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Method for enriching and sequencing protein terminal peptide fragment and reagent kit

A protein and kit technology, applied in the field of protein sequencing, to achieve a wide range of practical effects

Inactive Publication Date: 2007-09-26
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are some methods for enriching or identifying protein N-terminal or C-terminal peptides in the literature [1.Kosaka, T., Takazawa, T., Nakamura, T., Anal.Chem.2000, 72, 1179-1185; 2 .Sechi, S., Chait, B.T., Anal.Chem.2000, 72, 3374-3378; 3.Yamaguchi, M., Nakazawa, T., Kuyama, H., Obama, T.et al, Anal.Chem. 2005, 77, 645-651; 4. Gevaert, K., Goethals, M., Martens, L., Van Damme, J. et al, Nat.Biotechnol.2003, 21, 566-569.], but currently There is no simple and easy method to simultaneously enrich and efficiently sequence the N-terminal and C-terminal peptides of proteins from complex protein mixtures

Method used

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  • Method for enriching and sequencing protein terminal peptide fragment and reagent kit
  • Method for enriching and sequencing protein terminal peptide fragment and reagent kit
  • Method for enriching and sequencing protein terminal peptide fragment and reagent kit

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Enrichment and sequencing of terminal peptides in horse myoglobin

[0033] 1.1 Dissolve 1 mg of horse myoglobin in 1 mL of 0.2M borate solution at pH 8, add 20 mM o-sulfobenzoic anhydride, react at room temperature for 1 hour, then add 100 mM Tris / HCl to terminate the reaction. Myoglobin does not contain cysteine ​​residues, so the reductive alkylation step can be omitted. Add 20ug trypsin, digest at 37°C for 12 hours.

[0034] 1.2 On the Elite P230 liquid chromatograph (Dalian Elite Company), use a C18-reversed-phase chromatographic column (200×4.6mm, 5um, 300 Ȧ, Dalian Elite Company) and a strong cation exchange column (200×4.6 mm, 5um, 300 Ȧ, Dalian Elite Company) in series for the enrichment of terminal peptides. Equilibrate the chromatographic system with 0.1% trifluoroacetic acid (TFA) first, wash the column with 10 times the column volume of 0.1% trifluoroacetic acid (TFA) for desalting after loading the sample, then wash the column with 60% methanol ...

Embodiment 2

[0036] Example 2 Enrichment and sequencing of bovine lactoglobulin terminal peptides

[0037]2.1 Take 1mg bovine lactoglobulin, dissolve it in 0.2mL 0.2M pH 8 borate solution (containing 6M guanidine hydrochloride), add 20mM o-sulfobenzoic anhydride, react at room temperature for 1 hour, then add 100mM Tris / HCl to stop reaction. Add 10mM dithiothreitol (DTT), react at 37°C for 1 hour, dilute 6 times with water, add 20ug trypsin, and digest at 37°C for 12 hours.

[0038] 2.2 On the Elite P230 liquid chromatograph (Dalian Elite Company), use a C18-reversed-phase chromatographic column (200×4.6mm, 5um, 300 Ȧ, Dalian Elite Company) and a strong cation exchange column (200×4.6 mm, 5um, 300 Ȧ, Dalian Elite Company) in series for the enrichment of terminal peptides. Equilibrate the chromatographic system with 0.1% trifluoroacetic acid (TFA) first, wash the column with 10 times the column volume of 0.1% trifluoroacetic acid (TFA) for desalting after loading the sample, and then wash...

Embodiment 3

[0040] Example 3 Enrichment and sequencing of bovine insulin terminal peptide

[0041] 3.1 Take 1mg bovine insulin, dissolve it in 0.2mL 0.2M pH 8 borate solution (containing 6M guanidine hydrochloride), add 20mM o-sulfobenzoic anhydride, react at room temperature for 1 hour, then add 100mM Tris / HCl to stop the reaction . Add 10mM dithiothreitol (DTT), react at 37°C for 1 hour, dilute 6 times with water, add 20ug trypsin, and digest at 37°C for 12 hours.

[0042] 3.2 On the Elite P230 liquid chromatograph (Dalian Elite Company), use a C18-reversed-phase chromatographic column (200×4.6mm, 5um, 300 Ȧ, Dalian Elite Company) and a strong cation exchange column (200×4.6 mm, 5um, 300 Ȧ, Dalian Elite Company) in series for the enrichment of terminal peptides. Equilibrate the chromatographic system with 0.1% trifluoroacetic acid (TFA) first, wash the column with 10 times the column volume of 0.1% trifluoroacetic acid (TFA) for desalting after loading the sample, then wash the column...

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Abstract

This invention provides one collection and testing sequence for protein end enriching and testing method and test case, which comprises the following steps: protein end amidogen base decorating, alkali restoring base and tryptone enzyme slices, end peptide enrich end and mass spectrum test sequence. The invention through enzyme slice peptide mixture and end peptide section enriching and testing to get the protein end sequence information for protein testing and end analysis for protein group study. This invention also describes this method agent case.

Description

field of invention [0001] The invention relates to a protein sequencing method. Specifically, it involves the method of chromatographic enrichment and mass spectrometry sequencing of terminal peptides. The invention also relates to kits for use in the method. Background of the invention [0002] It is well known that the terminal information of proteins is very important for the structure determination and function elucidation of proteins. On the one hand, the terminal sequence tags of proteins are highly specific. Statistically, 43% to 83% of proteins have a unique N-terminal 4-residue tag and 74% to 97% of proteins have a unique C-terminal 4-residue tag (varies by species) [Wilkins, M.R., Gasteiger, E ., Tonella, L., Ou, K. et al, J. Mol. Biol. 1998, 278, 599-608.]. On the other hand, the N-terminal sequence of the protein is helpful to confirm the N-terminal processing of the protein, and the C-terminal sequence is helpful to understand the signal recognition domain o...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06C12Q1/68B01D15/08
Inventor 钱小红周春喜张养军
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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