# US 2022/0034901 A1 · Quantitation of gla proteins by mass spectrometric analysis

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- Number: US 2022/0034901 A1
- Document ID: US20220034901A1
- Status: Published application
- Kind: A1
- Application number: 17/386,234
- Filed: 2021-07-27
- Published: 2022-02-03
- Earliest claimed: 2020-07-28
- Assignees: University of Washington; Washington State University
- Applicants: University of Washington; Washington State University
- Inventors: Bhagwat Prasad; Kenneth E. Thummel; Abdul Basit Shaikh; Allan E. Rettie
- Web page: https://patentgrep.com/patent/US20220034901A1

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 2333/745: Assays involving non-enzymic blood coagulation factors
- 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

Methods of LC-MS/MS quantification of γ-carboxylated proteins in plasma, serum, or blood, including dried blood spots, are disclosed. The methods can be used to determine patient-specific dosing of anticoagulant drugs and diagnosis of liver diseases, such as hepatocellular carcinoma.

## Claims

### Claim 1

A method for quantifying one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins in a biological sample, comprising:
(a) extracting a sample comprising one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins from the biological sample having a first concentration, to provide a solution having a second concentration comprising one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins;
(b) contacting the γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins in the solution having the second concentration with a protease, to provide a solution of peptides comprising one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides; and
(c) determining the quantity of the one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides in the solution of peptides.

### Claim 2 (depends on 1)

The method of claim 1, wherein the one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins are derived from one or more vitamin K-dependent blood clotting factors.

### Claim 3 (depends on 1)

The method of claim 1, wherein determining the quantity of the one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides comprises mass spectrometric (MS) analysis.

### Claim 4 (depends on 3)

The method of claim 3, wherein the mass spectrometric analysis comprises tandem mass spectrometric (MS/MS) analysis.

### Claim 5 (depends on 3)

The method of claim 3, wherein the mass spectrometric analysis comprises liquid chromatographic mass spectrometric (LC-MS) analysis.

### Claim 6 (depends on 3)

The method of claim 3, wherein the mass spectrometric analysis comprises multiple reaction monitoring of a fragmentation profile of each of the one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins are derived from prothrombin (Factor II), Factor VII, Factor IX, Factor X, the anticoagulant proteins C and S, the factor X-targeting protein Z, osteocalcin, the calcification-inhibiting matrix Gla protein (MGP), the growth arrest specific gene 6 protein (GAS6), periostin, transmembrane Gla proteins (TMGPs), transthyretin (thyroxin binding protein), and proline-rich Gla-proteins (PRGPs).

### Claim 8 (depends on 1)

The method of claim 1, wherein the biological sample is plasma, serum, blood, or a dried blood spot.

### Claim 9 (depends on 8)

The method of claim 8, wherein the plasma, serum, or blood has a volume of about 5 uL to about 100 uL.

### Claim 10 (depends on 2)

The method of claim 2, wherein the one or more vitamin K-dependent blood clotting factors is prothrombin (Factor II).

### Claim 11 (depends on 1)

The method of claim 1, wherein the protease is trypsin or chymotrypsin.

### Claim 12 (depends on 1)

The method of claim 1, wherein the γ-carboxylated peptides comprise one or more peptide sequences,
wherein the peptide sequence for prothrombin comprises ANTFL[E][E]VRK (SEQ ID NO: 5) and GNL[E]R (SEQ ID NO: 7); Factor VII comprises ANAFL[E][E]LRPGSL[E]R (SEQ ID NO: 8), [E]IFKDA[E]R (SEQ ID NO: 9), and PGSL[E]R (SEQ ID NO: 51); Factor IX comprises L[E][E]FVQGNL[E]R (SEQ ID NO: 10), [E]CM[E][E]K (SEQ ID NO: 11), CSF[E][E]AR (SEQ ID NO: 12), [E]VF[E]NT[E]R (SEQ ID NO: 13) and TT[E]FWK (SEQ ID NO: 14); Factor X comprises ANSFL[E][E]MK (SEQ ID NO: 1), [E]VF[E]DSDK (SEQ ID NO: 15), TN[E]FWNK (SEQ ID NO: 16), and GHL[E]R (SEQ ID NO: 59); Protein S comprises ANSLL[E][E]TK (SEQ ID NO: 17), QGNL[E]R (SEQ ID NO: 18) and [E]CI[E][E]LCNK (SEQ ID NO: 19); Protein C comprises ANSFL[E][E]LR (SEQ ID NO: 20), HSSL[E]R (SEQ ID NO: 21), and [E]IFQNVDDTLAFWSK (SEQ ID NO: 22); osteocalcin comprises YLYQWLGAPVPYPDPL[E]PR (SEQ ID NO: 23) and [E]VC[E]LNPDCD[E]LADHIGFQ[E]AYR (SEQ ID NO: 24); and MGP comprises SKPVH[E]LNR (SEQ ID NO: 25) and [E]ACDDYR (SEQ ID NO: 26).

