Mass Spectrometry-Based Methodologies for Targeted and Untargeted Identification of Protein Covalent Adducts (Adductomics): Current Status and Challenges

Abstract

Protein covalent adducts formed upon exposure to reactive (mainly electrophilic) chemicals may lead to the development of a wide range of deleterious health outcomes. Therefore, the identification of protein covalent adducts constitutes a huge opportunity for a better understanding of events underlying diseases and for the development of biomarkers which may constitute effective tools for disease diagnosis/prognosis, for the application of personalized medicine approaches and for accurately assessing human exposure to chemical toxicants. The currently available mass spectrometry (MS)-based methodologies, are clearly the most suitable for the analysis of protein covalent modifications, providing accuracy, sensitivity, unbiased identification of the modified residue and conjugates along with quantitative information. However, despite the huge technological advances in MS instrumentation and bioinformatics tools, the identification of low abundant protein covalent adducts is still challenging. This review is aimed at summarizing the MS-based methodologies currently used for the identification of protein covalent adducts and the strategies developed to overcome the analytical challenges, involving not only sample pre-treatment procedures but also distinct MS and data analysis approaches.

The Mercapturomic Profile of Health and Non-Communicable Diseases

“…One of the main limitations to assess the global mercapturomic profile is the fact that the mercapturate pathway-related metabolites are often minor metabolites [105]. Despite the enormous technological advances in MS instrumentation, the identification of this minor adducts is still challenging.…”

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

“…Toward this end, we incubated the human histone octamer and recombinant human histone H4 with 12-mesyloxy-NVP ( 5 , Scheme 1 ), a synthetic surrogate for the reactive electrophilic metabolite 3 and a synthetic precursor of metabolite 4 , both derived from 12-hydroxy-NVP. Multiple sites of histone modification by NVP were identified through the use of an MS-based bottom-up proteomics approach [ 41 ].…”

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

“…Namely, human serum albumin (HSA) adducts occur at 0.1 mol% levels, or less, in vivo (reviewed by Sabbioni and Turesky, 2017). A frequent strategy to overcome this issue is to monitor the formation of covalent adducts in targeted (hot-spots) residues of proteins, using multiple reaction monitoring (MRM) acquisition to target specific parent and fragments ions (reviewed by Nunes et al, 2019). Despite the indisputable role of such approaches for the identification of biomarkers of exposure (reviewed by Carlsson et al, 2019), they are ineffective in providing information on the underlying mechanisms of the chemically-induced adverse reactions.…”

“…The major trend in such investigations is to adopt the MS-based shotgun proteomics workflows that traditionally rely on the chromatographic separation of digested peptides followed by a data dependent analysis (DDA), where MS and MS/MS data of selected precursors are afforded in a single run, thereby allowing subsequent adduct identification using database search engines that compare experimental and theoretical MS/MS spectra (reviewed by Gan et al, 2016; Tailor et al, 2016; Sabbioni and Turesky, 2017). Despite this workflow has been successfully applied to adductomics studies for the identification of high-abundant covalent adducts (reviewed by Nunes et al, 2019), it is easy to understand the failure of this strategy in the identification of low-abundant adducted peptides in vivo and ex vivo . In fact, by DDA methods only 10% of detectable peptides are identified and these methods are linked with low reproducibility across runs (Michalski et al, 2011).…”