Amino acid sequence determination method based on quasi-isobaric double labeling at two ends of polypeptide

An amino acid, double-labeling technology, applied in measurement devices, biological testing, material analysis by electromagnetic means, etc., can solve the problems of inability to simplify the spectrum, low B ion response, complex spectrum, etc., to achieve reaction efficiency and selectivity High, improved accuracy, and improved specificity

Active Publication Date: 2021-06-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, relying on a single example series often cannot greatly simplify the spectrum, not to mention the low response of some B ions in CID mode
In addition, these strategies usually need to enlarge the fragmentation window to more than 6Da. This setting will significantly increase the number of co-fragmented ions, which will make the spectrum more complex, which is not conducive to analysis and sequencing.

Method used

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  • Amino acid sequence determination method based on quasi-isobaric double labeling at two ends of polypeptide
  • Amino acid sequence determination method based on quasi-isobaric double labeling at two ends of polypeptide
  • Amino acid sequence determination method based on quasi-isobaric double labeling at two ends of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Isotope-labeled peptide ends based on free solution

[0025] Such as figure 1 As shown, mark according to the following process.

[0026] (1) Denaturation, reduction, alkylation and enzymatic hydrolysis of protein samples: 1 mg of bovine serum albumin (BSA) was dissolved in 1000 μl of 50 mM ammonium bicarbonate solution, with a final concentration of 1 mg / ml. Heat at 95°C for 15 minutes to denature BSA, add 8 μl of 1M DTT, mix well, heat at 56°C for 1 hour; then add 20 μl of 1M IAA, and store in the dark for 30 minutes. Add 20 μg of trypsin with a concentration of 1 mg / ml into the centrifuge tube, mix well, heat in a water bath at 37° C. for 12 hours, and add 2 μl of formic acid to stop the enzymatic hydrolysis. Use a C18 reverse-phase chromatographic column to desalt and freeze-dry to obtain trypsin-digested polypeptide powder.

[0027] (2) Labeling at both ends of the peptide in the free solution: take 100 μg of the above peptide powder, place it in a 500 μl cen...

Embodiment 2

[0031] Based on the immobilized enzyme reactor and chemically labeled protein enzymatic digestion polypeptide both ends of the label.

[0032] In this example, different methods of labeling the C-terminus of the polypeptide are adopted, and the methods for labeling, separating and identifying the N-terminus of the polypeptide are the same as those in Example 1 above.

[0033] 1) Denaturation, reduction, alkylation, enzymatic hydrolysis and C-terminal labeling of protein samples: 1 mg of protein was dissolved in 1000 μl of 50 mM ammonium bicarbonate solution, with a final concentration of 1 mg / ml. Heat at 95°C for 15 minutes to denature BSA, add 8 μl of 1M DTT, mix well, heat at 56°C for 1 hour; then add 20 μl of 1M IAA, and store in the dark for 30 minutes. The denatured and reductively alkylated protein was divided into two parts and freeze-dried. Use H respectively 2 16 O and H 2 18 O The concentration is 50 mM ammonium acetate aqueous solution to dissolve the powder, t...

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Abstract

The invention relates to an amino acid sequence de novo sequencing method of protein based on quasi-isobaric stable isotope labeling at two ends of polypeptide. According to the de novo sequencing method, trypsin is used for carrying out enzyme digestion on protein subjected to denaturation, reduction and alkylation, and a peptide fragment containing N-terminal alpha-amino and C-terminal alpha-carboxyl is formed. The method comprises the following steps: dividing a sample into two parts, carrying out derivative reaction on one part in 18O water by using trypsin, and then carrying out N-terminal dimethylation by using formaldehyde; directly carrying out N-terminal dimethylation labeling of the peptide fragment on the other part by using deuterated formaldehyde; and finally, mixing the two labeled samples, and separating and identifying peptide fragments by using high performance liquid chromatography-mass spectrometry. According to the method, the window of a mass spectrum is reduced, the influence of interference signals can be effectively reduced, and the selection specificity of fragment ions and the accuracy of polypeptide sequencing are improved. Meanwhile, b and y ions do not need to be distinguished, a de novo sequencing algorithm of the protein amino acid sequence is simplified, and the de novo sequencing speed is increased.

Description

technical field [0001] The invention relates to a method for de novo sequencing of protein amino acid sequences assisted by isotope labeling, which is a method for de novo sequencing of protein amino acid sequences for accurately identifying peptide fragment ions through the N-terminus and C-terminus of peptides produced by enzymatic hydrolysis, respectively, for stable isotope labeling method. Background technique [0002] With the advancement of mass spectrometry, the qualitative and quantitative analysis of proteins has developed rapidly, and the determination of protein sequences is an important part of protein qualitative. Protein sequencing plays a key role in the study of the biological function of proteins, the discovery of disease-related protein sequence mutants and the quality control of protein drugs such as antibodies. Protein sequencing mainly includes two methods based on database search and de novo sequencing. They all rely on mass spectrometry to crack the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N27/62
CPCG01N33/6848G01N33/6818Y02P20/55
Inventor 张丽华杨超单亦初杨开广张玉奎梁振
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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