Specific reversible newborn protein enrichment method

A protein and species-specific technology, applied in the field of protein enrichment, can solve the problems of reducing the ionization efficiency of peptides, interference of non-specifically adsorbed proteins, and affecting protein identification, etc., and achieves the effects of high efficiency, mass spectrometry compatibility, and convenient and reliable separation.

Active Publication Date: 2020-10-16
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems of this technique are as follows: (1) Due to the non-covalent interaction between biotin and streptavidin, relatively strong conditions cannot be used for washing, which often causes serious interference of non-specifically adsorbed proteins. If the conditions are severe, the recovery rate will be low; (2) After enrichment, the peptide has a biotin group, and the biotin molecule will affect the chromatographic separation and reduce the ionization efficiency of the peptide, which seriously affects the identification of the protein

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0080] Example 1: The method established based on the cleavable bioorthogonal reversible tag (CBOT) enriches the synthesized azido peptides. The enrichment principle of the CBOT tag is shown in figure 1 , the specific process of the experiment is as follows:

[0081] (1) Take 10 μL of commercial aldehyde resin, add 10 μg of synthetic alkynyl peptide GLAGLLAR (aPra), and add reducing agent NaCNBH3 to PBS with pH 7.4 to cause reductive alkylation reaction to obtain alkynyl functionalization Resin materials, referred to as alkyne-based resins;

[0082] (2) Take the synthesized alkyne-based resin, add 10 μg of azide peptide (m / z 860), and CuAAC catalyzes the covalent binding to the alkyne-based resin;

[0083] (3) Trypsin hydrolysis, the released target peptide was added with aPra group, detected by MALDI, the molecular weight increased to 112.12 (m / z 972).

[0084] figure 2MALDI spectrum for evaluating the capacity of alkyne groups on functionalized resin materials. After di...

Embodiment 2

[0087] Example 2: The cleavable label based on bioorthogonal reaction combined with trypsin digestion in Example 1 is used for the enrichment and identification of nascent proteins in 293T cells. The established enrichment and identification process for nascent proteins is shown in Figure 5 , the specific experimental procedure is as follows:

[0088] (1) AHA-labeled 293T cells, extract the whole sample: 1mM AHA, culture for 4 hours, collect cells to lyse and extract protein;

[0089] (2) Prepare 10 μL of alkyne-based resin according to step (1) in Example 1;

[0090] (3) According to step (2) in Example 1, 10 μg of azide peptides were replaced with 500 μg of enzymatic peptides of AHA-labeled whole protein to complete the CuAAC catalytic connection;

[0091] (4) Strong elution conditions to remove non-specifically adsorbed proteins. Wash sequentially with 200 times volume of elution reagents, respectively 1% SDS, 8M urea, PBS and 50% acetonitrile solution.

[0092] (5) Try...

Embodiment 3

[0096] Example 3: The process of CBOT enrichment of nascent proteins in Example 2 combined with light and heavy isotopes AHA / hAHA was used for quantitative research on nascent proteins in HEK293T cells. The specific experimental process is as follows:

[0097] (1) 293T cells were labeled with AHA and hAHA, incubated for 4 hours, and the cells were collected and lysed;

[0098] (2) Separately extract and quantify the protein, and mix the two samples with equal mass at 1:1;

[0099] (3) according to the flow process in protein embodiment 2 enrichment nascent protein;

[0100] (4) Added variable modifications: AHA(Met) and hAHA(Met) in the mass spectrometry library settings, and set mass changes +107.07 and +113.17 as variable modifications for protein identification and assignment. At the same time, the protein was quantitatively analyzed according to the AHA peptide and the corresponding hAHA peptide.

[0101] Figure 8 It is the omics result of using the CBOT method in Exa...

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Abstract

The invention belongs to the technical field of protein enrichment, and relates to a specific reversible newborn protein enrichment method which comprises steps: marking cells with AHA, and extractingtotal proteins; taking cell total proteins and an alkynyl resin to be connected through click reaction; washing non-specifically adsorbed proteins with different buffer solutions to remove the non-specifically adsorbed proteins; carrying out enzyme digestion by trypsin to release a target peptide fragment; collecting samples for mass spectrometry. Compared with the prior art, according to the method, high-selectivity enrichment of a newly synthesized low-abundance protein is achieved through biolabeling, selective covalent bonding, strict elution conditions and selective dissociation, large-scale analysis and identification of protein can be achieved by combining nano-LC-MS / MS, and the newly-generated protein can be further identified according to the added mass label. The method providesan efficient method for efficient enrichment and accurate identification of the newborn protein, and is widely applied to the research field of low-abundance new proteomics.

Description

technical field [0001] The invention belongs to the technical field of protein enrichment, in particular to a method for specific and reversible enrichment of nascent proteins. Background technique [0002] In the case of different pathological states or drug interference, the discovery of nascent proteins is of great significance in revealing the mechanism of disease and in explaining the process of drug action. However, in complex biological samples, nascent proteins are not labeled, and it is difficult to distinguish nascent proteins from existing proteins. At the same time, the abundance of nascent proteins is extremely low, and it is difficult to directly detect them by conventional methods. [0003] At present, BONCAT (PNAS, 2006, 103:9482-87; Nature Protocols, 2007, 2: 532-540) is the most widely used method for the enrichment of nascent proteins, and the later-developed QuaNCAT (Nature Methods, 2013, 10: 343-46 ) method combined with SILAC technology can enrich and ...

Claims

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Application Information

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IPC IPC(8): C08B37/12G01N1/40
CPCC08B37/0039G01N1/4044
Inventor 陆豪杰包慧敏邵钰银
Owner FUDAN UNIV
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