# US 12,181,484 B2 · Quantitation of insulin-like growth factor-I and insulin-like growth factor-II with high-resolution mass spectrometry

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- Number: US 12,181,484 B2
- Document ID: US12181484B2
- Status: Granted
- Kind: B2
- Application number: 18/132,009
- Filed: 2023-04-07
- Published: 2023-10-12
- Granted: 2024-12-31
- Earliest claimed: 2009-11-05
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments Incorporated
- Inventors: Cory Bystrom; Shijun Sheng; Nigel Clarke; Richard Reitz
- Web page: https://patentgrep.com/patent/US12181484B2

## CPC

- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- C07K 14/65: Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry}
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Methods are provided for determining the amount of an IGF-I and/or IGF-II protein in a sample using high resolution/high accuracy mass spectrometry. The methods generally comprise enriching an IGF-I and/or IGF-II protein in a sample, ionizing an IGF-I and/or IGF-II protein from the sample to generate IGF-I and/or IGF-II protein ions, and determining the amount of IGF-I and/or IGF-II protein ions with high resolution/high accuracy mass spectrometry.

## Claims

### Claim 1

A method for determining an amount of an insulin-like growth factor-II (IGF-II) protein in a serum or plasma sample, the method comprising:
subjecting IGF-II protein in the sample to ionization by electron ionization, chemical ionization, electrospray ionization, photon ionization, atmospheric pressure chemical ionization, photoionization, atmospheric pressure photoionization, fast atom bombardment, liquid secondary ionization, matrix assisted laser desorption ionization, field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization, inductively coupled plasma, or particle beam ionization to produce one or more IGF-II ions detectable by mass spectrometry; and
determining an amount of the one or more IGF-II ions by high resolution/high accuracy mass spectrometry, wherein the amount of the determined one or more IGF-II ions is related to the amount of the IGF-II protein in the sample, wherein:
the one or more IGF-II ions detectable by mass spectrometry are selected from the group consisting of IGF-II ions with mass to charge ratios of 934.69±2, 1068.07±2, 1245.92±2, and 1494.89±2, or
the one or more IGF-II ions detectable by mass spectrometry are selected from the group consisting of IGF-II ions with mass to charge ratios of 934.69±1, 1068.07±1, 1245.92±1, and 1494.89±1, or
the one or more IGF-II ions detectable by mass spectrometry are selected from the group consisting of IGF-II ions with mass to charge ratios of 1067.36±0.1, 1067.51±0.1, 1067.65±0.1, 1067.80±0.1, 1067.94±0.1, 1068.08±0.1, 1068.23±0.1, 1068.37±0.1, 1068.51±0.1, 1068.65±0.1, 1068.80±0.1, 1068.94±0.1, and 1069.08±0.1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the IGF-II protein is a human IGF-II protein.

### Claim 3 (depends on 1)

The method of claim 1, wherein the IGF-II protein is native to the sample, and/or wherein the IGF-II protein is an intact IGF-II protein.

### Claim 4 (depends on 1)

The method of claim 1, wherein the IGF-II protein is chemically modified prior to ionization.

### Claim 5 (depends on 4)

The method of claim 4, wherein the chemical modification comprises reduction of one or more disulfide bridges in the IGF-II protein, or wherein the chemical modification comprises alkylation of one or more cysteines in the IGF-II protein.

### Claim 6 (depends on 4)

The method of claim 4, wherein said sample is further purified by HPLC and/or SPE prior to ionization and said SPE and/or HPLC are conducted with on-line processing.

### Claim 7 (depends on 1)

The method of claim 1, wherein the IGF-II protein from said sample is purified with solid phase extraction (SPE) prior to ionization, and/or wherein the IGF-II protein from said sample is purified by high performance liquid chromatography (HPLC) prior to ionization.

### Claim 8 (depends on 1)

The method of claim 1, wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap mass spectrometer or with a time of flight mass spectrometer, or with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to 20,000 and an accuracy of less than or equal to 10 ppm, or with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to 20,000 and an accuracy of less than or equal to 5 ppm.

### Claim 9 (depends on 1)

The method of claim 1, wherein said determining the amount of one or more IGF-II comprises collecting spectrometric data from one or more peaks with each peak resulting from an isotopic form of an ion.

