CN-122003607-A - Method for simultaneous detection and quantification of insulin, proinsulin metabolic intermediate, C-peptide and C-peptide variants by mass spectrometry

Abstract

Methods for determining the amount of an analyte in a sample are described, wherein the analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or a mixture of any two or more thereof. Provided herein are mass spectrometry methods for detecting and quantifying analytes (e.g., insulin, proinsulin, metabolic intermediates of proinsulin, C-peptide, mutant C-peptide, or mixtures of any two or more thereof) in a biological sample using enrichment and/or purification methods coupled with mass spectrometry techniques.

Inventors

• T. S. Collier
• R. W. Pierce
• M. J. mcpsch

Assignees

• 奎斯特诊断投资有限公司

Dates

Publication Date

20260508

Application Date

20241007

Priority Date

20231011

Claims (20)

1. 1. A method of determining the amount of an analyte in a sample by mass spectrometry, the method comprising: immunocapture of said analyte from said sample; subjecting the immunocaptured analyte to an ionization source under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of the one or more ions by mass spectrometry, and Determining the amount of the analyte in the sample from the amount of the one or more ions; Wherein the method comprises the steps of The analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or a mixture of any two or more thereof; The metabolic intermediate of proinsulin is des-31, 32-proinsulin, des-64, 65-proinsulin or mixture thereof, and The mutant C-peptide is a C-peptide having one additional arginine residue at the C-terminus (C-peptide +R), a C-peptide having two additional arginine residues at the C-terminus (C-peptide +RR), or a mixture thereof.
2. 2. The method of claim 1, wherein the analyte comprises proinsulin.
3. 3. The method of claim 2, wherein the one or more ions comprise one or more proinsulin ions.
4. 4. The method of claim 3, wherein the one or more proinsulin ions comprise proinsulin precursor ions having a mass to charge ratio (m/z) of 1342.20 ± 0.5.
5. 5. The method of claim 3, wherein the one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions having m/z of 183.30±0.5, 120.20 ±0.5, and 219.30 ±0.5.
6. 6. The method of claim 1, wherein the analyte comprises a metabolic intermediate of proinsulin.
7. 7. The method of claim 6, wherein the metabolic intermediate of proinsulin comprises des-31, 32-proinsulin.
8. 8. The method of claim 7, wherein the one or more ions comprise one or more des-31, 32-proinsulin ions.
9. 9. The method of claim 8, wherein the one or more des-31, 32-proinsulin ions comprise a des-31, 32-proinsulin precursor ion having an m/z of 1299.50 ± 0.5.
10. 10. The method of claim 8, wherein the one or more des-31, 32-proinsulin ions comprise one or more des-31, 32-proinsulin fragment ions selected from the group consisting of ions having m/z 129.20 ± 0.5, 226.20 ± 0.5 and 183.25 ± 0.5.
11. 11. The method of claim 6, wherein the metabolic intermediate of proinsulin comprises des-64, 65-proinsulin.
12. 12. The method of claim 11, wherein the one or more ions comprise one or more des-64, 65-proinsulin ions.
13. 13. The method of claim 12, wherein the one or more des 64, 65-proinsulin ions comprise des 64, 65-proinsulin precursor ions having an m/z of 1304.30 ± 0.5.
14. 14. The method of claim 12, wherein the one or more des 64, 65-proinsulin ions comprise one or more des 64, 65-proinsulin fragment ions selected from the group consisting of ions having m/z 147.15 ± 0.5, 130.15 ± 0.5 and 183.20 ± 0.5.
15. 15. The method of claim 1, wherein the analyte comprises a mutant C-peptide.
16. 16. The method of claim 15, wherein the mutant C-peptide comprises a C-peptide (C-peptide+r) having one additional arginine residue at the C-terminus.
17. 17. The method of claim 16, wherein the one or more ions comprise one or more C-peptide + R ions.
18. 18. The method of claim 17, wherein the one or more C-peptide +r ions comprise a precursor C-peptide +r ion having an m/z of 1059.20 ±0.5.
19. 19. The method of claim 17, wherein the one or more C-peptide +r ions comprise one or more C-peptide +r fragment ions selected from the group consisting of ions having m/z of 966.40 ±0.5, 260.00 ±0.5, and 844.30 ±0.5.
20. 20. The method of claim 15, wherein the mutant C-peptide comprises a C-peptide having two additional arginine residues at the C-terminus (C-peptide + RR).

