In-Gel Detection of S-Nitrosated Proteins Using Fluorescence Methods

Kettenhofen, Nicholas J.; Wang, Xunde; Gladwin, Mark T.; Hogg, Neil

doi:10.1016/s0076-6879(08)01204-4

Cited by 36 publications

(34 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Optimal muscle length (L o) was determined from isometric twitch contractions (47), and forces were recorded on a PowerLab running Chart (6,7). Light from the dissection lamp diffused through the isolated muscle, and, therefore, is likely to break any S-nitrosylation bonds that may form, unlike indirect exposure to white light from laboratory lighting (24). Contracted muscles were then stimulated (pulse durations: 600 ms at a frequency of 60 Hz for 25 contractions/min) during the final 10 min of incubation.…”

Section: Methodsmentioning

confidence: 99%

Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction

Merry

Lynch

McConell

2010

American Journal of Physiology-Regulatory, Integrative and Comp

View full text Add to dashboard Cite

Merry TL, Lynch GS, McConell GK. Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction. Am J Physiol Regul Integr Comp Physiol 299: R1656 -R1665, 2010. First published October 13, 2010 doi:10.1152/ajpregu.00433.2010.-There is evidence that nitric oxide (NO) is required for the normal increases in skeletal muscle glucose uptake during contraction, but the mechanisms involved have not been elucidated. We examined whether NO regulates glucose uptake during skeletal muscle contractions via cGMP-dependent or cGMP-independent pathways. Isolated extensor digitorum longus (EDL) muscles from mice were stimulated to contract ex vivo, and potential NO signaling pathways were blocked by the addition of inhibitors to the incubation medium. Contraction increased (P Ͻ 0.05) NO synthase (NOS) activity (ϳ40%) and dichlorofluorescein (DCF) fluorescence (a marker of oxidant levels; ϳ95%), which was prevented with a NOS inhibitor N G -monomethyl-L-arginine (L-NMMA), and antioxidants [nonspecific antioxidant, N-acetylcysteine (NAC); thiol-reducing agent, DTT], respectively. L-NMMA and NAC both attenuated glucose uptake during contraction by ϳ50% (P Ͻ 0.05), and their effects were not additive. Neither the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, which prevents the formation of cGMP, the cGMPdependent protein (PKG) inhibitor Rp-8-bromo-␤-phenyl-1,N2-ethenoguanosine 3=,5=-cyclic monophosphorothioate sodium salt nor white light, which breaks S-nitrosylated bonds, affects glucose uptake during contraction; however, DTT attenuated (P Ͻ 0.05) contractionstimulated glucose uptake (by 70%). NOS inhibition and antioxidant treatment reduced contraction-stimulated increases in protein Sglutathionylation and tyrosine nitration (P Ͻ 0.05), without affecting AMPK or p38 MAPK phosphorylation. In conclusion, we provide evidence to suggest that NOS-derived oxidants regulate skeletal muscle glucose uptake during ex vivo contractions via a cGMP/PKG-, AMPK-, and p38 MAPK-independent pathway. In addition, it appears that NO and ROS may regulate skeletal muscle glucose uptake during contraction through a similar pathway.reactive oxygen species; peroxynitrite; S-glutathionylation; AMPactivated protein kinase BOTH INSULIN STIMULATION AND contraction of skeletal muscle facilitate GLUT4 translocation to the cell membrane and glucose uptake into muscle fibers (10). Although the mechanisms through which contraction stimulates glucose uptake have not yet been completely elucidated, they differ from that of insulin signaling (37). Possible signaling intermediates of contractionstimulated skeletal muscle glucose uptake include AMPK (17), nitric oxide (NO) (42), reactive oxygen species (ROS) (44), and CaMK (51).NO and ROS are highly interrelated molecules (9), and their generation in skeletal muscle is elevated during contraction (4, 38, 44). We have evidence that NO synthase (NOS) inhibition attenuates increases in skeletal muscle glucose uptake during exercise in humans (8), in situ con...

show abstract

Section: Methodsmentioning

confidence: 99%

Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction

Merry

Lynch

McConell

2010

American Journal of Physiology-Regulatory, Integrative and Comp

View full text Add to dashboard Cite

show abstract

“…The cyanide dye-labeling (CyDye)-switch assay utilizes CyDye for in-gel fluorescence scanning, enabling direct comparative proteomic analysis of S-nitrosoproteins between two or more samples (71). Modeled closely after the biotinswitch assay principle, the CyDye-switch method incorporates difference gel electrophoresis (DIGE) technology to visualize differences in protein S-nitrosylation profiles between control and treated (i.e., S-nitrosylated) samples (71).…”

Section: Two-dimensional Difference Gel Electrophoresismentioning

confidence: 99%

“…Modeled closely after the biotinswitch assay principle, the CyDye-switch method incorporates difference gel electrophoresis (DIGE) technology to visualize differences in protein S-nitrosylation profiles between control and treated (i.e., S-nitrosylated) samples (71). With this protocol, following the thiol blocking and reduction steps (as outlined in the biotin-switch method), free thiols in both control and experimental samples are labeled with a succinimidyl ester of the cyanine dyes, Cy3 (green) or Cy5 (red) (Fig.…”

