# US 11,906,522 B2 · Glycan analysis method

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- Number: US 11,906,522 B2
- Document ID: US11906522B2
- Status: Granted
- Kind: B2
- Application number: 16/347,639
- Filed: 2016-11-07
- Published: 2019-10-17
- Granted: 2024-02-20
- Earliest claimed: 2016-11-07
- Assignees: Shimadzu Corporation
- Applicants: Shimadzu Corporation
- Inventors: Takashi Nishikaze
- Web page: https://patentgrep.com/patent/US11906522B2

## CPC

- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- H01J 49/0027: {Methods for using particle spectrometers}
- H01J 49/004: {Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn}
- G01N 2400/10: Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- 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)
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)

## Abstract

Glycans having a branched structure are labeled with a labeling agent, such as 2-aminobenzoic acid, having one site that is easily negatively charged. At this time, reduction which is usually performed in labeling by reduction amination is not performed. A sample for mass spectrometry containing the labeled form of glycans thus obtained is prepared, and is subjected to MS/MS analysis in negative ion mode. In the MS/MS spectra obtained by the MS/MS analysis, peaks of E ions, D ions and the like which reflect the branched structure clearly appear. As a result, structural analysis of an entirety of the glycan including the branched structure can be easily performed.

## Claims

### Claim 1

A glycan analysis method for analyzing a structure of glycan using mass spectrometry, the glycan analysis method comprising:
a) a sample preparation step of preparing a sample by labeling glycan to be analyzed without reduction, using an acidic labeling agent having at least one site stably presenting a negative charge in a molecule with the acid labeling agent bound to the glycan; and
b) an analysis execution step of subjecting the sample to MS/MS analysis in negative ion mode, where a deprotonated ion of the glycan labeled in step a is used as a precursor ion;
c) a detecting step of detecting a product ion of D ion or E ion which reflects a branched structure of the glycan from a result of the MS/MS analysis; and
d) an estimating step of estimating a structure of the glycan based on step c,
wherein the site that stably presents a negative charge in the acidic labeling agent is a carboxy group.

### Claim 2 (depends on 1)

The glycan analysis method according to claim 1, wherein
the labeling agent has one site that stably presents a negative charge in a molecule.

### Claim 3 (depends on 1)

The glycan analysis method according to claim 1, wherein
the labeling agent has an amino group, a hydrazide group, an aminooxy group, or a corresponding basic functional group, and binds to a reducing terminal of the glycan.

### Claim 4 (depends on 1)

The glycan analysis method according to claim 1, wherein
the labeling agent reacts with an amino group of a glycosylamine structure in the glycan.

### Claim 5 (depends on 1)

The glycan analysis method according to claim 1, wherein
the analysis execution step executes MS/MS analysis using a deprotonated form of the glycan as a precursor ion.

### Claim 6 (depends on 2)

The glycan analysis method according to claim 2, wherein
the analysis execution step executes MS/MS analysis using a deprotonated form of the glycan as a precursor ion.

### Claim 7 (depends on 3)

The glycan analysis method according to claim 3, wherein
the analysis execution step executes MS/MS analysis using a deprotonated form of the glycan as a precursor ion.

### Claim 8 (depends on 4)

The glycan analysis method according to claim 4, wherein
the analysis execution step executes MS/MS analysis using a deprotonated form of the glycan as a precursor ion.

### Claim 9 (depends on 1)

The glycan analysis method according to claim 1, wherein
the acidic labeling agent has only one site capable of stably presenting the negative charge in the molecule.