Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

S-sulfhydration/desulfhydration and S-nitrosylation/denitrosylation: a common paradigm for gasotransmitter signaling by H2S and NO.

Sulfhydryl groups on protein Cys residues undergo an array of oxidative reactions and modifications, giving rise to a virtual redox zip code with physiological and pathophysiological relevance for modulation of protein structure and functions. While over two decades of studies have established NO-dependent S-nitrosylation as ubiquitous and fundamental for the regulation of diverse protein activities, proteomic methods for studying H2S-dependent S-sulfhydration have only recently been described and now suggest that this is also an abundant modification with potential for global physiological importance. Notably, protein S-sulfhydration and S-nitrosylation bear striking similarities in terms of their chemical and biological determinants, as well as reversal of these modifications via group-transfer to glutathione, followed by the removal from glutathione by enzymes that have apparently evolved to selectively catalyze denitrosylation and desulfhydration. Here we review determinants of protein and low-molecular-weight thiol S-sulfhydration/desulfhydration, similarities with S-nitrosylation/denitrosylation, and methods that are being employed to investigate and quantify these gasotransmitter-mediated cell signaling systems.

Methods for detection and characterization of protein S-nitrosylation.

Reversible protein S-nitrosylation, defined as the covalent addition of a nitroso moiety to the reactive thiol group on a cysteine residue, has received increasing recognition as a critical post-translational modification that exerts ubiquitous influence in a wide range of cellular pathways and physiological processes. Due to the lability of the S-NO bond, which is a dynamic modification, and the low abundance of endogenously S-nitrosylated proteins in vivo, unambiguous identification of S-nitrosylated proteins and S-nitrosylation sites remains methodologically challenging. In this review, we summarize recent advancements and the use of state-of-art approaches for the enrichment, systematic identification and quantitation of S-nitrosylation protein targets and their modification sites at the S-nitrosoproteome scale. These advancements have facilitated the global identification of >3000 S-nitrosylated proteins that are associated with wide range of human diseases. These strategies hold promise to site-specifically unravel potential molecular targets and to change S-nitrosylation-based pathophysiology, which may further the understanding of the potential role of S-nitrosylation in diseases.

Direct methods for detection of protein S-nitrosylation.

S-nitrosylation of protein cysteine residues is known to be an important mechanism for nitric oxide signaling. However, the detection of protein S-nitrosylation is still challenging due to technical limitations of current methods. This chapter provides a brief review on recent developments of methods, which directly target S-nitroso moieties for detection. We also describe in detail the protocol of an organophosphine-based biotin labeling of protein S-nitroso moieties.

Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.

The proteomic analysis of S-nitrosylated protein (SNO-proteins) has long depended on the biotin switch technique (BST), which requires blocking of free thiols, ascorbate-based denitrosylation of SNO-Cys, biotinylation of nascent thiol and avidin-based affinity isolation. A more recent development is resin assisted-capture of SNO-proteins (SNO-RAC), which substitutes thiopropyl Sepharose (TPS) for biotin-avidin, thus reducing the number of steps required for enrichment of S-nitrosylated proteins. In addition, SNO-RAC facilitates on-resin proteolytic digestion following SNO-protein capture, greatly simplifying the purification of peptides containing sites of S-nitrosylation ("SNO-sites"). This resin-based approach has also now been applied to detection of alternative Cys-based modifications, including S-palmitoylation (Acyl-RAC) and S-oxidation (Ox-RAC). Here, we review the important steps to minimize false-positive identification of SNO-proteins, give detailed methods for processing of protein-bound TPS for mass spectrometry (MS) based analysis, and discuss the various quantitative MS methods that are compatible with SNO-RAC. We also discuss strategies to overcome the current limitations surrounding MS-based SNO-site localization in peptides containing more than one potential target Cys residue. This article therefore serves as a starting point and guide for the MS-focused exploration of SNO-proteomes by SNO-RAC.

