Method for rapidly detecting protein isomers

A protein and isomer technology, applied in the field of protein separation and purification, can solve the problems of complicated detection and analysis, increased analysis time, inability to distinguish isomers, etc., to achieve rapid separation and identification, the method is convenient and simple, and highly practical effect of value

Inactive Publication Date: 2019-09-27
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have certain limitations
First, they cannot distinguish between isoforms, such as phosphorylated protein isoforms with different phosphorylation sites and the same molecular mass
Second, the simultaneous detection of a large number of protein isoforms inevitably complicates the associated detection analysis
Finally, analysis of glycosylated and phosphorylated proteins requires enzymatic digestion, which reduces protein integrity
In addition, this will also increase its analysis time, and it will also lead to the loss of some low-abundance isomers and the increase of some artificial errors, such as some non-enzymatic PTMs that do not appear in the protein modification process will appear in the analysis process

Method used

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  • Method for rapidly detecting protein isomers
  • Method for rapidly detecting protein isomers
  • Method for rapidly detecting protein isomers

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Embodiment 1

[0020] This embodiment discloses a method for rapidly detecting protein isomers. Taking α-lactalbumin as an example, the specific steps are:

[0021] 1. Purification of α-lactalbumin: Equilibrate a size exclusion chromatography (SEC) column (TSKgel G3000SWXL, Tosoh, Japan) with 50mM ammonium acetate solution, and then dissolve α-lactalbumin in 50mM ammonium acetate solution In, filtering with a 0.22 μm filter and then injecting into high performance liquid chromatography (HPLC). The setting parameters are: detection wavelength 280nm, flow rate 0.75mL / min, time 20min. Finally, collect the protein solution of each elution peak for later use.

[0022] 2. Mass spectrometry identification of α-lactalbumin isomers: Undecafluorohexanoic acid was used to calibrate the Fourier transform ion cyclotron resonance-Mass Spectrometry (FTICR-MS), and then the HPLC Connected to the mass spectrometer, equilibrate the ion exchange chromatography (IXC) column (ProPac SAX-10, Thermo Fisher Scientific...

Embodiment 2

[0025] This embodiment discloses a method for rapidly detecting protein isomers. Taking β-lactoglobulin as an example, the specific steps are:

[0026] 1. Purification of β-lactoglobulin: Equilibrate a size exclusion chromatography (SEC) column (TSKgel G3000SWXL, Tosoh, Japan) with 50mM ammonium acetate solution, and then dissolve β-lactoglobulin in 50mM acetic acid The ammonium solution was filtered with a 0.22 μm filter and then injected into high performance liquid chromatography (HPLC). The setting parameters are: detection wavelength 280nm, flow rate 0.75mL / min, time 20min. Finally, collect the protein solution of each elution peak for later use.

[0027] 2. Mass spectrometric identification of β-lactoglobulin isomers: Fourier transform ion cyclotron resonance-Mass Spectrometry (FTICR-MS) was calibrated with undecafluorohexanoic acid, and then The HPLC was connected to the mass spectrometer, and the ion exchange chromatography (IXC) column (ProPac SAX-10, Thermo Fisher Scien...

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Abstract

The invention discloses a method for rapidly detecting protein isomers, and method uses the proteins to be detected as a raw material, separates the proteins to be detected by molecular exclusion chromatography, and then performs mass spectrometry identification on the protein isomers by on-line chromatography-mass spectrometry. The method provided by the invention is simple and convenient, does not require pretreatment such as chemical or enzymatic hydrolysis, and only requires one ion exchange chromatography to identify isomers in proteins. Therefore, the technology can achieve rapid separation and identification of proteins, and has high practical value in many fields such as biopharmaceuticals, medicine and chemistry.

Description

Technical field [0001] The invention belongs to the technical field of protein separation and purification, and particularly relates to a method for rapidly detecting protein isomers. Background technique [0002] In nature, protein production is regulated by precise genetics, but most proteins show significant structural heterogeneity, which is usually caused by post-translational modifications (PTMs). Because many PTMs have a greater impact on the behavior of proteins in the body and their physical and chemical properties, the detection and characterization of protein isomers is a frequently encountered problem in many fields such as biology, medicine and chemistry. The detailed location and characterization of all PTMs in a given protein sample is usually a huge and arduous task that requires a lot of resources and time investment. However, in many applications, especially in the field of biotechnology, the discovery of PTMs is not necessary. The focus is on detection. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72G01N33/68
CPCG01N30/02G01N30/7233G01N33/6848
Inventor 刘俊涂宗财沙小梅邵艳红
Owner JIANGXI NORMAL UNIV
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