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Preparation method, product and application of nano-calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification

A nano-calcium phosphate and polyphosphoric acid technology, which is applied in the preparation of test samples, chemical instruments and methods, phosphorus compounds, etc., can solve problems such as in-depth research on the biological function of unfavorable phosphorylated proteins, and achieve broad application prospects and preparation methods. Simple, easy to synthesize effects

Active Publication Date: 2022-07-26
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with the research on the separation and enrichment of phosphopeptides from the mixture of non-phosphopeptides, the types of functional materials involved in the enrichment of polyphosphopeptides in the existing reports are very limited and most of them are composite materials.
(Nature Communications, 2013, 4: 1656.) The production of composite materials often requires special experimental equipment and superb synthesis technology, which will greatly limit its normal use in laboratories in the field of biological and medical research on phosphoproteomics. It is very unfavorable for the in-depth study of the biological function of phosphorylated proteins

Method used

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  • Preparation method, product and application of nano-calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification
  • Preparation method, product and application of nano-calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification
  • Preparation method, product and application of nano-calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Prepare an ammonia solution with pH=11;

[0036] Add 10mL of the above pH ammonia solution, 100uL of 0.5MCaCl to the conical flask 2 Solution, magnetic stirring (500rpm) 4min to obtain A system;

[0037] In another conical flask, add 10mL of the above pH ammonia solution, 120uL of 0.25M Na 2 HPO 4 Solution, magnetic stirring (500rpm) 4min to obtain B system;

[0038] The B system was added dropwise to the A system at 5 mL / min under magnetic stirring, and the magnetic stirring (500 rpm) was carried out for 10 min. After the reaction is completed, the water-soluble calcium phosphate nanomaterial is obtained by freeze-drying.

Embodiment 2

[0040] Application of Nano-Calcium Phosphate in Polyphosphopeptide Enrichment

[0041] 1. Eluent: 5mL ACN+4900uL H 2 O+100uL volume fraction 10% TFA; eluent: volume fraction 10% ammonia solution.

[0042] 2. The protease cleavage product of typical phosphorylated protein β-casein (40pmol) or the peptide mixture of 40pmol monophosphopeptide and 40pmol polyphosphopeptide (monophosphopeptide: CSKNQI(p-S)TLDFS; polyphosphopeptide: CRGVHHID(p-Y)( p-Y)KKTSN, represented by molar ratio: 1:1), was dissolved in 10uL of eluent, and 1 mg of nano-calcium phosphate material prepared in Example 1 was added to it and vortexed for 30min; For 2 min, the precipitate was obtained by centrifugation; finally, the eluent was added to the precipitate and ultrasonicated for 10 min, vortexed for 8 min to elute, and the supernatant was centrifuged to obtain the polyphosphopeptide.

[0043] When β-casein digested peptides were not subjected to direct mass spectrometry analysis by material enrichment, on...

Embodiment 3

[0046] The preparation method of Example 3 is the same as that of Example 1, and the difference is that the ammonia solution with pH=8 is prepared.

[0047] The transmission electron microscope of the nano calcium phosphate material prepared in this example is as follows: figure 1 As shown in a, it can be seen from the figure that the calcium phosphate of the present invention is a lamellar nanomaterial under this condition;

[0048] The X-ray diffraction pattern of the nano calcium phosphate material prepared in this example is as follows figure 2 As shown, the results compared with the calcium phosphate standard card indicated that the samples were calcium phosphate nanocrystals.

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Abstract

The invention discloses a preparation method, product and application of nano calcium phosphate used for polyphosphopeptide enrichment and phosphorylation site identification. The preparation method comprises the following steps: adding calcium salts in sequence in an alkaline solution The aqueous solution and the phosphate aqueous solution are stirred, and dried after the reaction to obtain nano calcium phosphate material. In the present invention, due to the strong interaction between calcium ions and phosphate radicals, nano-calcium phosphate can effectively achieve selective enrichment of polyphosphopeptides and identification of phosphorylation sites; at the same time, the calcium phosphate nanomaterials prepared by the method of the present invention are A biofunctional material with simple synthesis, low cost and high stability has broad application prospects in phosphorylation proteomics research.

Description

technical field [0001] The present invention relates to a polyphosphopeptide selective enrichment material, in particular to a preparation method, product and application of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification. Background technique [0002] Protein phosphorylation is one of the most important post-translational modifications in organisms, regulating many important processes in life activities, including cell proliferation, differentiation, apoptosis and metabolism. Protein phosphorylation includes not only single phosphorylation modifications but also polyphosphorylation modifications, among which the identification of polyphosphorylation modifications and their sites has a more significant impact on the biological activity of proteins. For example, the tubulin Tau in the adult brain is an important polyphosphorylated protein, and its longest isoform (Tau441) contains a total of 80 serine (Ser) and threonine (Thr) ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C01B25/32G01N1/40G01N27/623
CPCC01B25/325G01N1/40G01N27/62C01P2002/72C01P2004/04C01P2004/20C01P2004/32
Inventor 刘海龙王鑫慧华明丽余浩瀚周林周家宏沈健
Owner NANJING NORMAL UNIVERSITY
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