Mass spectrometry-based identification of S-nitrosocysteine in vivo using organic mercury assisted enrichment.

Protein S-nitrosylation is considered as one of the molecular mechanisms by which nitric oxide regulates signaling events and protein function. The present review presents an updated method which allows for the site-specific detection of S-nitrosylated proteins in vivo. The method is based on enrichment of S-nitrosylated proteins or peptides using organomercury compounds followed by LC-MS/MS detection. Technical aspects for determining the reaction and binding efficiency of the mercury resin that assists enrichment of S-nitrosylated proteins are presented and discussed. In addition, emphasis is given to the specificity of the method by providing technical details for the generation of four chemically distinct negative controls. Finally it is provided an overview of the key steps for generation and evaluation of mass spectrometry derived data.

Ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction.

This present work describes the ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction based on a novel adsorbent TiO(2)-graphene nanocomposite. 1,3,5,7-Tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence is used as fluorescent probe for S-nitrosothiols label. The procedure is based on the fluorescent probe selective reaction with S-nitrosothiols to form highly fluorescent product, its extraction to the TiO(2)-graphene-packed SPE cartridge and spectrofluorimetric determination. The experimental variables affecting the extraction procedure, such as the type of the eluent and its volume, sample pH, and sample volume, have been studied. Under the optimized extraction conditions, the method showed good linearity in the range of 0.5-100 nM. The limit of detection was 0.08 nM (signal-to-noise ratio=3). Relative standard deviation was 2.5%. The developed method was applied to the determination of S-nitrosothiols in human blood samples with recoveries of 92.0-104.0%. This work revealed the great potentials of TiO(2)-graphene as an excellent sorbent material in the analysis of biological samples.

Quantitative analysis of S-nitrosylated proteins.

Protein S-nitrosylation is the covalent binding of nitric oxide to specific cysteine residues in proteins. This modification influences a large number of cellular events and signaling processes. As this process is finely regulated in vivo, the level of nitrosylation changes in response to different stimuli. Since its introduction, the biotin-switch technique (BST) is the most used indirect method for the study of S-nitrosylation both in vivo and in vitro and its coupling with mass spectrometry-based proteomics lead to the identification of the S-nitroso proteome in different organisms. However, this method does not give any information about the posttranslational modification level on the same residue in different biological conditions. Quantitative proteomic methods can assess the relative change in S-nitrosylation for hundreds sites in a single experiment. Stable isotope labeling by aminoacids in cell culture (SILAC) is one of the most used and accurate quantitative techniques in MS-based proteomics. Here we present a SILAC-based method for the quantification of endogenously S-nitrosylated proteins in RAW 264.7 cells.

The determination of S-nitrosothiols in biological samples--procedures, problems and precautions.

Despite the considerable number of published studies in the field of S-nitrosothiols (RSNO), the determination of these compounds in biological samples still represents an analytical challenge, due to several technical obstacles and often long sample preparation procedures. Other problems derive from the intrinsic lability of RSNO and the absence of certified reference material, analytically validated methods or suitable internal standards. Also, thiols and nitrites are usually present at high concentrations in biological matrices, and all precautions must be adopted in order to prevent artifactual formation of RSNO. Preanalytical steps (sampling, preservation and pre-treatment of samples) are particularly critical for the obtainment of reliable measurements. Three main mechanisms have been identified capable of compromising the assays: metal-catalyzed RSNO decomposition, reduction of the S-NO bond by thiols (transnitrosylation reactions) and enzymatic degradation of S-nitroso-glutathione (GSNO) by endogenous γ-glutamyltransferase (GGT) activity possibly present in the sample. If not adequately controlled, these factors likely contribute to the wide dispersion of values reported in the literature for RSNO and GSNO concentration in biological fluids, blood in the first place. The use of metal chelators, thiol reagents and GGT inhibitors appears therefore mandatory.