Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases.

Trong Khoa Pham,Mark O Collins,Pamela J Shaw,Richard J Mead,Weronika A Buczek

doi:10.3389/fnmol.2021.678837

Abstract

Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms.

Highlights

Post-translational modifications (PTMs) of cysteine (Cys) residues play a crucial role in protein function and cellular homeostasis
We have summarized the main proteomic approaches for different types of oxidized cysteine residues (Oxi-Cys) PTM analyses in Figure 2, and their applications, advantages and disadvantages are shown in Table 1 and Supplementary Table 1, respectively
The global quantitative Oxi-Cys analysis can be performed based on the ratio of Cyscontaining peptide extracted ion chromatogram (XIC) intensities detected by mass spectrometry (MS) between samples

Summary

INTRODUCTION

Post-translational modifications (PTMs) of cysteine (Cys) residues play a crucial role in protein function and cellular homeostasis. The global quantitative Oxi-Cys analysis can be performed based on the ratio of Cyscontaining peptide XIC (extracted ion chromatogram) intensities detected by MS (either reduced or oxidized Cys intensities) between samples This type of analysis includes two different alkylating steps (e.g., IAM and NEM), stable isotopic labeling (SICyLIA), resin-assisted capture (RAC) combined with isobaric labeling reagents (TMT, iTRAQ, and iCAT). It combines two different strategies; tandem orthogonal proteolysis (TOP) and activity-based protein profiling and has been used to predict functions of cysteine residues in proteomes (Weerapana et al, 2007) This approach consists of three main steps, alkylating reduced Cys in proteins with a probe, click chemistry-based incorporation of isotopicallylabeled cleavable tags, protein purification and on-bead digestion (Weerapana et al, 2007). Various probes recently introduced (summarized in Figure 5C) include (i) IA-alkyne based probes such as chloropyridine (RB2) and fluoronitrobenzene (ERW3) (Shannon et al, 2014), a photocaged bromomethyl ketone (CBK) (Abo and Weerapana, 2015), iodomethyl ketone

Method used

Findings

DISCUSSION AND FUTURE