Method and product for preparing a protein-containing sample for analysis by mass spectrometry

Patent metadata

Field	Value
Publication number	US-11906408-B2
Application number	US-202117394609-A
Country	US
Kind code	B2
Filing date	Aug 5, 2021
Priority date	Sep 15, 2017
Publication date	Feb 20, 2024
Grant date	Feb 20, 2024

Abstract

A method for preparing a protein-containing sample for analysis by mass spectrometry includes introducing the sample into a reaction vessel. The reaction vessel contains a reagent mixture including pre-measured quantities of an immobilized proteolytic enzyme, a reducing agent and an alkylating agent. The contents of the reaction vessel are activated by heating or by sonication.

First claim

What is claimed is:

1. A product for preparing a protein-containing sample for mass spectrometry analysis, comprising:
a closeable reaction vessel having an internal volume; and
a reagent mixture disposed within the internal volume of the closeable reaction vessel and comprising:
an immobilized proteolytic enzyme;
a reducing agent; and
an alkylating agent, wherein the reducing agent is at a concentration between 1 mM and 5 mM, and the alkylating agent is at a concentration between 10 mM and 25 mM.

2. The product of claim 1 , wherein the proteolytic enzyme is selected from the group consisting of trypsin, LysC, LysN, AspN, GluC, ArgC and chymotrypsin.

3. The product of claim 2 , wherein the reagent mixture further comprises a cell lysing agent.

4. The product of claim 3 , wherein the cell lysing agent is selected from the group consisting of: a detergent in an amount up to 1% (vol/vol); an organic solvent in an amount up to 10% (vol/vol); and urea up to 1M as calculated after the sample is added.

5. The product of claim 3 , wherein the reagent mixture further comprises an isobaric labeling reagent.

6. The product of claim 1 , wherein the reducing agent is tris(2-carboxyethyl) phosphine (TCEP) and the alkylating agent is one of the following: chloroacetamide (CAA), iodacetic acid (IAC), and Iiodoacetamide (IAA).

7. The product of claim 1 , wherein the reagent mixture further comprises calcium chloride (CaCl 2 )).

8. A method for manufacturing the product of claim 1 , comprising:
providing a closeable reaction vessel having an internal volume;
introducing the reagent mixture into the internal volume of the closeable reaction vessel.

9. The method of claim 8 , wherein the proteolytic enzyme is selected from the group consisting of trypsin, LysC, LysN, AspN, GluC, ArgC and chymotrypsin.

10. The method of claim 8 , further comprising introducing a cell lysing agent into the internal volume of the reaction vessel.

11. The method of claim 10 , wherein the cell lysing agent is selected from the group consisting of: a detergent in an amount up to 1% (vol/vol); an organic solvent in an amount up to 10% (vol/vol); and urea up to 1M as calculated after the sample is added.

12. The method of claim 8 , further comprising a formulation buffered to store the mixture.

13. The method of claim 8 , wherein the reducing agent is tris(2-carboxyethyl) phosphine (TCEP) and the alkylating agent is chloroacetamide (CAA) or Iodacetic acid (IAC) or Iodoacetamide (IAA).

14. The method of claim 8 , wherein the mixture further comprises calcium chloride (CaCl 2 )).

15. The method of claim 8 , comprising storing the vessel at a temperature between −80° C. and 24° C.

16. A method for manufacturing a product for preparing a protein-containing sample for mass spectrometry analysis, comprising:
providing a closeable reaction vessel having an internal volume;
introducing a reagent mixture into the internal volume of the closeable reaction vessel, wherein the reagent mixture comprises:
an immobilized proteolytic enzyme;
a reducing agent; and
an alkylating agent, wherein the reducing agent is tris(2-carboxyethyl) phosphine (TCEP) at a concentration between 1 mM and 5 mM, and wherein the alkylating agent is one of: chloroacetamide (CAA) at a concentration between 10 mM and 25 mM; iodacetic acid (IAC) at a concentration between 1 mM and 5 mM; and Iodoacetamide (IAA) at a concentration between 1 mM and 15 mM.

17. The method of claim 16 , wherein the reagent mixture further comprises CaCl 2 ) at a concentration between 0.1 mM and 1 mM.

18. A product for preparing a protein-containing sample for mass spectrometry analysis, comprising:
a closeable reaction vessel having an internal volume; and
a reagent mixture disposed within the internal volume of the closeable reaction vessel and comprising:
an immobilized proteolytic enzyme;
a reducing agent; and
an alkylating agent, wherein the reducing agent is tris(2-carboxyethyl) phosphine (TCEP) at a concentration between 1 mM and 5 mM, and wherein the alkylating agent is one of: chloroacetamide (CAA) at a concentration between 10 mM and 25 mM; iodacetic acid (IAC) at a concentration between 1 mM and 5 mM; and Iodoacetamide (IAA) at a concentration between 1 mM and 15 mM.

19. The product of claim 18 , wherein the reagent mixture further comprises CaCl 2 ) at a concentration between 0.1 mM and 1 mM.

Assignees

• Thermo Finnigan Llc

Inventors

• Lopez Ferrer Daniel
• Robitaille Aaron M
• Krawitzky Michael O
• Foster Greg A

Classifications

• G01N1/4044 Primary

  by chemical techniques; Digestion; Chemical decomposition · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title