Development of a Method and Validation for the Quantitation of FruArg in Mice Plasma and Brain Tissue Using UPLC–MS/MS

Aged garlic extract (AGE) is a popular nutritional supplement and is believed to promote health benefits by exhibiting antioxidant and anti-inflammatory activities and hypolipidemic and antiplatelet effects. We have previously identified <i>N</i>-α-(1-deoxy-d-fructos-1-yl)-l-arginine (FruArg) as a major contributor to the bioactivity of AGE in BV-2 microglial cells whereby it exerted a significant ability to attenuate lipopolysaccharide-induced neuroinflammatory responses and to regulate the Nrf2-mediated antioxidant response. Here, we report on a sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) protocol that was validated for the quantitation of FruArg in mouse plasma and brain tissue samples. Solid-phase extraction was used to separate FruArg from proteins and phospholipids present in the biological fluids. Results indicated that FruArg was readily absorbed into the blood circulation of mice after intraperitoneal injections. FruArg was reliably detected in the subregions of the brain tissue postinjection, indicating that it penetrates the blood–brain barrier in subnanomolar concentrations that are sufficient for its biological activity

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

One activation gene regulatory module in the fifth data set.

<p>The gene regulatory module was constructed for the comparison between Col (Wild-Type) and <i>hae</i>-3 <i>hsl</i>2-3 (mutant), and the expression correlation score of the module was 0.85. The decision tree on the middle top illustrates how four putative transcription factors (AT3G59580, AT1G28050, AT1G52890, AT1G56650) may collaboratively regulate the cluster of co-expressed genes in the middle bottom, where each row denotes a gene listed in the bottom left box and each column denotes one of six biological replicates of two samples (i.e. Col and <i>hae-3 hsl2-3</i>). The levels of gene expression values were represented by different colors ranging from lowest (green) to highest (red). The expression of the genes in the cluster under each sample is predicted to be regulated according to the expression levels of transcription factors listed on top of the condition. For example, under the first replicate of Col, the low expression of AT3G59580 and the low expression of AT1G28050 caused the repression of the group of genes.</p

Differential spots detection by the SameSpots software.

<p>A total of 1,925 protein spots on the 24-cm 2D-DIGE gels were detected using SameSpots analysis. Compared to untreated conditions, 25 protein spots showed significant protein level changes (ratio >1.3, p<0.05) after treatment with LPS. In comparison to LPS treatment, 20 and 21 protein spots were altered by AGE and FruArg, respectively. All of these differential spots were indicated on a representative 2D-DIGE gel (<b>A</b>). Quantification results of two example spots, #825 (left) and #1737 (right), were shown (<b>B</b>).</p

The workflow of predicting gene functions.

<p>The blue box denotes the tool used in the step of predicting gene functions. The tools of PSI-BLAST and HHSearch used in MULTICOM-PDCN are listed inside the blue box. The external input information is represented by brown boxes and the output information is represented by green boxes. The information flow between these components is denoted by arrows.</p