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A method for protein identification and imaging mass spectrometry based on efficient in situ enzymatic hydrolysis of immobilized enzyme

An immobilized enzyme, mass spectrometry imaging technology, applied in the field of biochemical analysis, can solve problems such as affecting the detection of low-abundance proteins, ineffective enzyme digestion, low protein content, etc., and achieves good aqueous solution stability, simple operation and simple steps. Effect

Inactive Publication Date: 2014-08-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the conditions of this method still need to be optimized: self-cleavage peptides produced by too high trypsin amount will affect the detection of low-abundance proteins
However, if the amount of trypsin is too low, the slice thickness is very thin and the protein content is very low, so it cannot be digested effectively

Method used

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  • A method for protein identification and imaging mass spectrometry based on efficient in situ enzymatic hydrolysis of immobilized enzyme
  • A method for protein identification and imaging mass spectrometry based on efficient in situ enzymatic hydrolysis of immobilized enzyme
  • A method for protein identification and imaging mass spectrometry based on efficient in situ enzymatic hydrolysis of immobilized enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Investigation and Experiment of Graphene Oxide Material Interfering with Mass Spectrometry Detection Signal

[0050] A bovine serum albumin enzymatic peptide solution with a concentration of 5 ng / μL and its mixed solution with the graphene oxide material dispersion (prepared at a ratio of 1:1) were prepared respectively. Take 1 μL of the samples and spot them on the MALDI target plate, and then spot an equal volume of CHCA matrix solution (0.1% trifluoroacetic acid 50% acetonitrile aqueous solution) after drying. MALDI-TOF / MS analysis was carried out after drying and crystallization, the results are as follows figure 2 shown.

[0051] A bovine serum albumin enzymatic peptide solution with a concentration of 10 ng / μL and its mixed solution with the graphene oxide material dispersion (prepared at a ratio of 1:1) were prepared respectively. Take 1 μL of the samples and spot them on the MALDI target plate, and spot the samples on ten target spots continuously. After dryin...

Embodiment 2

[0053] Experiment on the Adsorption Ability of Graphene Oxide Material to Trypsin

[0054] Prepare graphene oxide material dispersion and trypsin solution with a final concentration of 400 ng / μL in 20 mM phosphate buffer solution (pH5), mix and incubate in equal proportions for a certain period of time, then centrifuge for 5 minutes to take the supernatant. The concentration of unfixed trypsin in the supernatant was determined using the BCA protein concentration assay. According to the concentration of trypsinogen and the concentration of unfixed trypsin in the supernatant, the adsorption amount of trypsin by the graphene oxide material was obtained. The result is as Figure 4 shown.

[0055] The above experiments were repeated under different pH conditions (pH 4-10). The result is as Figure 5 shown.

Embodiment 3

[0057] Study on the effect of immobilized enzyme and free enzyme on enzymolysis of standard protein target

[0058] After fixation of trypsin (pH 5) as described in Example 2, it was diluted 10-fold in 25 mM ammonium bicarbonate buffer.

[0059] Prepare an aqueous solution of α-casein (10ng / μL), take 1μL of the above solution and spot on the MALDI target plate, after drying, spot the free enzyme or immobilized enzyme diluted with 25mM ammonium bicarbonate on the corresponding target , and placed in an incubator at 37 degrees Celsius for enzymatic hydrolysis. After the enzymatic hydrolysis, 1 μ LDHB matrix solution (0.1% trifluoroacetic acid 50% acetonitrile aqueous solution) was spotted on the MALDI target plate, dried and crystallized for MALDI-TOF / MS analysis, the results are as follows Figure 7 shown.

[0060] Bovine serum albumin and horse heart blood myoglobin (10ng / μL) were enzymatically hydrolyzed by the above method and then searched by mascot database search softwa...

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Abstract

The invention belongs to the field of biochemical analysis, and relates to a new method for protein identification of a tissue section using immobilized enzyme (in situ enzymatic hydrolysis) and for application of results in imaging mass spectrometry. The method uses graphene oxide whose surface has hydrophilic groups such as hydroxyls and carboxyls to immobilize trypsins, realizes the efficient in situ enzymatic hydrolysis and identification of the protein in the tissue section by improving efficiency of the enzymatic hydrolysis, and applies the result to the imaging mass spectrometry technology for determining the distribution of the protein in the tissue section. The selected carrier material which is graphene oxide has strong adsorption ability, and the graphene oxide is capable of adsorbing the trypsins in a short time, and thus greatly improves efficiency of enzymatic hydrolysis of the trypsins by the confinement effect, and effectively promotes the enzymatic hydrolysis of the tissue section and the identification of protein. The method has characteristics of simple steps, of convenient operation, and of high speed and efficiency, and the method can be widely applied in related fields of proteomics.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to a method for protein identification and mass spectrometry imaging based on an immobilized enzyme in situ high-efficiency enzymatic hydrolysis method, in particular to a method for immobilized enzyme in situ enzymatic hydrolysis of tissue sections to realize protein identification and apply to mass spectrometry imaging new method. Background technique [0002] The prior art discloses that tissue imaging based on biological mass spectrometry is rapidly developing as a new field in mass spectrometry. It directly scans the surface of the tissue, combined with professional image processing software, and processes the ion signals generated by the scan through data processing and image reconstruction to obtain the composition, relative abundance and distribution of compounds in the tissue. This technology has been widely used to detect the distribution of small molecules, lipids, pept...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N1/28
Inventor 陆豪杰焦竞张莹蔡炎杨芃原
Owner FUDAN UNIV
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