Section: Two-dimensional Difference Gel Electrophoresismentioning

confidence: 99%

“…It is important to note that Cy labeling induces a subtle shift in the molecular mass of the target protein, which is responsible for an elevated false identification rate of excised spots on LC-MS analysis (108,135). Thus, poststain treatment with the SYPRO Ruby dye is often necessary to enhance the accuracy of target band excision before LC-MS analysis (71,108,135,150).…”

Section: Two-dimensional Difference Gel Electrophoresismentioning

confidence: 99%

See 1 more Smart Citation

S-Nitrosothiols and theS-Nitrosoproteome of the Cardiovascular System

Maron

Tang²,

Loscalzo³

2013

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Significance: Since their discovery in the early 1990's, S-nitrosylated proteins have been increasingly recognized as important determinants of many biochemical processes. Specifically, S-nitrosothiols in the cardiovascular system exert many actions, including promoting vasodilation, inhibiting platelet aggregation, and regulating Ca 2+ channel function that influences myocyte contractility and electrophysiologic stability. Recent Advances: Contemporary developments in liquid chromatography-mass spectrometry methods, the development of biotin-and His-tag switch assays, and the availability of cyanide dye-labeling for S-nitrosothiol detection in vitro have increased significantly the identification of a number of cardiovascular protein targets of S-nitrosylation in vivo. Critical Issues: Recent analyses using modern S-nitrosothiol detection techniques have revealed the mechanistic significance of S-nitrosylation to the pathophysiology of numerous cardiovascular diseases, including essential hypertension, pulmonary hypertension, ischemic heart disease, stroke, and congestive heart failure, among others. Future Directions: Despite enhanced insight into S-nitrosothiol biochemistry, translating these advances into beneficial pharmacotherapies for patients with cardiovascular diseases remains a primary as-yet unmet goal for investigators within the field. Antioxid. Redox Signal. 18, 270-287.

show abstract

“…A significant advancement was made with the development of the biotin switch assay that allows identification of protein S-nitrosothiols using a combination of thiol-blocking, RSNO-reducing strategies: immunoblotting with LC-MS-based protein identification (45). Several method-focused texts about the pros and cons of the biotin switch assay have been compiled and thus not discussed in this review (19,55). We would like to underscore, however, how this approach can be used, in combination with quantitative approaches outlined above, to determine the position of an RSNO on a protein, and the amount of this modification.…”

Section: No and H 2 S Measurementsmentioning

confidence: 99%

Working with nitric oxide and hydrogen sulfide in biological systems

Yuan

Patel

Kevil

2015

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

(H 2S) are gasotransmitter molecules important in numerous physiological and pathological processes. Although these molecules were first known as environmental toxicants, it is now evident that that they are intricately involved in diverse cellular functions with impact on numerous physiological and pathogenic processes. NO and H 2S share some common characteristics but also have unique chemical properties that suggest potential complementary interactions between the two in affecting cellular biochemistry and metabolism. Central among these is the interactions between NO, H 2S, and thiols that constitute new ways to regulate protein function, signaling, and cellular responses. In this review, we discuss fundamental biochemical principals, molecular functions, measurement methods, and the pathophysiological relevance of NO and H 2S. thiol; oxidation; redox; pulmonary; cardiovascular HISTORICALLY CONSIDERED ONLY as industrial pollutants, nitric oxide (NO) and hydrogen sulfide (H 2 S) are now appreciated as two key physiologically produced signaling molecules that control multiple cellular functions. Appreciation that dysfunction in how these solvated gases affect these functions, through either deficient formation or downstream reactivity, has opened up new therapeutic avenues for a host of diseases in all major organ systems including the lung. Our understanding of NO homeostasis mechanisms are relatively advanced compared with H 2 S, primarily because of the latter's discovery as a biologically relevant entity being postdated by a decade or more compared with NO. Although they are chemically distinct, there are intriguing parallel mechanisms/concepts in NO and H 2 S biology that include overlapping functions, technical challenges in how each of these gases are measured in biological milieu, appreciation of the mechanisms and factors that modulate how metabolism of each of these is regulated, and therapeutic potential for either inhibiting or repleting NO or H 2 S. In this article, and within the framework outlined above, we provide a general overview of NO and H 2 S biology. Nitric Oxide Formation: Enzymatic and Nonenzymatic SourcesEnzymatic sources. Nitric oxide is synthesized by one of three different isoforms of nitric oxide synthase (NOS) that differ in enzymatic activity, in how they are regulated (transcriptional, translational, and posttranslational mechanisms), and in how they are expressed/compartmentalized in tissues as well as the subcellular level. These are called NOS I, II, and III, corresponding to the inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) isoforms, typically with high, mid, and low activities, respectively. In the systemic and pulmonary vasculature, eNOS plays a key role in controlling blood flow and maintaining an antithrombotic and anti-inflammatory luminal surface. On the other hand, iNOS is induced by inflammatory stimuli in leukocytes, airway epithelial cells, and alveolar macrophages to mediate pathogen killing. However, production of NO at higher concentrations in ...

show abstract

In-Gel Detection of S-Nitrosated Proteins Using Fluorescence Methods

Cited by 36 publications

References 20 publications

Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction

Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction

S-Nitrosothiols and theS-Nitrosoproteome of the Cardiovascular System

Working with nitric oxide and hydrogen sulfide in biological systems

Contact Info

Product

Resources

About