Method for obtaining peptides

Patent metadata

Field	Value
Publication number	US-10407711-B2
Application number	US-201816223615-A
Country	US
Kind code	B2
Filing date	Dec 18, 2018
Priority date	May 3, 2012
Publication date	Sep 10, 2019
Grant date	Sep 10, 2019

Abstract

The present invention relates to a method of obtaining peptides from procaryotic and/or eucaryotic cells.

First claim

The invention claimed is:

1. A method of obtaining peptides from procaryotic and/or eucaryotic cells, the method comprising:
a) lysis of the procaryotic and/or eucaryotic cells and recovery of the proteins thus obtained;
b) denaturation of the proteins using at least one denaturing agent;
c) alkylation of the denatured proteins using at least one alkylating agent;
d) enzymatic proteolysis of the proteins obtained at the end of step c) using at least one proteolytic enzyme;
e) recovery of the peptides obtained at the end of the enzymatic proteolysis step d), in which the lysis of the procaryotic and/or eucaryotic cells in step a) is a lysis at a low concentration of chaotropic agent(s);
and in which at least steps a) and b) are performed simultaneously;
the lysis at a low concentration of chaotropic agent(s) being carried out at a concentration of chaotropic agent(s) less than or equal to 1 M; and
the method being implemented under a pressure below 100 bar.

2. The method according to claim 1 , in which the lysis at a low concentration of chaotropic agent(s) is carried out in the absence of non-saline chaotropic agent(s).

3. The method according to claim 1 , in which the lysis at a low concentration of chaotropic agent(s) is a lysis by sonication performed by means of an ultrasound probe.

4. The method according to claim 3 , in which the ultrasound probe is not subjected to a preheating step prior to lysis by sonication.

5. The method according to claim 1 , in which the proteolytic enzyme is a serine protease selected from the group consisting of trypsin, chymotrypsin and elastase.

6. The method according to claim 1 , in which the denaturing agent is a thiol-reducing agent, selected from 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithioerythritol, tributylphosphine and dithiothreitol.

7. The method according to claim 1 , in which the alkylating agent is selected from the group formed by N-ethylmaleimide, iodoacetamide and M-biotin.

8. The method according to claim 1 , in which the minimum duration corresponding to the step of proteolysis d) is approximately 15 minutes.

9. The method according to claim 1 , in which at least steps a)-c) are performed simultaneously.

10. The method according to claim 1 , in which steps a)-d) are performed simultaneously.

11. The method according to claim 9 , in which:
the denaturing agent is a thiol-reducing agent selected from tris(2 carboxyethyl)phosphine and dithiothreitol; and
the alkylating agent is iodoacetamide.

12. The method according to claim 1 , the method being implemented under atmospheric pressure.

13. The method of analysis of peptides of procaryotic and/or eucaryotic cells comprising:
i) obtaining peptides from the procaryotic and/or eucaryotic cells using the method according to claim 1 ; and
ii) analysis of the peptides thus obtained, the analysis being performed using an analysis means of mass spectrometry type.

14. The method according to claim 1 , in which the concentration of chaotropic agent(s) is less than or equal to 100 mM.

15. The method according to claim 1 , in which the pressure is less than 50 bar.

16. The method according to claim 1 , in which the pressure is less than 10 bar.

Assignees

• Biomerieux Sa

Inventors

• Charles Marie-Hélène
• Charrier Jean-Philippe
• Cubizolles Myriam-Laure
• Dupont-Filliard Agnès
• Lanet Véronique
• Rivera Florence
• Veron Laurent
• Baujard-Lamotte Lucie
• Peponnet Christine

Classifications

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K1/12 Primary

  by hydrolysis {, i.e. solvolysis in general} · CPC title