Quantitation of GLA proteins by mass spectrometric analysis
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.
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 proteins selected from the group consisting of γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins from the biological sample with a solvent, wherein the sample is a protein precipitate and extracting the sample with the solvent provides a liquid extract and a residual protein precipitate, wherein the residual protein precipitate is enriched with the one or more proteins relative to the sample;
(b) separating the liquid extract from the residual protein precipitate;
(c) dissolving the residual protein precipitate in an aqueous medium to provide a solution comprising the one or more proteins;
(d) contacting the solution comprising the one or more proteins 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
(e) 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.
2. 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.
3. 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.
4. The method of claim 3 , wherein the mass spectrometric analysis comprises tandem mass spectrometric (MS/MS) analysis.
5. The method of claim 3 , wherein the mass spectrometric analysis comprises liquid chromatographic mass spectrometric (LC-MS) analysis.
6. 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.
7. 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).
8. The method of claim 1 , wherein the biological sample is plasma, serum, blood, or a dried blood spot.
9. The method of claim 8 , wherein the plasma, serum, or blood has a volume of about 5 uL to about 100 uL.
10. The method of claim 2 , wherein the one or more vitamin K-dependent blood clotting factors is prothrombin (Factor II).
11. The method of claim 1 , wherein the protease is trypsin or chymotrypsin.
12. 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).
13. 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).
14. 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.
15. The method of claim 1 , wherein the liquid extract comprises an albumin.