Absolute quantification method based on dimethylated multiple labeling and characteristic fragment ions

A dimethylation and absolute quantification technology, applied in the direction of measuring devices, material separation, instruments, etc., can solve the problems of labeling multiplicity limitation and high mass spectrometry resolution requirements, achieve high throughput of absolute quantification, save mass spectrometer time, Simple and cheap method

Active Publication Date: 2018-05-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, the above-mentioned methods have limitations such as labeling multiplicity limitations or high requirements for mass spectrometry resolution.

Method used

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  • Absolute quantification method based on dimethylated multiple labeling and characteristic fragment ions
  • Absolute quantification method based on dimethylated multiple labeling and characteristic fragment ions
  • Absolute quantification method based on dimethylated multiple labeling and characteristic fragment ions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Protein enzymatic hydrolysis

[0037] Dissolve seven standard proteins including ovalbumin, β-casein, lysozyme C, cytochrome C, myoglobin, transferrin, and bovine serum albumin and E.coli protein extracts in 8M urea solution, Add dithiothreitol to a final concentration of 10mM, denature and reduce at 56°C for 1 hour, add iodoacetamide to a final concentration of 20mM, and react in the dark for 30min, then dilute urea 10 times, according to 1:50 (enzyme / protein, w / w) Trypsin was added and hydrolyzed overnight at 37°C. Use a C18 trapping column to desalt and evaporate to dryness, and redissolve in water.

[0038] 2. Peptide dimethylation labeling

[0039] Six-fold dimethylation labeling (Dim30L, Dim30H, Dim32L, Dim32H, Dim34L, Dim34H) was performed on the peptides obtained from the enzymatic hydrolysis of each sample. The specific labeling methods are as follows:

[0040] The first heavy labeling: add 50μL volume concentration of 4% to 1mL pH 8.0 phosphate buffer so...

Embodiment 2

[0059] Use the five-fold labeling in this protocol to draw the internal standard curve, and use the remaining three-fold labeling to achieve simultaneous absolute quantitative analysis of three samples. That is, five-fold labeling is performed on the target protein standard, and then the corresponding target protein standard is diluted according to the absolute amount range of the corresponding protein in the sample to be tested, and the internal standard curve is prepared by using five-fold labeling. Add the remaining triple-labeled three samples to be tested to prepare quantitative analysis samples, and other processes are the same as in Example 1 for absolute quantitative analysis.

Embodiment 3

[0061] Using this protocol to add a double-labeled internal standard, the simultaneous absolute quantitative analysis of seven samples was achieved. That is, perform one-fold labeling on the target protein standard, then dilute the corresponding target protein standard according to the absolute amount range of the corresponding protein in the sample to be tested, and use the remaining seven-fold labeling method to mark seven samples to be tested. The other processes are the same as in Example 1, and multi-sample absolute quantitative analysis.

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Abstract

The present invention relates to a multiple-sample multiple-target high-throughput accurate absolute quantification method of proteomes. A N-terminal and a lysine amino group of a peptide fragment canbe labeled by dimethylation reaction, and by organic combination of various isotopic forms of a dimethylation labeling reagent, multiple labeling of a to-be-tested protein and/or peptide fragment sample and a protein and/or peptide fragment in a target internal standard substance with a known amount can be realized. The labeled sample is analyzed by a LC/MS system. Peak areas of characteristic fragment ions in the secondary spectrum of the sample and the internal standard substance are extracted for quantifying to obtain the absolute amount of a target protein in the to-be-tested sample. Themethod has the advantages of high analysis throughput, great saving of analysis time, cheap labeling reagent, strong practical application, high quantitative accuracy, good precision, and wide dynamicrange, and can meet the needs of multiple-sample analysis based on the internal standard substance or multiple-target analysis based on a standard curve.

Description

technical field [0001] The invention relates to a method for absolute quantification of multiple labels, which can realize simultaneous quantification of multiple samples of multiple proteins or peptides and corresponding standard curves, and realize accurate absolute quantification of multiple samples and multiple targets. The invention has the advantages of simple and cheap method, high labeling efficiency, high quantitative accuracy, good precision, wide dynamic range, high absolute quantitative throughput, no optimization steps of absolute quantitative method, wide range of applicable samples, and strong practicability of the method. It has significant advantages in the absolute quantitative analysis of proteomes with large sample volumes. Background technique [0002] The change of protein content is closely related to the life process of organisms. The study of the absolute amount of protein plays an irreplaceable role in understanding the function of proteins in the i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 张丽华刘健慧周愿单亦初杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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