# US 11,906,408 B2 · Method and product for preparing a protein-containing sample for analysis by mass spectrometry

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- Number: US 11,906,408 B2
- Document ID: US11906408B2
- Status: Granted
- Kind: B2
- Application number: 17/394,609
- Filed: 2021-08-05
- Published: 2022-02-24
- Granted: 2024-02-20
- Earliest claimed: 2017-09-15
- Assignees: Thermo Finnigan LLC
- Applicants: Thermo Finnigan LLC
- Inventors: Daniel Lopez Ferrer; Aaron M. Robitaille; Michael O. Krawitzky; Greg A. Foster
- Web page: https://patentgrep.com/patent/US11906408B2

## CPC

- G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 3)

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.

### Claim 5 (depends on 3)

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

### Claim 6 (depends on 1)

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).

### Claim 7 (depends on 1)

The product of claim 1, wherein the reagent mixture further comprises calcium chloride (CaCl2)).

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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

### Claim 11 (depends on 10)

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.

### Claim 12 (depends on 8)

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

### Claim 13 (depends on 8)

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).

### Claim 14 (depends on 8)

The method of claim 8, wherein the mixture further comprises calcium chloride (CaCl2)).

### Claim 15 (depends on 8)

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

### Claim 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.

### Claim 17 (depends on 16)

The method of claim 16, wherein the reagent mixture further comprises CaCl2) at a concentration between 0.1 mM and 1 mM.

### Claim 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.

### Claim 19 (depends on 18)

The product of claim 18, wherein the reagent mixture further comprises CaCl2) at a concentration between 0.1 mM and 1 mM.