Sample preparation method for proteomic analysis

A proteomics, protein technology, applied in the field of proteomics analysis, can solve the problem of insufficient recovery liquid-mass spectrometry analysis and other problems, achieve the effect of reducing the chance of sample contamination, improving the identification and quantification effect, and widening the scope of use

Inactive Publication Date: 2020-09-08
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

However, due to the limitation of 96-well plates, an obvious limitation of this method is that the maximum amount of each sample used is 150 microliters. Since the protein concentration in some clinical samples, especially urine, is very low, when the urine volume is 1.5 When L / d is lower than 100mg / L, the final peptide recovery is not enough for subsequent liquid-mass analysis.

Method used

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  • Sample preparation method for proteomic analysis
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  • Sample preparation method for proteomic analysis

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preparation example Construction

[0035] Specifically, the present invention relates to a sample preparation method for proteomics analysis, the method includes reducing and alkylating the protein, then adding pre-cooled acetone at -20°C for 30 minutes to precipitate, and the precipitate After the protein is reconstituted, it is transferred to the activated PVDF membrane pre-placed in a 96-well plate, so that the protein is adsorbed on the PVDF membrane, and the protein is rinsed and rinsed with tris solution on the membrane by using a suction filtration device. Replacement, followed by adding trypsin for enzymatic hydrolysis on the membrane, and finally the peptide solution on the membrane was eluted with acetonitrile methanol water solution, and the peptide was reconstituted for mass spectrometry analysis after draining. This sample processing method combines the steps of protein purification and 96-well plate membrane enzyme digestion. The purification step before protein membrane enzyme digestion provides a...

Embodiment 1

[0039] Example 1. Collection of protein-containing samples

[0040] Twelve normal subjects (6 males, 6 females) from Peking Union Medical College volunteers mixed morning urine. Urine samples were centrifuged at 4500 × g for 10 min at 4°C to remove cell debris, and the supernatant was aliquoted for subsequent analysis.

[0041] 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM, 4.5g / L glucose, 10% FCS, 2mM L-glutamine, 50mg / mL penicillin, 50mg / mL streptomycin) until the cell confluence was about 90%. Wash 3 times with 5 mL of ice-cold PBS. Cells were dissolved in 50 mM Tris-HCl with 8M urea, 1% NP-40, 5% SDS, and then incubated at 96°C for 5 minutes, followed by six 30s ultrasonic cycles (Bioruptor Pico, Diagenode, USA). The urea lysate was incubated at room temperature for 10 min, sonicated, and the supernatant protein solution after centrifugation was used for subsequent analysis.

[0042] The blood was centrifuged at 4000×g for 5 minutes, and the superna...

Embodiment 2

[0045] Example 2. Protein digestion

[0046] FASP method

[0047] Digestion was carried out according to the FASP method reported in the literature, see Wisniewski, J.R. et al., Universal sample preparation method for proteome analysis. Nat Methods, 2009.6(5): p.359-62. Specifically, the reducing agent DTT was added to the sample containing approximately 100 micrograms of protein (obtained in Example 1), and reacted in a water bath at 95°C for 5 minutes; after cooling, the alkylating agent IAM was added, and dark treatment was performed for 45 minutes. Add pre-cooled acetone 6 times the volume of the sample, vortex and mix well, put it in a -20°C refrigerator for half an hour, and then centrifuge at 12000g at 4°C for 10 minutes to obtain a protein precipitate. The protein was reconstituted with dissolving solution (2% SDS, 20mM Tris), transferred to a pre-washed 30KD ultrafiltration tube for centrifugation, and washed 3 times with 20mM Tris. According to the protein: enzyme ...

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Abstract

The invention relates to a sample preparation method for proteomic analysis. Specifically, the method comprises the following steps: reducing and alkylating proteins in a sample to be detected, addingacetone to precipitate the reduced and alkylated proteins, redissolving the proteins, adding the redissolved protein solution to a 96-well plate with a PVDF membrane, and carrying out enzymolysis onthe 96-well plate with the PVDF membrane. The sample obtained according to the method of the invention can be used for proteome analysis.

Description

technical field [0001] The method relates to the technical field of proteomics analysis. Specifically, the present invention relates to a sample preparation method for proteomic analysis. Background technique [0002] At present, clinical proteomics based on mass spectrometry is developing into the fields of post-translational modification and clinical research, so a set of stable and efficient sample pretreatment methods is very important. So far, many research groups are trying to develop enzymatic digestion methods for proteomic samples, the most widely used method is the filter-assisted sample preparation method (FASP), see Wisniewski, J.R. et al., Universal sample preparation method for proteomeanalysis.Nat Methods, 2009.6(5): pp. 359-62. In simple terms, in this method, protein precipitation will be enriched on the filter membrane of the ultrafiltration tube, and protein reduction, alkylation and digestion will be performed on the membrane, and the digested peptides ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/30G01N33/68
CPCC07K1/30C12P21/06G01N33/68
Inventor 孙伟汤晓悦孙海丹郭正光刘晓燕李京周冬冬
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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