Qualitative and quantitative method for thio thiolation post-translational modification peptide segment

A post-translational modification and peptide technology, applied in the direction of labels, measuring devices, and material inspection products used in chemical analysis, can solve the problems of R-SSH instability, loss of sulfhydrylation site information, false positives, etc. Achieve the effect of reducing the false positive rate of identification, improving detection efficiency and reliability

Active Publication Date: 2018-05-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a nucleophile, R-SSH has properties similar to R-SH; as an electrophile, R-SSH has properties similar to R-SS-R; coupled with the inherent instability of R-SSH, enrichment identification exists greater difficulty
At present, the enrichment and identification methods of thiothiolated proteins / peptides mostly use indirect methods, which lose the information of thiothiolated sites and cause certain false positives.

Method used

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  • Qualitative and quantitative method for thio thiolation post-translational modification peptide segment
  • Qualitative and quantitative method for thio thiolation post-translational modification peptide segment
  • Qualitative and quantitative method for thio thiolation post-translational modification peptide segment

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Experimental program
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Effect test

Embodiment 1

[0023] Route A:

[0024] Preparation of iodoacetamide polymer microspheres: Weigh 13 mg of iodoacetic acid-N-hydroxysuccinimide ester, dissolve in 400 μL of methanol and 200 μL of 50 mM, pH 8.0 phosphate buffered saline mixed solution, shake and dissolve with 10 mg , 5μm, The amino polymer microspheres were reacted in the dark for 6h. After the reaction is completed, wash with phosphate buffer saline and methanol three times respectively, and dry in vacuum to obtain iodoacetamide polymer microspheres.

[0025] Enrichment and elution of thiothiol-labeled peptides by iodoacetamide polymer microspheres: Weigh 1 mg of iodoacetamide polymer microspheres, wash with 50 mM, pH 8.0 phosphate buffered saline three times, and then disperse in 200 μL phosphate buffered saline buffer salts. Add 2 μg of sulfur-thiol-labeled peptide and thiol-labeled peptide mixture, wherein the mass ratio of thio-thiol-labeled peptide and thiol-labeled peptide is 2:3, and react in the dark for 6 hours. ...

Embodiment 2

[0028] Route B:

[0029] Use a cleavable isotope affinity tag (AB SCIEX Company, P / N: 4337337) to label bovine serum albumin and enzymatic hydrolysis of bovine serum albumin: weigh 100 μg of bovine serum albumin, dissolve in 100 μL, 50 mM trimethylol Aminomethane, pH 8.3, 6M urea, 5mM ethylenediaminetetraacetic acid and 1M, 1μL of tris(2-carboxyethyl)phosphine were added, and denatured and reduced at 37°C for 1h. Then add 20 μL of a cleavable isotope affinity tag (AB SCIEX company, P / N: 4337337), and react at 37° C. in the dark for 2 hours. After labeling, the protein solution was transferred to a filter membrane with a molecular weight cut-off of 10,000 Da, and was centrifuged several times to remove excess labeling reagent and urea. Finally, 100 μL of ammonium bicarbonate and 3 μg of trypsin were added, and the enzyme was hydrolyzed at 37° C. for 12 hours. The next morning, centrifuge to collect the digested peptides. Matrix-assisted laser desorption ionization time-of-fl...

Embodiment 3

[0032] Identification and quantification of thiothiol peptides in complex samples: Extract 1×10 in 300 μL, 50 mM Tris, pH 8.3, 6 M urea, 5 mM EDTA 7 The protein of individual neuroblastoma cells is divided into two routes after the protein concentration is measured.

[0033] Route A

[0034] 200 μg of protein was removed and transferred to a filter membrane. The solvent was replaced, 200 μL of ammonium bicarbonate and 8 μg of trypsin were added, and the enzyme was digested at 37° C. for 12 hours. The next morning, centrifuge to collect the digested peptides. The enzymatically hydrolyzed peptides were reacted with iodoacetamide polymer microspheres in 50 mM, pH 8.0 phosphate buffered saline for 6 h in the dark. After completion of the reaction, wash with 30% acetonitrile and 2M sodium chloride to remove non-specific adsorption, then add 100 μL of tris(2-carboxyethyl)phosphine with a final concentration of 5 mM and reduce at 37°C for 1 h to release the thiomercapto peptide. ...

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Abstract

The present invention relates to a qualitative and quantitative method for a thio thiolation post-translational modification peptide segment. The method is divided into two routes such as a route A and a route B, wherein the route A comprises that a peptide segment produced through proteolysis and iodoacetamide beads are incubated, the mercapto peptide segment and the thio mercapto peptide segmentare enriched, the disulfide bond of the thio mercapto peptide segment is reduced and released with a reducing agent, the released thio mercapto peptide segment can be labeled with a cleavable isotopeaffinity tag, and finally the linking arm of the cleavable isotope affinity tag is cleaved with a lysis liquid, and the route B comprises that protein containing mercapto and thio mercapto is labeledwith a cleavable isotope affinity tag, proteolysis is performed, the mercapto peptide segment and the thio mercapto peptide segment are subjected to affinity enrichment, and finally the linking arm of the cleavable isotope affinity tag linked to the mercapto peptide segment and the thio mercapto peptide segment is cleaved with a lysis liquid. According to the present invention, the qualitative and quantitative method has advantages of good selectivity, low false positive and the like, and can achieve the qualitation and the quantification of the scaled thio thiolation peptide segment in the complex proteome sample.

Description

technical field [0001] The invention relates to a qualitative and quantitative method for thiothiolated post-translationally modified peptides and its application in large-scale analysis of thiothiolated peptides in proteome samples such as cells, tissues and body fluids. Background technique [0002] In the late 1990s, H 2 S has been proved to be the third new type of endogenous gas molecule existing in the body, and it is widely involved in various physiological and pathological processes of the body. The current study shows that the post-translational modification of protein sulfhydrylation (conversion of active R-SH to R-SSH) is H 2 One of the important biological pathways of S signaling. Thiothiolation modification of proteins is ubiquitous in the body, and some important proteins have been confirmed to undergo thiothiolation modification, such as: Parkin-SSH that occurs under physiological conditions can enhance its ubiquitination activity, which is beneficial to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58
CPCG01N33/58G01N33/6851G01N2458/15G01N2570/00
Inventor 张丽华吴琼袁辉明杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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