A Novel Strategy for Global Analysis of the Dynamic Thiol Redox Proteome

Abstract

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations

“…Such correction has however rarely been implemented into previous thiol redox strategies, as reviewed in [2]. Most of the strategies attempting correction of modification levels by protein abundance changes rely on non-isobaric isotopic labelling which suffers from reduced sensitivity and multiplexing capability [23][24][25]. Furthermore, most of these protocols are laborious, requiring additional experiments employing alternative quantitation strategies and extensive fractionation to determine protein abundance changes [23,25].…”

“…The majority of current methods provide quantitative information, yet, correction of modification levels by protein abundance changes is still a rare practise. In fact there are only few existing methods which either inherently correct observed modification levels in such a way [15], or with additional workload provide such correction [23][24][25]. However, all of these approaches use non-isobaric isotopic labels which suffer from low throughput due to increased sample complexity, limited dynamic range of quantitation and typically can target only 2 samples/conditions due to isotopic limitations.…”

Thiol switches in mitochondria: operation and physiological relevance

“…The addition of H 2 O 2 to cells was reported to preferentially damage (or modify) mitochondrial proteins (Garcia et al, 2010;Perluigi et al, 2010;Qin et al, 2011;Martinez-Acedo et al, 2012;Stauch et al, 2014). In several investigations H 2 O 2 addition reduced respiratory activity and mitochondrial membrane potential.…”

¿Quo Vadis, Proteómica?

“…Estos subproteomas pueden obtenerse mediante una interminable lista de estrategias, que incluyen el fraccionamiento subcelular (proteoma mitocondrial, exosoma, secretoma7, "sheddoma") y la cromatografía de afinidad (fosfoproteoma), o utilizando las propiedades de unión específica de proteínas o anticuerpos (interactomas, quinomas). Otras aproximaciones ingeniosas combinan modificaciones químicas o enzimáticas con estrategias de marcaje y purificación selectivos que permiten enriquecer subpoblaciones de péptidos modificados o procesados de forma muy específica (nitrosiloma, degradoma, proteoma posicional, proteoma redox) (8). Y las ideas no acaban ahí.…”