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Preparation method of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification as well as product and application of nano calcium phosphate

A nano-calcium phosphate and phosphorylation technology, 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 functions of unfavorable phosphorylated proteins, and achieve broad application prospects and preparation methods Simple, Inexpensive Effects

Active Publication Date: 2021-03-16
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 of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification as well as product and application of nano calcium phosphate
  • Preparation method of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification as well as product and application of nano calcium phosphate
  • Preparation method of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification as well as product and application of nano calcium phosphate

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Embodiment 1

[0035] Preparation of ammonia solution with pH=11;

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

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

[0038] Add system B dropwise to system A at 5 mL / min under magnetic stirring, and stir magnetically (500 rpm) for 10 min. After the reaction is finished, the water-soluble calcium phosphate nanometer material is obtained by freeze-drying.

Embodiment 2

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

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

[0042] 2. Protease cleavage product of typical phosphorylated protein β-casein (40pmol) or peptide mixture of 40pmol monophosphopeptide and 40pmol polyphosphopeptide (monophosphopeptide: CSKNQI(p-S)TLDFS; polyphosphopeptide: CRGVHHID(p-Y)( p-Y) KKTSN, molar ratio: 1:1) as a representative, dissolve with 10uL eluent, add 1mg nano-calcium phosphate material prepared in Example 1 and vortex for 30min; then, elute with eluent 3 times, each Centrifuge for 2 minutes to obtain the precipitate; finally, add the eluent to the precipitate and sonicate for 10 minutes, vortex for 8 minutes to elute, and centrifuge to take the supernatant to obtain the polyphosphopeptide.

[0043] When the β-casein cleaved peptide was directly analyzed by mass spectrometry without material enrichment, only t...

Embodiment 3

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

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

[0048] The X-ray diffraction figure of the nano-calcium phosphate material prepared by the present embodiment is as follows figure 2 As shown, compared with the calcium phosphate standard card, the results show that the samples are calcium phosphate nanocrystals.

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Abstract

The invention discloses a preparation method of nano calcium phosphate for polyphosphopeptide enrichment and phosphorylation site identification as well as a product and application of the nano calcium phosphate, and the preparation method comprises the following steps: sequentially adding a calcium salt aqueous solution and a phosphate aqueous solution into a slightly alkaline solution, conducting stirring, and conducting drying after the reaction is finished, so as to obtain a nano calcium phosphate material. Due to strong interaction between calcium ions and phosphate radicals, nano calciumphosphate can effectively realize selective enrichment of polyphosphopeptides and identification of phosphorylation sites. Meanwhile, the calcium phosphate nano material prepared by the method is a biological functional material which is simple to synthesize, low in cost and high in stability, and has a wide application prospect in phosphorylated proteomics research.

Description

technical field [0001] The invention relates to a material for selectively enriching polyphosphopeptides, in particular to a method for preparing nano-calcium phosphate used for enriching polyphosphopeptides and identifying phosphorylation sites, as well as its products and applications. 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 modification but also multiple phosphorylation modification, and the identification of multiple phosphorylation modification and its site has a more significant impact on the biological activity of the protein. For example, tubulin Tau in the adult brain is an important polyphosphorylated protein, and its longest isoform (Tau441) contains a total of 80 serine (Ser) and thre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C01B25/32G01N1/40G01N27/62
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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