Vinyl sulfone silica: application of an open preactivated support to the study of transnitrosylation of plant proteins by S-nitrosoglutathione.

Juan C Begara-Morales,Francisco J Corpas,Mariano Ortega-Muñoz,F Javier López-Jaramillo,Juan B Barroso,Alfonso Carreras,Beatriz Sánchez-Calvo,Francisco Santoyo-González

doi:10.1186/1471-2229-13-61

Abstract

BackgroundS-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles. The high lability of the S-NO bond makes the study of proteins regulated by S-nitrosylation/denitrosylation a challenging task and most studies have focused on already S-nitrosylated proteins. We hypothesize that: i) S-nitrosoglutathione (GSNO) transnitrosylation is a feasible mechanism to account for the physiological S-nitrosylation of rather electropositive sulfur atoms from proteins, ii) affinity chromatography is a suitable approach to isolate proteins that are prone to undergo S-transnitrosylation and iii) vinyl sulfone silica is a suitable chromatographic bead.ResultsThe combination of vinyl sulfone silica with GSNO yielded an affinity resin that withstood high ionic strength without shrinking or deforming and that it was suitable to isolate potential GSNO transnitrosylation target candidates. Fractions eluted at 1500 mM NaCl resulted in a symmetrical peak for both, protein and S-nitrosothiols, supporting the idea of transnitrosylation by GSNO as a selective process that involves strong and specific interactions with the target protein. Proteomic analysis led to the identification of 22 physiological significant enzymes that differ with the tissue analyzed, being regulatory proteins the most abundant group in hypocotyls. The identification of chloroplastidic FBPase, proteasome, GTP-binding protein, heat shock Hsp70, syntaxin, catalase I, thioredoxin peroxidase and cytochrome P450 that have already been reported as S-nitrosylated by other techniques can be considered as internal positive controls that validate our experimental approach. An additional validation was provided by the prediction of the S-nitrosylation sites in 19 of the GSNO transnitrosylation target candidates.ConclusionsVinyl sulfone silica is an open immobilization support that can be turned ad hoc and in a straightforward manner into an affinity resin. Its potential in omic sciences was successfully put to test in the context of the analysis of post-translational modification by S-nitrosylation with two different tissues: mature pea leaves and embryogenic sunflower hypocotyls. The identified proteins reveal an intriguing overlap among S-nitrosylation and both tyrosine nitration and thioredoxin regulation. Chloroplastidic FBPase is a paradigm of such overlap of post-translational modifications since it is reversible modified by thioredoxin and S-nitrosylation and irreversibly by tyrosine nitration. Our results suggest a complex interrelation among different modulation mechanisms mediated by NO-derived molecules.

Highlights

S-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles
It is important to recall that GSNO affinity chromatography on S-nitrosoglutathione-sepharose (GSNO-sepharose) has been described and that as an affinity support GSNO-vinyl sulfone silica is closely related to GSNO-sepharose since both resins are based on glutathione covalently immobilized by the amine end, being the major difference the solid support [44,45]
GSNO affinity chromatography on GSNO-sepharose has been reported as an approach to screen proteins that may be modulated by Nitric oxide (NO) from a different conceptual hypothesis: GSNO does not yield S-nitrosylation but S-glutathionylation and this S-glutathionylation induced by GSNO has been proposed as a general mechanism by which GSNO modifies proteins [44,45]

Summary

Introduction

S-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles. The classical experimental approach consists of a separation step based on two dimensional protein electrophoresis (2-DE) followed by an identification step that involves the cleavage with sequence specific endopeptidases and mass spectrometry (MS) for the high-throughput identification [5]. In this context affinity chromatography has been included as part of the traditional workflow either before 2-DE to selectively concentrate the sample or prior to MS for either the purification of the peptides resulting from the digestion or for the isolation of proteins bearing post-translational modifications [6]. Commercial preactivated supports (agarose and polystyrene derivatives and, to a lesser extent, functionalized silicas) are available and there exist examples of their use [16,17,18,19], it is not the general approach for the study of posttranslational modifications

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Conclusion