# US 10,770,276 B2 · Techniques of mass spectrometry for isotopomer analysis and related systems and methods

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- Number: US 10,770,276 B2
- Document ID: US10770276B2
- Status: Granted
- Kind: B2
- Application number: 15/755,641
- Filed: 2016-08-30
- Published: 2020-07-23
- Granted: 2020-09-08
- Earliest claimed: 2015-08-31
- Assignees: Yale University
- Applicants: Yale University
- Inventors: Richard Kibbey; Tiago Cardoso Alves; Graeme F. Mason
- Web page: https://patentgrep.com/patent/US10770276B2

## CPC

- H01J 49/0045: {characterised by the fragmentation or other specific reaction}
- G01N 27/623: combined with mass spectrometry
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- H01J 49/0031: {Step by step routines describing the use of the apparatus (H01J49/0081 takes precedence)}
- H01J 49/0036: {Step by step routines describing the handling of the data generated during a measurement}
- H01J 49/421: {Mass filters, i.e. deviating unwanted ions without trapping}
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)
- 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

According to some aspects, a spectrometer is provided, the spectrometer configured to receive molecules of a plurality of metabolites including one or more molecules of a first metabolite, filter the received molecules to retain molecules of the first metabolite including a plurality of different mass isotopomers of the first metabolite, fragment molecules of a first mass isotopmer of the retained molecules to produce a first plurality of daughter ions, measure abundances of the first plurality of daughter ions as a function of daughter ion mass, fragment molecules of a second istopomer, different from the first mass isotopmer, of the retained molecules to produce a second plurality of daughter ions, and measure abundances of the second plurality of daughter ions as a function of the daughter ion mass.

## Claims

### Claim 1

A spectrometer, configured to:
receive molecules of a plurality of metabolites including one or more molecules of a first metabolite;
filter the received molecules to retain molecules of the first metabolite including a plurality of different mass isotopomers of the first metabolite;
fragment molecules of a first mass isotopomer of the retained molecules to produce a first plurality of daughter ions;
measure abundances of the first plurality of daughter ions as a function of daughter ion mass;
fragment molecules of a second mass isotopomer, different from the first mass isotopomer, of the retained molecules to produce a second plurality of daughter ions; and
measure abundances of the second plurality of daughter ions as a function of daughter ion mass.

### Claim 2 (depends on 1)

The spectrometer of claim 1, wherein the spectrometer is configured to perform said filtering based at least in part on a mobility of the received molecules.

### Claim 3 (depends on 2)

The spectrometer of claim 2, wherein the spectrometer is configured to perform said filtering via ion-mobility spectrometry.

### Claim 4 (depends on 3)

The spectrometer of claim 3, wherein the spectrometer is configured to perform said filtering via differential mobility spectrometry (DMS).

### Claim 5 (depends on 1)

The spectrometer of claim 1, wherein the spectrometer is configured to perform said fragmenting steps and said measuring steps via tandem mass spectrometry.

### Claim 6 (depends on 5)

The spectrometer of claim 5, wherein the spectrometer is configured to perform said fragmenting steps and said measuring steps via multiple reaction monitoring (MRM).

### Claim 7 (depends on 1)

A system comprising:
the spectrometer of claim 1; and
at least one processor configured to:
receive indications of the measured abundances of the first and second pluralities of daughter ions from the spectrometer; and
determine a metabolic flux for the first metabolite based at least in part on the received indications.

### Claim 8 (depends on 7)

The system of claim 7, wherein the at least one processor is further configured to perform a correction to the received indications of the measured abundances based on a process in which isocitrate dehydrogenase (ICDH) reacts with isocitrate.

### Claim 9 (depends on 7)

The system of claim 7, wherein the at least one processor is further configured to perform a correction to the received indications of the measured abundances based on a natural abundance of carbon-13.

### Claim 10 (depends on 7)

The system of claim 7, wherein the determined metabolic flux comprises a plurality of conversion rates to the first metabolite from metabolites of the plurality of metabolites other than the first metabolite.

### Claim 11 (depends on 7)

The system of claim 7, wherein determining the metabolic flux for the first metabolite comprises determining an abundance of a group of positional isotopomers of the first mass isotopomer.

### Claim 12

A method, comprising:
receiving molecules of a plurality of metabolites including one or more molecules of a first metabolite;
filtering the received molecules to retain molecules of the first metabolite including a plurality of different mass isotopomers of the first metabolite;
fragmenting molecules of a first mass isotopomer of the retained molecules to produce a first plurality of daughter ions;
measuring abundances of the first plurality of daughter ions as a function of daughter ion mass;
fragmenting molecules of a second mass isotopomer, different from the first mass isotopomer, of the retained molecules to produce a second plurality of daughter ions;
measuring abundances of the second plurality of daughter ions as a function of daughter ion mass.

### Claim 13 (depends on 12)

The method of claim 12, wherein the received molecules include citrate, pyruvate, aspartate, malate, succinate and glutamate.

### Claim 14 (depends on 12)

The method of claim 12, wherein the molecules of the first metabolite include a plurality of isotopologues of the first metabolite.

### Claim 15 (depends on 14)

The method of claim 14, wherein the molecules of the first metabolite include a plurality of carbon-13 enriched molecules.

### Claim 16 (depends on 12)

The method of claim 12, further comprising introducing a source of carbon-13 to the molecules of the plurality of metabolites.

### Claim 17 (depends on 16)

The method of claim 16, further comprising determining metabolic fluxes for the first metabolite at a plurality of time points subsequent to the introduction of the source of carbon-13.

### Claim 18 (depends on 12)

The method of claim 12, further comprising determining a metabolic flux for the first metabolite based at least in part on the measured abundances.

### Claim 19 (depends on 12)

The method of claim 12, further comprising introducing a plurality of effector molecules to the molecules of the plurality of metabolites.

### Claim 20 (depends on 19)

The method of claim 19, further comprising determining metabolic fluxes for the first metabolite at a plurality of time points subsequent to the introduction of the plurality of effector molecules.