Compositions and methods for detecting s-nitrosylation and s-sulfinylation

a technology of s-nitrosylation and s-sulfinylation, which is applied in the field of compositions and methods for detecting s-nitrosylation and s-sulfinylation, can solve the problems of unstable and transient modification, introduction of false positives, and insufficient investigation of the selectivity of organomercury enrichment, so as to reduce loading buffer, reduce sulfenic acid, and reduce the effect of sulfenic acid

Inactive Publication Date: 2016-03-24
RGT UNIV OF MICHIGAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0036]FIG. 12. Sulfenic acids are eliminated by ascorbate reduction or denaturing buffers. Lysates were labeled for 1 hour with 1 mM dimedone-alkyne, and precipitated with chloroform:methanol. The protein fraction was sonicated in PBS and diluted to 1 mg / mL, and incubated with 20 μM TAMRA-azide, 1 mM CuSO4, and 100 μM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) for 1 hour, boiled in reducing loading buffer, and separated by SDS-PAGE. After transfer and Cy5-streptavidin binding, the gel was visualized using a GE Typhoon fluorescence scanner. Dimedone alkyne (compound 14) detection of sulfenic acids in 293T cell lysates is most efficient in PBS. When the lysate is denatured in 6 M urea / PBS, dimedone labeling is nearly completely eliminated. Pre-treatment with sodium ascorbate (20 mM) for 30 minutes efficiently reduces sulfenic acids. These data demonstrates sulfenic acids are eliminated under the denaturing conditions used for biotin-SO2H labeling of endogenous S-nitrosylated proteins. Furthermore, ascorbate reduces sulfenic acids, and confirms non-selective enrichment of an additional cysteine oxidative modification by the biotin-switch method.

Problems solved by technology

These modifications are unstable and transient, and often act to temporarily inactivate functional cysteines (see, e.g., Nakamura, T. et al.
Similarly, the selectivity of organomercury enrichment has not been thoroughly explored, and may introduce false-positives after performic acid oxidation of disulfide-capped thiols, and oxidize other amino acid side chains.
J Am Chem Soc 135, 7693-704 (2013)), these methods have not been thoroughly validated in complex proteomes.

Method used

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  • Compositions and methods for detecting s-nitrosylation and s-sulfinylation
  • Compositions and methods for detecting s-nitrosylation and s-sulfinylation
  • Compositions and methods for detecting s-nitrosylation and s-sulfinylation

Examples

Experimental program
Comparison scheme
Effect test

example i

[0087]While exploring the interplay of cysteine post-translational modifications, a reported reaction between phenylsulfinic acid and S-nitrosocysteine was identified, leading to thiosulfonate formation in aqueous buffers at room temperature (see, e.g., Hart, T. W. Tetrahedron Letters 26, 2013-2016 (1985)) (FIG. 1a, FIG. 2). Thiosulfonates are readily exchangeable with thiols, serving as the basis for the cysteine capping agent methyl methanethiosulfonate (MMTS). To prevent such exchange, it was found that sulfinic acids do not react with iodoacetamide, enabling orthogonal alkylation of thiols without perturbing nitrosothiols or sulfinic acids (FIG. 3). Furthermore, it was found that sulfinic acids do not react with thiols (cysteine), disulfides (cystine or 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)), or aldehydes (benzaldehyde) (FIG. 4). Additionally, the thiosulfonate product is unaffected by ascorbate, but reduced by tris(2-carboxyethyl)phosphine (TCEP) (FIG. 5). Most S-sulfinyl...

example ii

[0096]This example describes synthetic methods pertaining to Example I.

[0097]All compounds were purchased from Sigma-Aldrich, unless otherwise noted. NMR analysis was performed using a Varian 400 MHz NMR instrument. Small molecule high-resolution mass spectrometry was performed using an electrospray Agilent Q-TOF mass spectrometer (accuracy 1-5 ppm). Low-resolution mass spectrometry was performed using an electrospray Micromass LCT time-of-flight mass coupled to a HPLC pump with a rheodyne loop injector. Compounds were purified by normal phase silica column chromatography or by semi-prep High-Performance Column Chromatography (HPLC). HPLC purifications were performed using a Waters semi-preparative 1525 binary pump system coupled to a photodiode array detector, an autosampler, and an automatic fraction collector. Separations were carried out on an Atlantis prep T3 C18 column (10×250 mm), in 95 / 5 water / acetonitrile 0.1% formic acid for 2 minutes, followed by a 40 minute gradient incr...

example iii

[0098]This example describes materials and methods pertaining to Examples I and II.

Rate Constant Determination.

[0099]S-nitrosoglutathione (GSNO, Cayman) and sodium phenylsulfinate (Sigma-Aldrich) were used for rate-determination studies at four pH values: pH 1.0 (0.2 N HCl / KCl buffer), pH 4.0 (0.1 M Sodium acetate / Acetic Acid buffer), pH 7.0 (0.1 M potassium phosphate buffer) and pH 10.0 (0.1 M Sodium bicarbonate / Sodium hydroxide buffer). The purity of S-nitrosoglutathione was calculated as 94±1.3% by absorbance at 334 nm using the molar extinction coefficient of 900 M−1 cm−1. Assays were performed using a plate reader (Tecan Infinite F500) monitoring absorbance of 2 mM GSNO at 340 nm over a course of 90 minutes in the presence of varying concentrations of sodium phenylsulfinate. Additional experiments confirmed thiosulfonate stability for >5 hours at pH 1, 4, and 7, but hydrolysis at pH 10. GSNO was stable in 6 M urea / PBS for >1 hour. Absorbance data was imported into KaleidaGraph ...

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Abstract

The present invention relates to methods for detecting protein S-sulfinylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to pending U.S. Provisional Patent Application No. 61 / 820,401, filed May 7, 2013, the contents of which are incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to methods for detecting protein S-nitrosylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.BACKGROUND OF THE INVENTION[0003]Reversible redox post-translational modifications on protein thiols have been implicated in several signaling pathways of biological importance. Protein S-nitrosylation and proteins S-sulfinylation are two of these modifications that play critical roles in maintaining the redox balance of proteins. Redox imbalance has recently been shown to play a crucial role in heart disease, neurodegeneration and cancer. Protein S-nitrosylation describes the reversible, post-translational modification of select thiols with nitric oxide (NO) and / or its oxidize...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/68
CPCG01N33/6848G01N2560/00G01N2458/15G01N2570/00
Inventor MARTIN, BRENT, R.MAJMUDAR, JAIMEEN, D.
Owner RGT UNIV OF MICHIGAN
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