title: Vinyl sulfone silica: application of an open preactivated support to the study of transnitrosylation of plant proteins by S-nitrosoglutathione.
abstract: BACKGROUND
  S-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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Vinyl 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.
authors.affiliation: 1Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Departamento de Bioquímica y Biología Molecular, Universidad de Jaén, de Jaén, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Begara-Morales
authors.forename: F Javier
authors.initials: FJ
authors.lastname: López-Jaramillo
authors.forename: Beatriz
authors.initials: B
authors.lastname: Sánchez-Calvo
authors.forename: Alfonso
authors.initials: A
authors.lastname: Carreras
authors.forename: Mariano
authors.initials: M
authors.lastname: Ortega-Muñoz
authors.forename: Francisco
authors.initials: F
authors.lastname: Santoyo-González
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.forename: Juan B
authors.initials: JB
authors.lastname: Barroso
mesh_terms: D020816:Amino Acid Motifs; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006368:Helianthus / Q000737:chemistry / Q000378:metabolism*; D018532:Pisum sativum / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D012822:Silicon Dioxide / Q000737:chemistry; D013450:Sulfones / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D013450:Sulfones; D026422:S-Nitrosoglutathione; C009873:divinyl sulfone; D012822:Silicon Dioxide