Profiling Dopamine-Induced Oxidized Proteoforms of β-synuclein by Top-Down Mass Spectrometry.

Giuseppe Legname,PierLuigi Mauri,Elena De Cecco,Carlo Santambrogio,Rita Grandori,Erika Ponzini,Arianna Luise,Martina Sollazzo,Frank Sobott

doi:10.3390/antiox10060893

Abstract

The formation of multiple proteoforms by post-translational modifications (PTMs) enables a single protein to acquire distinct functional roles in its biological context. Oxidation of methionine residues (Met) is a common PTM, involved in physiological (e.g., signaling) and pathological (e.g., oxidative stress) states. This PTM typically maps at multiple protein sites, generating a heterogeneous population of proteoforms with specific biophysical and biochemical properties. The identification and quantitation of the variety of oxidized proteoforms originated under a given condition is required to assess the exact molecular nature of the species responsible for the process under investigation. In this work, the binding and oxidation of human β-synuclein (BS) by dopamine (DA) has been explored. Native mass spectrometry (MS) has been employed to analyze the interaction of BS with DA. In a second step, top-down fragmentation of the intact protein from denaturing conditions has been performed to identify and quantify the distinct proteoforms generated by DA-induced oxidation. The analysis of isobaric proteoforms is approached by a combination of electron-transfer dissociation (ETD) at each extent of modification, quantitation of methionine-containing fragments and combinatorial analysis of the fragmentation products by multiple linear regression. This procedure represents a promising approach to systematic assessment of proteoforms variety and their relative abundance. The method can be adapted, in principle, to any protein containing any number of methionine residues, allowing for a full structural characterization of the protein oxidation states.

Highlights

The oxidation of proteins at methionine residues (Met) to form methionine sulfoxides (MetO) is a reversible post-translational modification (PTM) known to regulate different cellular functions, ranging from signaling to enzymatic activity and protein degradation [1,2,3,4].The control of important biological processes is ensured by a subtle tuning of Met redox status, thanks to the action of enzymes such as monooxygenases of the MICAL family or methionine sulfoxide reductases (Msrs) [2,3,4]
Biomedical research in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) has mainly focused on the study of AS aggregation and the factors affecting the formation of fibrils and oligomers
A new approach has been described here to tackle the complexity deriving from the combination of multiple PTMs

Summary

Introduction

The control of important biological processes is ensured by a subtle tuning of Met redox status, thanks to the action of enzymes such as monooxygenases of the MICAL family or methionine sulfoxide reductases (Msrs) [2,3,4]. Besides this involvement in physiological conditions, MetO formation is commonly related to the excessive production of reactive oxygen species characterizing several conditions of cellular oxidative stress [5]. The synuclein family consists of three intrinsically disordered proteins (IDPs): α-synuclein (AS), β-synuclein (BS) and γ-synuclein (GS) [10]

Methods

Results

Conclusion