# US 11,029,316 B2 · Multiplex proteome quantification method based on isobaric dimethyl labeling

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,029,316 B2
- Document ID: US11029316B2
- Status: Granted
- Kind: B2
- Application number: 15/754,212
- Filed: 2015-11-20
- Published: 2018-08-30
- Granted: 2021-06-08
- Earliest claimed: 2015-08-26
- Assignees: Dalian Institute of Chemical Physics, Chinese Academy of Sciences
- Applicants: Dalian Institute of Chemical Physics, Chinese Academy of Sciences
- Inventors: Lihua Zhang; Jianhui Liu; Yuan Zhou; Kaiguang Yang; Yukui Zhang
- Web page: https://patentgrep.com/patent/US11029316B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 30/02: Column chromatography
- G01N 30/04: Preparation or injection of sample to be analysed
- G01N 30/06: Preparation
- G01N 30/72: Mass spectrometers {(mass spectrometers per se H01J49/00)}
- G01N 33/6827: {Total protein determination, e.g. albumin in urine}
- G01N 30/7233: {interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04)}
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- 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 multiplex proteome quantification method based on isobaric dimethyl labeling implements dimethyl labeling of peptide N-terminal in an acidic condition and C-terminal in an alkaline condition one after another by means of Hall the property that a dimethylation reaction has different rates on an amino group at the peptide N-terminal and an amino group on a Lysine side chain at the peptide C-terminal in the acidic condition. Multiplex labeling of peptide samples is implemented by means of the organic combination of various isotope forms of a dimethyl labeling reagents. The mass-to-charge ratios in MS1 of peptides after multiplex labeling are completely the same, the mass-to-charge ratios of the fragment ions in MS2 are different, and multiplex quantitative analyses are carried out by extracting the intensity values of corresponding fragment ions in the MS2.

## Claims

### Claim 1

A multiplex proteome quantification method, comprising:
digesting a protein into a plurality of peptides using a protease that cleaves at a carboxyl side of lysine; and
performing dimethyl labeling of a peptide among the plurality of peptides to produce a plurality of labeled peptides, wherein the dimethyl labeling comprises:
performing a first dimethyl labeling to a peptide N-terminal under a first acidic condition using a first dimethyl labeling reagent and the second dimethyl labeling to a peptide C-terminal on a Lysine side chain under a first alkaline condition using a second dimethyl labeling reagent to form a first labeled peptide;
performing a third dimethyl labeling to a peptide N-terminal under a second acidic condition using a third dimethyl labeling reagent and a fourth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a second alkaline condition using a fourth dimethyl labeling reagent to form a second labeled peptide;
performing a fifth dimethyl labeling to a peptide N-terminal under a third acidic condition using a fifth dimethyl labeling reagent and a sixth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a third alkaline condition using a sixth dimethyl labeling reagent to form a third labeled peptide,
wherein each of the first dimethyl labeling reagent, the second dimethyl labeling reagent, the third dimethyl labeling reagent, the fourth dimethyl labeling reagent, the fifth dimethyl labeling reagent, and the sixth dimethyl labeling reagent comprises a first compound selected from CH2O, 13CH2O, CD2O, and 13CD2O, and a second compound selected from NaBH3CN and NaBD3CN, and
the plurality of labeled peptides have a same mass-to-charge ratio;
ionizing the plurality of labeled peptides and acquiring ionized forms of the plurality of labeled peptides in MS1 of a mass spectrometer;
separating and fragmenting the ionized forms of the plurality of labeled peptides and acquiring fragment ions in MS2 of the mass spectrometer; and
performing multiplex quantitative analysis based on intensities of the fragment ions in the MS2.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the plurality of labeled peptides further comprises a fourth labeled peptide, or the fourth labeled peptide and a fifth labeled peptide, or the fourth labeled peptide, the fifth labeled peptide, and a sixed labeled peptide,
wherein the first to the sixth dimethyl labeling reagents are selected from a reagent comprising 13CH2O and NaBH3CN, a reagent comprising CD2O and NaBD3CN, a reagent comprising CD2O and NaBH3CN, a reagent comprising 13CH2O and NaBD3CN, a reagent comprising 13CD2O and NaBH3CN, a reagent comprising CH2O and NaBD3CN, a reagent comprising CH2O and NaBD3CN, a reagent comprising 13CD2O and NaBH3CN, a reagent comprising 13CH2O and NaBD3CN, a reagent comprising CD2O and NaBH3CN, a reagent comprising CD2O and NaBD3CN, and a reagent comprising 13CH2O and NaBH3CN.

### Claim 3 (depends on 2)

The analysis method according to claim 2, comprising displaying a spectrum of each of the plurality of labeled peptides on a same spectrum of MS2;
extracting and summing up intensity values of fragment ions of a, b and y for each of the plurality of labeled peptides on the MS2;
assigning the sum of intensity values of fragment ions of a, b and y as an intensity of the corresponding labeled peptide; and comparing the intensity of each of the plurality of labeled peptides.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the protein is denatured, reduced, alkylated and then is incubated by the protease.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the mass spectrometer comprises an Orbitrap analyzer, or a TOF analyzer, or an FT-ICR analyzer.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the protease is a Lys-C protease.

### Claim 7 (depends on 1)

The method according to claim 1, performing a first dimethyl labeling proceeds the second dimethyl labeling.

### Claim 8 (depends on 1)

The method according to claim 1, wherein, in each of the first acidic condition, the second acidic condition, and the third acidic condition, a pH value is 2.0 to 5.0 and a labeling time is 5 to 120 min.

### Claim 9 (depends on 1)

The method according to claim 1, wherein, in each of the first alkaline condition, the second alkaline condition, and the third alkaline condition, a pH value is 7.5 to 12 and a labeling time is 5 to 120 min.