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Protein quantification method based on dimethyl labeling DIA strategy

A dimethylation and protein technology, applied in the direction of measuring devices, material inspection products, instruments, etc., can solve the problems of low throughput and affecting quantitative accuracy, etc., achieve good reproducibility, increase quantitative coverage, and high efficiency Effect

Active Publication Date: 2019-01-04
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, due to the large number of fragment ions in each DIA secondary spectrum, it affects the quantitative accuracy, so it is of great significance and challenge to improve the accuracy of DIA data analysis
In addition, the current common quantification method is based on a label-free strategy (Mol.Cell.Proteomics 2012,11,O111 016717.), and the labeling strategy is currently only based on NeuCoDIA labeling technology (Anal Chem.2015,87,2570.), But current throughput is generally low

Method used

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  • Protein quantification method based on dimethyl labeling DIA strategy
  • Protein quantification method based on dimethyl labeling DIA strategy
  • Protein quantification method based on dimethyl labeling DIA strategy

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Effect test

Embodiment 1

[0029] 1. Protein extraction and sample pretreatment

[0030] E. coli samples were washed three times with 1×PBS. 8M urea + 0.1% protease inhibitor (cocktail, Sigma-Aldrich, MO) was used as the lysate to suspend the cell line. The cell lines were sonicated for 100 s with a tissue disruptor at a speed of 10,000. The cell extract was centrifuged at 25,000 g to obtain the supernatant, precipitated with acetone, discarded the supernatant, and evaporated to dryness. After reconstitution with 8M urea, the protein concentration was determined by Bradford method. Store at -20°C until use.

[0031] 1 mg E.coli protein extract was denatured and reduced with 2 μL 1M DTT at 56°C for 1 hour, then added 4 μL 1M IAA, and reacted in the dark for 40 minutes. Urea was then diluted to 1 M with 50 mM phosphate buffer (pH 8.0). Add trypsin to Ecoli according to 1:50 (enzyme / protein, mass ratio), and digest at 37°C overnight. Use a C18 trapping column to desalt and evaporate to dryness for la...

Embodiment 2

[0045] This strategy is suitable for absolute quantitative analysis based on internal standards. One labeling method is used to label the internal standard of the target protein, and the other labeling method is used to label the sample to be tested. The other processes are the same as in Example 1. The absolute value of the target protein of the sample to be tested is obtained according to the relative quantification results between the two markers of the target protein, and then the absolute quantification of the target protein based on the DIA strategy is realized. This strategy achieves high-accuracy absolute quantification of target proteins.

Embodiment 3

[0047] High-throughput DIA quantification can be achieved by dimethylated quintuple labeling. The five samples to be tested for relative quantitative comparison were respectively marked with dimethylation, and the marking method was: sample1:CH 2 O+NaBH 3 CN(Dim28); sample2: CH 2 O+NaBD 3 CN(Dim30); sample3: 13 CH 2 O+NaBD 3 CN(Dim32); sample4: 13 cd 2 O+NaBH 3 CN(Dim34); sample5: 13 cd 2 O+NaBD 3 CN(Dim36). DIA data collection is carried out after mixing, and other processes are the same as in Example 1, and the parent-child ion pairs (Dim28, Dim30, Dim32, Dim34, Dim36) of the fivefold label are respectively extracted for comparison, that is to say, simultaneous DIA of 5 samples in one experiment Quantitative analysis has obtained high-accuracy quantitative information of large-scale proteomes.

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Abstract

The invention relates to a labeling-based DIA quantification method, which uses a dimethyl reaction to label peptides and achieve multiple labelling of a peptide segment samples by organic combinationof various isotope forms of the dimethyl labeling reagents. The data is collected by utilizing a Data Independent Acquisition (DIA) mode, and the ion chromatograms of multiple parent ions and daughter ions in each corresponding DIA data are extracted. The extracted peak area is used for multiple quantitative analysis of the protein. The DIA quantification method has the advantages that the quantification accuracy is high, the repeatability is good, the marking efficiency is high, the method is simple, the reproducibility requirement on the liquid quality system is low, the multi-dimensional separation of the samples is easy to carry out, and the throughput of the protein group quantitative analysis is high. The method improves the quantitative accuracy and throughput of the DIA strategy by labeling, and has obvious advantages in large sample protein groups.

Description

technical field [0001] The invention relates to a label-based DIA quantification method, which improves the quantitative accuracy and throughput of the DIA strategy through the label. The method has the advantages of high quantitative accuracy, good reproducibility, high labeling efficiency, simple method, low requirements on the reproducibility of the liquid-mass system, easy multi-dimensional separation of samples, and high throughput of quantitative proteome analysis. It has obvious advantages in proteome analysis of large samples. Background technique [0002] In recent years, biology and precision medicine have increasingly required proteomics data, and the large sample size has put forward higher and higher requirements for the reproducibility and throughput of proteomic methods. In the conventional data-dependent acquisition mode, the randomness of precursor ion acquisition will lead to poor reproducibility of peptide identification. Therefore, data-independent (DIA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6848
Inventor 张丽华刘健慧单亦初杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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