A Metabolomics-Inspired Strategy for the Identification of Protein Covalent Modifications

Abstract

Identification of protein covalent modifications (adducts) is a challenging task mainly due to the lack of data processing approaches for adductomics studies. Despite the huge technological advances in mass spectrometry (MS) instrumentation and bioinformatics tools for proteomics studies, these methodologies have very limited success on the identification of low abundant protein adducts. Herein we report a novel strategy inspired on the metabolomics workflows for the identification of covalently-modified peptides that consists on LC-MS data preprocessing followed by statistical analysis. The usefulness of this strategy was evaluated using experimental LC-MS data of histones isolated from HepG2 and THLE2 cells exposed to the chemical carcinogen glycidamide. LC-MS data was preprocessed using the open-source software MZmine and potential adducts were selected based on the m/z increments corresponding to glycidamide incorporation. Then, statistical analysis was applied to reveal the potential adducts as those ions are differently present in cells exposed and not exposed to glycidamide. The results were compared with the ones obtained upon the standard proteomics methodology, which relies on producing comprehensive MS/MS data by data dependent acquisition and analysis with proteomics data search engines. Our novel strategy was able to differentiate HepG2 and THLE2 and to identify adducts that were not detected by the standard methodology of adductomics. Thus, this metabolomics driven approach in adductomics will not only open new opportunities for the identification of protein epigenetic modifications, but also adducts formed by endogenous and exogenous exposure to chemical agents.

Covalent Histone Modification by an Electrophilic Derivative of the Anti-HIV Drug Nevirapine

“…A larger number of adduction sites were identified in histone H2B. This preference was also observed with 4-oxo-2-nonenal-[ 35 ], as well as furan- and glycidamide-induced [ 40 , 44 ] covalent modifications, thereby suggesting that histone 2B is a major target for non-enzymatic adduction. The majority of the NVP modifications ( Figure 2 ) occurred in lysine residues, which is consistent with the fact that lysines are the most abundant nucleophilic amino acids of histones.…”

“…Supplementary Materials , Figure S1 ). In contrast, the 2 h trypsin digestion with a 1:10 enzyme/histone ratio, which we previously used successfully for the identification of covalent furan- and glycidamide-derived histone modifications, in vivo and in vitro in cells, respectively [ 40 , 44 ], allowed the identification of NVP-derived modifications in all histone samples analyzed.…”

In vitro biological activity of extracts from marine bacteria cultures against Toxoplasma gondii and Mycobacterium tuberculosis

“…Raw data from UHPLC‐MS/MS run were converted to.mzXML format with Bruker's DataAnalysis software (Moro et al, 2018). LC‐MS data were preprocessed with the open‐source software MZmine and consisted of peak detection, isotopes removal, peak matching and peak filling (Nunes et al, 2019). Peak detection was performed in three steps: (i) mass detection with noise value = 10,000; (ii) chromatogram builder with minimum time span = 0.01 min, minimum height = 5000 and m/z tolerance = 5 ppm and (iii) deconvolution with peak width = 0.01–0.5 min, noise = 5000.…”

Bottom-up proteomics analysis for adduction of the broad-spectrum herbicide atrazine to histone

“…Proteins can covalently adduct with xenobiotic compounds from exposure to endogenous or exogenous chemicals, such as drugs, pesticides, or their metabolites, at active amino acid residues [ 1 – 8 ]. Research over the past half century has demonstrated that these protein adducts might lead to multiple health issues, including cancer and immune system effects [ 1 , 5 , 7 , 9 – 13 ].…”

“…Proteins can covalently adduct with xenobiotic compounds from exposure to endogenous or exogenous chemicals, such as drugs, pesticides, or their metabolites, at active amino acid residues [ 1 – 8 ]. Research over the past half century has demonstrated that these protein adducts might lead to multiple health issues, including cancer and immune system effects [ 1 , 5 , 7 , 9 – 13 ]. Therefore, identification of xenobiotics adducted to key proteins and identification of the sites of adduction within the protein are important to better understand the events underlying diseases and chemically induced adverse reactions.…”