Proteomics has expanded to include not only protein expression profiling [1], but the analysis of post-translational modifications [2, 3] and protein-protein interactions [4]. 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].
Many studies of post-translational modifications rely on commercial resins conjugated to the proper ligand to target the particular post-translational modification being analyzed. For example beads-bound anti-phosphotyrosine antibodies have been used in phosphoproteomics [7, 8] and beads coupled to lectins or saccharides in glycomics [9–12]. However this approach is constrained by the availability of the resin with the proper ligand. Supports that exploit more general affinities are also available and proteinA-IgG affinity and poly-histidine-Ni2+ affinity have been used for the analysis of ubiquitinated proteins, the latter implying the preparation of cells in which ubiquitin genes were replaced by a His-tag-ubiquitin coding plasmid [13–15]. A greater degree of flexibility can be achieved by exploiting the reactivity of the naturally present functional groups in biomolecules (i.e. amino, thiol and hydroxyl groups) towards classical derivatizations such as the Schiff base (reductive amination) method, N-hydroxy-succinimide method, carbonyldiimidazole method, epoxy (bisoxirane) method, ethyl dimethylaminopropyl carbodiimide method, maleimide method or hydrazide method. Commercial preactivated supports (agarose and polystyrene derivatives and, to a lesser extent, functionalized silicas) are available and there exist examples of their use [16–19], although it is not the general approach for the study of post-translational modifications.
Among the different post-translational modifications, S-nitrosylation, the addition of a nitric oxide group to cysteine thiols, is raising an increasing interest. Nitric oxide (NO) metabolism in cells has a relative short history but it has been found to be involved in a wide range of physiological and phytopatological functions. Nitric oxide exerts these functions directly or through a group of derived molecules designated as reactive nitrogen species (RNS) which are involved in post-translational modifications in cell signalling including binding to metal centres, nitration of tyrosine and S-nitrosylation of thiol groups [20]. In animals it is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles and in many cases pathophysiology correlates with dysregulation of S-nitrosylation [21, 22]. As a classical post-translational protein modifications, S-nitrosylation is a reversible mechanism. It is not strictly dependent on enzymatic catalysis, although enzymes with nitrosylase and denitrosylase activity have been reported. In plants, evidences also support the importance of S-nitrosylation as a physiological mediator and NO is produced in most tissues [23–26]. At cellular level NO is generated and/or diffuses to cytosol, chloroplast, mitochondria, peroxisomes as well as nucleus. Nitrate reductase and L-arginine dependent nitric oxide synthase are both well recognized source of NO in plant cells [27]. Additionally NO can be produced nonenzymatically from nitrite and polyamines induce the biosynthesis of NO [26].
The high lability of the S-NO bond makes the study of proteins regulated by S-nitrosylation/denitrosylation a challenging task. Most studies have focused on already S-nitrosylated proteins and have relied on the lability of the S-NO bond to isolate them. Two different strategies are commonly assayed: i) tag derivatization and ii) resin assisted capture. The tag derivatization strategy is based on the selective derivatization of S-NO bonds with a stable tag that confers affinity for a specific resin. Thus, the biotin switch technique (BST) yields the selective biotinylation of S-nitrosylated Cys, isolation of the biotinylated proteins (i.e. S-nitrosylated proteins) by affinity chromatography on a streptavidin bound bead and their analysis by tryptic digestion and MS [28]. Variants of this technique are SNOSID (SNO-Cys Site Identification) [29] and His-tag Switch [30]. The resin-assisted capture strategy of S-nitrosylated proteins (SNO-RAC) uses the labile S-NO bond not to attach a tag and confer affinity for a resin but as a source of thiol groups that react with a thiol reactive resin [31]. Also the combination of selective fluorescent labeling of the S-NO bond and bidimensional electrophoresis has been used for the comparative study of biological samples in a variant of the DIGE technique named SNO-DIGE [32].
From a chemical point of view, S-nitrosylation reactions can be understood as the electrophilic attack of a nitro-sonium cation (NO+) to a thiolate [33]. In physiological conditions S-nitrosylation agents such as peroxynitrite (ONOO–), hyponitrite anión (NO–) and metal-NO complexes as well as other processes such as reaction of thiyl radicals (SO•) with nitric oxide radical (•NO) may account for the S-nitrosylation of the relatively electropositive sulfur atoms present in protein [23]. Particularly transnitrosylation (i.e. the exchange of NO+ from a S-nitrosylated specimen to a reactive thiolate of a target protein) seems to be an important process for in vivo protein S-nitrosylation [33]. In fact S-nitrosylated glutathione (GSNO) originated by reaction of NO with GSH is considered a natural reservoir of NO [24, 34], being widely used for in vitro protein S-nitrosylation. Moreover, trans-nitrosylase activity has been reported for some proteins, among them hemoglobin [35] and protein disulfide isomerase [36], and particularly human thioredoxin 1 (Tdx1) that has been proposed as a “master regulator” capable of protein modulation via both redox reactions and S-nitrosylation [37]. Thus the identification of proteins that interact with transnitrosylating agents provides an additional insight complementary to that obtain by the approaches that focus on already S-nitrosylated proteins. To our knowledge such transnitrosylation studies in plants have not been published.
The availability of pre-activated materials that can be turned into an affinity support by direct reaction with the desired NO donor is a key element in the search for transnitrosylation target candidates. In this context, we have reported the biotechnological application of the reactivity of vinyl sulfone group in mild conditions toward both amino and thiol groups naturally present in biomolecules [38–40]. In the particular case of silica, the functionalization with vinyl sulfone yields a “ready to use” pre-activated material that upon reaction with both amino and thiol groups naturally present in biomolecules can be turned into an affinity support with the advantage of being obtained ad hoc by direct incubation with the desired macromolecule in mild conditions that preserves the biological function [40]. Thus the reaction with glutathione turned the vinyl sulfone resin into a glutathione-S-transferase (GST) affinity support that led to the one step purification of the GST and that can be used for the purification of GST tag fused proteins [40]. As an additional proof of concept, the reaction with thioredoxins (Tdx) h1 and h2 yielded two affinity chromatographic supports with different specificity in the capturing of proteins targets in vitro: Tdx h1 captured a transcription factor whereas Tdx h2 interacted with classical antioxidant proteins [41].
In this paper we further explore the use of the vinyl sulfone silica as an open support to address a specific proteomic problem: identification of S-nitrosylated proteins from a mature tissue (i.e. leaves from mature Pisum sativun) and from an embryogenic tissue (i.e. hypocotyls from Helianthus annuus). We hypothesize that: i) transnitrosylation by GSNO is a feasible mechanism to account for S-nitrosylation of rather electropositive sulfur atoms from proteins, ii) affinity chromatography is a good approach to isolate proteins that are prone to undergo S-transnitrosylation and iii) vinyl sulfone silica is a suitable support to be functionalized with GSNO and carry out such affinity chromatography. Our results suggest a complex interrelation among different post-translational modifications mediated by RNS.