### Claim 13 (depends on 1)

The method of claim 1, wherein the des-carboxylated peptides comprise one or more peptide sequences,
wherein the peptide sequence for prothrombin comprises ANTFLEEVRK (SEQ ID NO: 3) and GNLER (SEQ ID NO: 6); Factor VII comprises ANAFLEELRPGSLER (SEQ ID NO: 27), EIFKDAER (SEQ ID NO: 28), and PGSLER (SEQ ID NO: 53); Factor IX comprises LEEFVQGNLER (SEQ ID NO: 29), ECMEEK (SEQ ID NO: 30), CSFEEAR (SEQ ID NO: 31), EVFENTER (SEQ ID NO: 32) and TTEFWK (SEQ ID NO: 33); Factor X comprises ANSFLEEMK (SEQ ID NO: 34), EVFEDSDK (SEQ ID NO: 35), TNEFWNK (SEQ ID NO: 36), and GHLER (SEQ ID NO: 60); Protein S comprises ANSLLEETK (SEQ ID NO: 37), QGNLER (SEQ ID NO: 38), and ECIEELCNK (SEQ ID NO: 39); Protein C comprises ANSFLEELR (SEQ ID NO: 40), HSSLER (SEQ ID NO: 41), and EIFQNVDDTLAFWSK (SEQ ID NO: 42); osteocalcin comprises YLYQWLGAPVPYPDPLEPR (SEQ ID NO: 43) and EVCELNPDCDELADHIGFQEAYR (SEQ ID NO: 44); and MGP comprises SKPVHELNR (SEQ ID NO: 45) and EACDDYR (SEQ ID NO: 46).

### Claim 14 (depends on 1)

The method of claim 1, wherein the γ-carboxylated peptide proteoforms comprise one or more peptide sequences, wherein at least one glutamate or glutamic acid residue is γ-carboxylated, and wherein fewer than all glutamate or glutamic acid residues are γ-carboxylated.

### Claim 15

A method for quantifying one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins in a biological sample, comprising:
(a) extracting a sample comprising one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins from the biological sample having a first concentration, to provide a solution having a second concentration comprising one or more of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins;
(b) contacting the γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins in the solution having the second concentration with a protease, to provide a solution of peptides comprising one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides;
(c) derivatizing the one or more of γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides of the solution of peptides, to provide one or more derivatized γ-carboxylated peptides, derivatized γ-carboxylated peptide proteoforms, and derivatized des-carboxylated peptides; and
(d) determining the quantity of the one or more of derivatized γ-carboxylated peptides, derivatized γ-carboxylated peptide proteoforms, and derivatized des-carboxylated peptides in the solution of peptides.

### Claim 16 (depends on 15)

The method of claim 15, wherein the derivatization comprises esterification, alkylation, or amidation.

### Claim 17 (depends on 15)

The method of claim 15, further comprising determining the quantity of the one or more derivatized γ-carboxylated peptides, derivatized γ-carboxylated peptide proteoforms, and derivatized des-carboxylated peptides by mass spectrometric (MS) analysis.

### Claim 18 (depends on 17)

The method of claim 17, wherein the mass spectrometric analysis comprises tandem mass spectrometric (MS/MS) analysis.

### Claim 19 (depends on 17)

The method of claim 17, wherein the mass spectrometric analysis comprises liquid chromatographic mass spectrometric (LC-MS) analysis.

### Claim 20 (depends on 18)

The method of claim 18, wherein the mass spectrometric analysis comprises multiple reaction monitoring of a fragmentation profile of each of the one or more derivatized γ-carboxylated peptides, derivatized γ-carboxylated peptide proteoforms, and derivatized des-carboxylated peptides.