Differential alkylation-based redox proteomics--Lessons learnt.
Cysteine is one of the most reactive amino acids. This is due to the electronegativity of sulphur atom in the side chain of thiolate group. It results in cysteine being present in several distinct redox forms inside the cell. Amongst these, reversible oxidations, S-nitrosylation and S-sulfenylation are crucial mediators of intracellular redox signalling, with known associations to health and disease. Study of their functionalities has intensified thanks to the development of various analytical strategies, with particular contribution from differential alkylation-based proteomics methods. Presented here is a critical evaluation of differential alkylation-based strategies for the analysis of S-nitrosylation and S-sulfenylation. The aim is to assess the current status and to provide insights for future directions in the dynamically evolving field of redox proteomics. To achieve that we collected 35 original research articles published since 2010 and analysed them considering the following parameters, (i) resolution of modification site, (ii) quantitative information, including correction of modification levels by protein abundance changes and determination of modification site occupancy, (iii) throughput, including the amount of starting material required for analysis. The results of this meta-analysis are the core of this review, complemented by issues related to biological models and sample preparation in redox proteomics, including conditions for free thiol blocking and labelling of target cysteine oxoforms.
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Katarzyna
K
Wojdyla
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark. Electronic address: adelinar@bmb.sdu.dk.
Adelina
A
Rogowska-Wrzesinska
D000478:Alkylation; D000818:Animals; D018053:Arsenites / Q000737:chemistry; D001205:Ascorbic Acid / Q000737:chemistry; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D005057:Eukaryotic Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry; D008954:Models, Biological; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
D018053:Arsenites; D001205:Ascorbic Acid; D003545:Cysteine; D004229:Dithiothreitol; D005033:Ethylmaleimide; D007460:Iodoacetamide; D010720:Phosphines; D013438:Sulfhydryl Compounds; C080938:tris(2-carboxyethyl)phosphine; C015001:arsenite
Cysteine oxidation; Differential alkylation; Modification site occupancy; Quantitative redox proteomics