitation, the protein was digested by trypsin/Glu-C and the resulting peptides were analyzed by high-resolution mass spectrometry using an ultra-high-performance liquid chromatograph interfaced with a quadrupole Exactive-Orbitrap mass spectrometer. The resulting tryptic/Glu-C peptide of DTNLCAIHAK from calf thymus histone H3.1 or human histone H3.3 was identified with an accurate mass shift of +179.117 Da in atrazine incubated samples. It is deduced that a chemical group with an elemental composition of C8H13N5 (179.1171 Da) from atrazine adducted with calf thymus histone H3.1 or human histone H3.3. It was confirmed by MS/MS analysis that the adduction position was at its cysteine 110 residue. Time- and concentration-dependent assays also confirmed the non-enzymatic covalent modification of histone H3.3 by atrazine in vitro. Thus, the potential exists that atrazine adduction may lead to the alteration of histones that subsequently disturbs their normal function.
