Carbonyl containing compound high-throughput quantitative analysis using isobaric multiplex reagents

Patent metadata

Field	Value
Publication number	US-11535576-B2
Application number	US-201916252464-A
Country	US
Kind code	B2
Filing date	Jan 18, 2019
Priority date	Jan 19, 2018
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The present invention provides a set of novel isobaric chemical tags, also referred herein as SUGAR (Isobaric Multiplex Reagents for Carbonyl Containing Compound). These labeling tags are compact and easy to synthesize at high yield and purity in just a few steps using commercially available starting materials. The tagging reagents of the present invention comprise: a) a reporter group, having at least one atom that is optionally isotopically labeled; b) a balancing group, also having at least one atom that is optionally isotopically labeled, and c) an aldehyde, ketone, or carboxylic acid reactive group. The multiplex SUGAR tags are able to react with an aldehyde, ketone, or carboxylic acid group of the molecule to be tagged, which offers the capability for labeling and quantitation of glycans, proteins/peptides, and fatty acids.

First claim

We claim:

1. A method of analyzing a target molecule having an aldehyde, ketone, or carboxylic acid group, said method comprising the steps of:
a) providing the target molecule;
b) labeling the target molecule with a tagging reagent, thereby generating a labeled molecule, wherein said tagging reagent comprises: i) a reporter group, ii) an aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group, and iii) a balancing group located between the reporter group and the aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group,
wherein labeling the target molecule comprises reacting the aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group with an aldehyde, ketone, or carboxylic acid of the target molecule to generate the labeled molecule,
wherein one or more atoms in the reporter group, the balancing group, or both, are isotopically heavy versions of the one or more atoms, said tagging reagent having the formula of:

wherein,
R 1 is the aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group, wherein the aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group is any functional group able to react with an aldehyde, ketone, or carboxylic acid of the target molecule;
R 2 and R 3 , independently of one another, are selected from the group consisting of branched and unbranched C i to C 12 alkyl groups, C 4 to C 12 cycloalkyl groups, C 2 to C 12 alkenyl groups, C 5 to C 12 cycloalkenyl groups, C 6 to C 12 aryl groups and C 7 to C 12 arylalkyl groups, wherein each of R 2 and R 3 optionally contain one or more 13 C atoms and one or more deuterium atoms;
R 4 , R 5 and R 6 , independently of one another, are selected from the group consisting of hydrogen, deuterium, branched and unbranched C 1 to C 12 alkyl groups, C 4 to C 12 cycloalkyl groups, C 2 to C 12 alkenyl groups, C 5 to C 12 cycloalkenyl groups, C 6 to C 12 aryl groups and C 7 to C 12 arylalkyl groups, wherein each of R 4 , R 5 and R 6 optionally contain one or more 13 C atoms and one or more deuterium atoms;
C V and C x , independently of one another, are 12 C or 13 C,
O U and O Y , independently of one another, are 16 O or 18 O; and
N z and N W , independently of one another, are 14 N or 15 N;
c) fragmenting the labeled molecule to generate an immonium ion from the labeled molecule; and
d) detecting and analyzing fragments of the labeled molecule.

2. The method of claim 1 wherein R 1 is a hydrazine.

3. The method of claim 1 wherein R 2 and R 3 , independently of one another, are CH 3 , 13 CH 3 , CDH 2 , 13 CDH 2 ,CD 2 H, 13 CD 2 H, CD 3 or 13 CD 3 .

4. The method of claim 1 wherein at least one of: a) R 2 or R 3 contains a deuterium atom, b) N z is 15 N, or c) N W is 15 N.

5. The method of claim 1 wherein R 6 is hydrogen or deuterium.

6. The method of claim 1 wherein R 6 is hydrogen or deuterium and R 5 is selected from the group consisting of:
a) a methyl group containing one or more deuterium atoms and wherein the carbon is 12 C or 13 C;
b) hydrogen;
c) deuterium;
d) an isopropyl group containing one or more deuterium atoms and one or more 13 C atoms; and
e) a butyl group containing one or more deuterium atoms and one or more 13 C atoms.

7. The method of claim 1 wherein the labeling step comprises reacting the tagging reagent with a carboxylic acid group of the target molecule.

8. The method of claim 1 wherein the step of labeling the target molecule with a tagging reagent has a labeling efficiency of greater than 90%.

9. The method of claim 1 wherein the tagging reagent has a formula selected from the following:

10. The method of claim 1 , wherein
providing the target molecule in step a) comprises providing two or more samples, each sample comprising an amount of the target molecule;
labeling the target molecule in step b) comprises labeling the target molecule in the two or more samples with two or more tagging reagents of said tagging reagent, wherein each sample is labeled with a different tagging reagent; and
the reporter group of each of said two or more tagging reagents has a different mass due to differently isotopically labeled atoms in each reporter group, the balancing group of each of said two or more tagging reagents has a different mass due to differently isotopically labeled atoms in each balancing group, and the aggregate mass of each of said two or more tagging reagents is the same.

11. The method of claim 10 further comprising quantifying amounts of labeled molecule in each sample.

12. The method of claim 10 wherein at least one sample is a biological sample taken from a patient before a treatment is administered to the patient, and one or more samples are biological samples taken from the patient at one or more time periods after the treatment has been administered to the patient.

13. The method of claim 12 wherein the treatment is a cancer treatment.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Li Lingjun
• Feng Yu
• Chen Bingming
• Yu Qinying
• Zhong Xuefei
• Li Miyang

Classifications

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• C07B2200/05

  Isotopically modified compounds, e.g. labelled · CPC title

• G01N2458/15

  Non-radioactive isotope labels, e.g. for detection by mass spectrometry · CPC title

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title