Description

Method for simultaneous detection and quantification of insulin, proinsulin metabolic intermediate, C-peptide and C-peptide variants by mass spectrometry Cross Reference to Related Applications The present application claims priority from U.S. provisional patent application No. 63/589,367 filed on 10/11 of 2023, the entire disclosure of which is incorporated herein by reference for any and all purposes. Background Insulin is a hormone that is critical in regulating carbohydrate and fat metabolism in the body. Abnormal insulin levels indicate the presence of dysglycemia and/or insulin resistance syndrome, such as diabetes. Diabetes and its complications are marked by significant public health problems. Proinsulin is a prohormone precursor of insulin. The most common method for measuring proinsulin is an immunoassay, but this method has the problem of cross-reacting with proinsulin intermediates such as des 31, 32-proinsulin and des 64, 65-proinsulin. Thus, there remains a need for new measurement methods with improved reliability and accuracy. Disclosure of Invention In one aspect, provided herein is a method of determining the amount of an analyte in a sample by mass spectrometry, the method comprising: immunocapture of said analyte from said sample; subjecting the immunocaptured analyte to an ionization source under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of the one or more ions by mass spectrometry, and Determining the amount of the analyte in the sample from the amount of the one or more ions; Wherein the method comprises the steps of The analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or a mixture of any two or more thereof; The metabolic intermediate of proinsulin is des-31,32-proinsulin, des-64,65-proinsulin or a mixture thereof, and The mutant C-peptide is a C-peptide having one additional arginine residue at the C-terminus (C-peptide +R), a C-peptide having two additional arginine residues at the C-terminus (C-peptide +RR), or a mixture thereof. In another aspect, provided herein is a method of determining the amount of proinsulin in a sample, the method comprising: immunocapture of proinsulin from said sample; subjecting the immunocaptured proinsulin to an ionization source under conditions suitable for generating one or more proinsulin ions detectable by mass spectrometry; Determining the amount of the one or more proinsulin ions by mass spectrometry, and Determining the amount of proinsulin in the sample from the amount of the one or more proinsulin ions. In another aspect, provided herein is a method of determining the amount of a metabolic intermediate of proinsulin in a sample, the method comprising: Immunocapture of said metabolic intermediate from said sample; Subjecting the immunocaptured metabolic intermediate to an ionization source under conditions suitable for generating one or more metabolic intermediate ions detectable by mass spectrometry; Determining the amount of the one or more metabolic intermediate ions by mass spectrometry, and Determining the amount of metabolic intermediate of the proinsulin in the sample from the amount of the one or more metabolic intermediate ions; Wherein the method comprises the steps of The metabolic intermediate of proinsulin is des-31, 32-proinsulin, des-64, 65-proinsulin or mixture thereof. In another aspect, provided herein is a method of determining the amount of a mutant C-peptide in a sample, the method comprising: Immunocapture of said mutant C-peptide from said sample; Subjecting the immunocaptured mutant C-peptide to an ionization source under conditions suitable for generating one or more mutant C-peptide ions capable of detection by mass spectrometry; Determining the amount of the one or more mutant C-peptide ions by mass spectrometry, and Determining the amount of the mutant C-peptide in the sample from the amount of the one or more mutant C-peptide ions; Wherein the method comprises the steps of The mutant C-peptide is a C-peptide having one additional arginine residue at the C-terminus (C-peptide +R), a C-peptide having two additional arginine residues at the C-terminus (C-peptide +RR), or a mixture thereof. In some embodiments, the analyte comprises proinsulin. In some embodiments, the one or more ions comprise one or more proinsulin ions. In some embodiments, the one or more proinsulin ions comprise proinsulin precursor ions having a mass to charge ratio (m/z) of 1342.20 ±0.5. In some embodiments, the one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions with m/z of 183.30±0.5, 120.20 ±0.5, and 219.30 ±0.5. In some embodiments, the analyte comprises a metabolic intermediate of proinsulin. In some embodiments, the metabolic intermediate of proinsulin comprises des-31, 32-proinsulin. In some embodiments, the one or more ions comprise one or more d