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

Abstract

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 auto-sampler 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 minutes, 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.

Low Doses of Arsenic in a Mouse Model of Human Exposure and in Neuronal Culture Lead to S-Nitrosylation of Synaptic Proteins and Apoptosis via Nitric Oxide

“…First, we quantified S-nitrosoglutathione (GSNO) in the brains of these mice. Using chemical probe-based mass spectrometry (MS) for targeted GSNO quantification [22], we found a statistically significant upregulation of GSNO in the cortices of the SA-treated mice compared to the controls ( Figure 4A). Next, we examined brain regions [23] with potential elevation of Ntyr levels.…”

“…The quantification of GSNO internal standard synthesis in the cortex, sample preparation for GSNO quantification, and ESI +/− QqQ-MS analyses were carried out as described by us previously [22]. Briefly, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of GSNO.…”

Shank3 mutation in a mouse model of autism leads to changes in the S-nitroso-proteome and affects key proteins involved in vesicle release and synaptic function

“…To test whether the nNOS activity was altered, we measured S-nitrosoglutathione (GSNO) levels [72] in all WT and KO samples (Fig. 4g) and identified an increase of GSNO levels in the KO compared to the WT.…”

“…For GSNO internal standard synthesis, sample preparation for GSNO quantification, and ESI + -QqQ-MS analyses, see reference [72] and SI.…”

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis

“…A derivatizing‐reagent‐modified polymer monolithic column [poly(methacrylic acid‐ co ‐ethylene dimethacrylate)] was prepared by Wang, Garcia, Gong, Wishnok, and Tannenbaum (2018) and adapted for on‐line SPD–LC–MS analysis of S ‐nitrosothiols (a circulating endogenous reservoir of nitric oxide), based on the sequence of reactions depicted in Scheme 6. According to this schema the cation exchange polymer monolith is derivatized with 2‐[1‐(dimethylamino)ethanethioate] triphenylphosphine at a suitable pH value.…”