### Claim 10 (depends on 9)

The method of claim 9, wherein two or more peaks each resulting from a different isotopic form of an ion are used to confirm the identity of the IGF-II protein, or wherein spectrometric data from a peak resulting from a single isotopic form is used to determine the amount of the IGF-II protein in said sample, or wherein spectrometric data from two or more peaks each resulting from a different isotopic form are used to determine the amount of the IGF-II protein in said sample.

### Claim 11 (depends on 1)

The method of claim 1, wherein said sample comprises a biological fluid, or wherein said sample comprises plasma or serum.

### Claim 12 (depends on 1)

The method of claim 1, wherein said one or more IGF-II ions detectable by mass spectrometry comprise one or more IGF-II ions in a 8+, 7+, 6+, or 5+ charge state.

### Claim 13

A method for determining an amount of an insulin-like growth factor-II (IGF-II) protein in a serum or plasma sample, the method comprising:
subjecting IGF-II protein in the sample to ionization by electron ionization, chemical ionization, electrospray ionization, photon ionization, atmospheric pressure chemical ionization, photoionization, atmospheric pressure photoionization, fast atom bombardment, liquid secondary ionization, matrix assisted laser desorption ionization, field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization, inductively coupled plasma, or particle beam ionization to produce one or more IGF-II ions detectable by mass spectrometry; and
determining an amount of the one or more IGF-II ions by high resolution/high accuracy mass spectrometry;
wherein the amount of the determined one or more IGF-II ions is related to the amount of the IGF-II protein in the sample; and
the one or more IGF-II ions detectable by mass spectrometry are selected from the group consisting of IGF-II ions with mass to charge ratios of 1067.94±0.1 and 1068.08±0.1.

### Claim 14 (depends on 13)

The method of claim 13, wherein the IGF-II protein is a human IGF-II protein.

### Claim 15 (depends on 13)

The method of claim 13, wherein the IGF-II protein is native to the sample, and/or wherein the IGF-II protein is an intact IGF-II protein.

### Claim 16 (depends on 13)

The method of claim 13, wherein the IGF-II protein is chemically modified prior to ionization.

### Claim 17 (depends on 16)

The method of claim 16, wherein the chemical modification comprises reduction of one or more disulfide bridges in the IGF-II protein, or wherein the chemical modification comprises alkylation of one or more cysteines in the IGF-II protein.

### Claim 18 (depends on 16)

The method of claim 16, wherein said sample is further purified by HPLC and/or SPE prior to ionization and said SPE and/or HPLC are conducted with on-line processing.

### Claim 19 (depends on 13)

The method of claim 13, wherein the IGF-II protein from said sample is purified with solid phase extraction (SPE) prior to ionization, and/or wherein the IGF-II protein from said sample is purified by high performance liquid chromatography (HPLC) prior to ionization.

### Claim 20 (depends on 13)

The method of claim 13, wherein said high resolution/high accuracy mass spectrometry is conducted with an orbitrap mass spectrometer or with a time of flight mass spectrometer, or with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to 20,000 and an accuracy of less than or equal to 10 ppm, or with an orbitrap or time of flight mass analyzer capable of a FWHM of greater than or equal to 20,000 and an accuracy of less than or equal to 5 ppm.

### Claim 21 (depends on 13)

The method of claim 13, wherein said determining the amount of one or more IGF-II comprises collecting spectrometric data from one or more peaks with each peak resulting from an isotopic form of an ion.

### Claim 22 (depends on 21)

The method of claim 21, wherein two or more peaks each resulting from a different isotopic form of an ion are used to confirm the identity of the IGF-II protein, or wherein spectrometric data from a peak resulting from a single isotopic form is used to determine the amount of the IGF-II protein in said sample, or wherein spectrometric data from two or more peaks each resulting from a different isotopic form are used to determine the amount of the IGF-II protein in said sample.

### Claim 23 (depends on 13)

The method of claim 13, wherein said sample comprises a biological fluid, or wherein said sample comprises plasma or serum.

### Claim 24 (depends on 13)

The method of claim 13, wherein said one or more IGF-II ions detectable by mass spectrometry comprise one or more IGF-II ions in a 8+, 7+, 6+, or 5+ charge state.