# US 10,119,974 B2 · Isobaric tandem mass tags for quantitative proteomics and peptidomics

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- Number: US 10,119,974 B2
- Document ID: US10119974B2
- Status: Granted
- Kind: B2
- Application number: 15/182,329
- Filed: 2016-06-14
- Published: 2016-12-08
- Granted: 2018-11-06
- Earliest claimed: 2011-09-15
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Lingjun Li; Feng Xiang; Tyler Greer; Dustin Frost; Zhidan Liang
- Web page: https://patentgrep.com/patent/US10119974B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- C07D 207/46: with hetero atoms directly attached to the ring nitrogen atom
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)

## Abstract

Compositions and methods of tagging peptides and other molecules using novel isobaric tandem mass tagging reagents, including novel N, N-dimethylated amino acid 8-plex and 16-plex isobaric tandem mass tagging reagents. The tagging reagents 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 amine reactive group. The tagging reagents disclosed herein serve as attractive alternatives for isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency and improved fragmentation efficiency.

## Claims

### Claim 1

A kit comprising two or more tagging reagents, wherein each tagging reagent comprises a reporter group, an amine reactive group, and a balancing group located between the reporter group and amine reactive group, wherein one or more atoms in the reporter group, balancing group, or both, are isotopically heavy versions of the atom, wherein, for each isotopically heavy version of the atom, heavy isotopes are present in excess of the naturally occurring isotopic abundance;
wherein the reporter group of each tagging reagent has a mass different than the reporter groups of the other tagging reagents, the balancing group of each tagging reagent has a mass different than the balancing groups of other tagging reagents, and the aggregate mass of the reporter group plus the balancing group for each tagging reagent is the same;
wherein a first tagging reagent has the formula:
(CH3)2-AA1-AA2-R1 (formula 16)
where R1 is an amine reactive group;
AA1 is a first amino acid having an N-terminus;
AA2 is a second amino acid having an N-terminus wherein AA1 and AA2 cannot be the same amino acid; and
the two CH3 groups are attached to the N-terminus of AA1; and
wherein at least one tagging reagent has the formula:
(CH3)2-AA2-AA1-R1 (formula 17)
where AA1 and AA2 are the same amino acids as in the first tagging reagent with the exception that the amino acids may contain different isotopes; and
the two CH3 groups are attached to the N-terminus of AA2;
wherein the reporter group of each tagging reagent has the formula:
wherein R2 and R3, independently of one another, are CH3, 13CH3, CDH2, 13CDH2, CD2H, 13CD2H, CD3 or 13CD3;
wherein the balancing group of each tagging reagent has the formula:
wherein R4 and R5 are, independently from one another, selected from the group consisting of hydrogen, deuterium, a methyl group optionally containing one or more deuterium atoms and a 13C atom; an isopropyl group optionally containing one or more deuterium atoms and one or more 13C atoms; and a butyl group optionally containing one or more deuterium atoms and one or more 13C atoms;
R6 is hydrogen or deuterium;
CV and Cx, independently of one another, are 12C or 13C,
OU and Oy, independently of one another, are 16O or 18O;
NW is 14N or 15N; and
wherein at least one of: R2 or R3 contains a deuterium atom, Nz is 15N, or NW is 15N; and at least one of OU or Oy is 18O.

### Claim 2 (depends on 1)

The kit of claim 1 wherein AA1 and AA2, independently from one another, are selected from the group consisting of leucine, isoleucine, alanine, glycine, and valine with the provision that AA1 and AA2 cannot have the same mass.

### Claim 3 (depends on 1)

The kit of claim 1 wherein the reporter group of each tagging reagent has a mass that differs from one another by two or more Daltons.

### Claim 4 (depends on 1)

The kit of claim 1 comprising from 2 to 8 tagging reagents having the formula (CH3)2-AA1-AA2-R1 and from 2 to 8 tagging reagents having the formula (CH3)2-AA2-AA1-R1.

### Claim 5 (depends on 1)

The kit of claim 1 wherein the reporter group of each tagging reagent has a mass that differs from one another by one or more Daltons.