16α-Hydroxyestrone: mass spectrometry-based methodologies for the identification of covalent adducts formed with blood proteins.

Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of "bottom up" proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted on the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodologies might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1, but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.