# US 12,216,125 B2 · Method and kit for isotope-labelling of a glutathione-containing biological sample for mass spectrometry

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- Number: US 12,216,125 B2
- Document ID: US12216125B2
- Status: Granted
- Kind: B2
- Application number: 17/270,813
- Filed: 2019-08-28
- Published: 2021-06-24
- Granted: 2025-02-04
- Earliest claimed: 2018-08-28
- Assignees: Technische UniversitäT Wien
- Applicants: Technische UniversitäT Wien
- Inventors: Matthias Schittmayer-Schantl; Ruth Birner-Grünberger; Tamara Tomin
- Web page: https://patentgrep.com/patent/US12216125B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6815: {containing sulfur, e.g. cysteine, cystine, methionine, homocysteine}
- 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 of stabilising a biological sample including glutathione (GSH) and glutathione disulfide (GSSG), including a) providing the biological sample having GSH and GSSG; b) contacting GSH and GSSG of the sample with a maleimide to obtain maleimide-alkylated GSH; c) separating excess maleimide from maleimide-alkylated GSH and GSSG; d) contacting maleimide-alkylated GSH and GSSG with a reducing agent such as TCEP under conditions which allow reduction of GSSG by the reducing agent such as TCEP to obtain further GSH; and e) contacting maleimide-alkylated GSH and GSH with a heavy isotopologue of the maleimide to obtain a heavy isotopologue of the maleimide-alkylated GSH. A stabilised biological sample is provide containing maleimide-alkylated GSH and a heavy isotopologue thereof, as well as a mass-spectrometric method for quantifying maleimide-alkylated GSH and a heavy isotopologue thereof in a sample and a kit for stabilising a biological sample including GSH and GSSG for mass spectrometric analysis.

## Claims

### Claim 1

A method of stabilising a biological sample comprising glutathione (GSH) and glutathione disulfide (GSSG), the method comprising the following steps:
a) providing the biological sample comprising GSH and GSSG;
b) contacting GSH and GSSG of the biological sample with a maleimide under conditions which allow alkylation of the sulfhydryl group of GSH with the maleimide to obtain maleimide-alkylated GSH, wherein the maleimide is N-ethyl maleimide (NEM);
c) separating excess maleimide from maleimide-alkylated GSH and GSSG by solvent extraction, wherein the solvent is an inorganic solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, hexane, diethyl ether, and mixtures thereof;
d) contacting maleimide-alkylated GSH and GSSG after step c) with a reducing agent selected from the group consisting of tris(2-carboxyethyl) phosphine (TCEP), tributylphosphine, dithiothreitol (DTT), dithioerythritol (DTE) and β-mercaptoethanol, under conditions which allow reduction of GSSG by the reducing agent to obtain further GSH; and
e) contacting maleimide-alkylated GSH and GSH after step d) with a heavy isotopologue of the maleimide under conditions which allow alkylation of the sulfhydryl group of GSH with the heavy isotopologue of the maleimide to obtain a heavy isotopologue of the maleimide-alkylated GSH;
whereby a stabilised biological sample, comprising the maleimide-alkylated GSH and the heavy isotopologue of the maleimide-alkylated GSH, is obtained;
wherein the heavy isotopologue of the maleimide is N-ethyl-D5-maleimide (D5-NEM).

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample is a blood sample, wherein step b) occurs in a blood collection tube pre-filled with the maleimide.

### Claim 3 (depends on 2)

The method of claim 2, wherein the blood collection tube is further pre-filled with another heavy isotopologue of the maleimide-alkylated GSH for use as an internal standard.

### Claim 4 (depends on 1)

The method of claim 1, wherein the reducing agent is TCEP.

### Claim 5 (depends on 1)

The method of claim 1, wherein the biological sample further comprises protein and the method further comprising separating protein from maleimide-alkylated GSH and GSSG by solvent precipitation of the protein wherein the solvent is ethanol, methanol, acetonitrile or a mixture thereof; and wherein the separating is conducted before step c).

### Claim 6 (depends on 1)

The method of claim 1, further comprising a step of concentrating and/or drying the sample after step e), comprising the maleimide-alkylated GSH and the heavy isotopologue of the maleimide-alkylated GSH.