Quantitative detection method of plant histone variant H3.3 based on MRM

A quantitative detection method and quantitative detection technology, applied in the field of chemical detection, can solve problems such as mutual interference, insufficient ionic strength, modification positioning and quantitative accuracy reduction, etc., to improve the signal-to-noise ratio, reduce chemical background, increase accuracy and The effect of reliability

Pending Publication Date: 2021-10-29
SHANGHAI STOMATOLOGICAL HOSPITAL FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Mass spectrometry has become a key technique for histone analysis. However, the difficulty in the analysis of post-translational modifications on histone variant H3.3 lies in the diversity of histone post-translational modification states, mutual interference between different variants, and the simultaneous Precise mapping of modification status and modification sites requires high-quality tandem spectrum information
For example, there are only two important modification sites K27 and K36 on the peptide segment K27-R40 on H3, corresponding to 20 different combinations of modifications (5 on K27 and 4 on K36). In the mass spectrometry acquisition mode, many low-abundance modified peptides are filtered out due to insufficient ionic strength. Secondly, different variants and different modified peptides often have problems of co-elution and mixed spectra, resulting in accurate positioning and quantification of modifications. greatly reduced
At present, there is no accurate quantitative detection method for the post-translational modification of plant histone variants. Based on the status of the existing technical means, the inventors of the present application intend to provide a high-throughput, high-sensitivity, and high-reliability Reliable MRM quantification method for histone variant H3.3, this method can solve the problem of accurate quantification of post-translational modifications on plant histone variant H3.3, and can be applied in fields such as botany and epigenetics

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  • Quantitative detection method of plant histone variant H3.3 based on MRM
  • Quantitative detection method of plant histone variant H3.3 based on MRM
  • Quantitative detection method of plant histone variant H3.3 based on MRM

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Establishment and optimization of MRM-based quantitative detection method for plant histone variant H3.3 K27 / K36

[0037] 1. Synthesis of Plant Histone H3.3

[0038] The biological company synthesized the following sequence:

[0039] (ARTKQTARKSTGGKAPRKQLATKAARKSAPTTGGVKKPHRYRPGTVALREIRKYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSHAVLALQEAAEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA) K27-R40 peptides of 20 different combinations were dissolved in ddH 2 In O, until the final concentration is 100 μM, it is the mother (reservoir) solution;

[0040] 2. Dilute 1 μL of the mother solution to 100nM with 0.1% formic acid, enter the mass spectrometry, analyze the fragment ion response of each peptide, and select 3-5 product ions. The parent ion selection is good responsivity and has 3+ or 4+ charges, and the product ion selection is: 1) good responsivity; 2) product ion contains modification sites (K27 and K36); 3) between variants Product ions do not interfere.

[0041] 3. Optim...

Embodiment 2

[0049] Example 2 Quantitative detection and analysis of histone variant H3.3 K27 / K36 in wild-type Arabidopsis

[0050] 1. Collect Arabidopsis thaliana seedlings about 15 days old, and extract core histones by acid extraction;

[0051] 2. Perform SDS-PAGE gel electrophoresis on the extracted crude histone, in-gel propionylation derivatization and enzymatic hydrolysis, as follows:

[0052] 1) Perform SDS-PAGE gel electrophoresis (12.5% ​​or 15% gel) on the extracted crude histone, stain the protein with Coomassie Brilliant Blue overnight, and then decolorize until the background is almost transparent and colorless, and the bands are clear;

[0053] 2) Cut off the 15KD protein, mainly H3. Cut the strips into cubes as small as possible (1mm 3 left and right), placed in a centrifuge tube;

[0054] 3) Use about 500μL of ddH 2 O Low-speed shaking and washing for 15 minutes, repeat twice;

[0055] 4) Add 500 μL of decolorization solution (25mM NH 4 HCO 3 / 50%ACN) shake at room ...

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Abstract

The invention discloses a quantitative detection method of plant histone variant H3.3 based on MRM, and belongs to the technical field of chemical detection. The quantitative detection method comprises the following steps of: identifying peptide fragments modified by different forms of a histone variant H3.3K27-R40, synthesizing standard peptide fragments modified by different forms of a histone variant H3.3K27-R40, and carrying out mass spectrum quantitative condition optimization on the standard peptide fragments by utilizing a mass spectrum multi-reaction monitoring technology so as to realize quantitative detection on the peptide fragments modified by different forms of the histone variant H3.3K27-R40. According to the quantitative detection method, rapid, accurate and high-throughput quantitative detection of the histone H3.3 and the modified peptide fragment thereof in the plant body based on the MRM is realized.

Description

technical field [0001] The invention relates to the field of chemical detection, in particular to an MRM-based quantitative detection method of plant histone variant H3.3. Background technique [0002] Histones are components of chromatin, and their post-translational modifications play an important role in regulating the structure and function of chromatin. It is generally believed that the basic unit of eukaryotic chromatin is the nucleosome, which is composed of 147 base pairs of DNA wrapped around a histone octamer (assembled by four histones H2A, H2B, H3 and H4) Composition, nucleosomes are connected through histone H1 to gradually form their higher-level structures. In addition to the conventional four histones, it also includes many variants of histones. Different variants of histones may carry completely different biological information. For example, histone H3.3 is mainly related to transcriptional activation. Histone variants and their modifications are also join...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62G01N33/68
CPCG01N27/62G01N33/6848
Inventor 胡亚君金红
Owner SHANGHAI STOMATOLOGICAL HOSPITAL FUDAN UNIV
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