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The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS

  • Lisa E. Kilpatrick ORCID logo EMAIL logo , Roger Bouillon , W. Clay Davis , Clark M. Henderson , Andrew N. Hoofnagle , Steven Pauwels , Dirk Vanderschueren , Etienne Waelkens , Hans Wildiers , James H. Yen and Karen W. Phinney
Published/Copyright: October 24, 2022
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 61 Issue 1

Abstract

Objectives

Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy.

Methods

VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories.

Results

Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV.

Conclusions

VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Keywords: LC-MS/MS; proteoform; quantification; reference value; vitamin D-binding protein
Corresponding author: Lisa E. Kilpatrick, Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899, USA, Phone: 301-975-5323, Fax: 301-975-8505, E-mail:

Acknowledgments

The authors would like to thank Dr. Stephen Wise and Dr. Adam Kuszak for their advice and critical review of the manuscript.

  1. Research funding: This work was partially funded by the UW Nutrition Obesity Research Center (P30DK035816), UW Diabetes Research Center (P30DK017047), and the NIH-ODS through an interagency agreement.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2018), and has been approved by the authors’ Institutional Review Boards (Committee on Medical Ethics at the University Hospitals Leuven and KU Leuven and the NIST Research Protections Office).

  6. Disclaimer: Certain commercial instruments, software or materials are identified in this document. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the products identified are necessarily the best available for the purpose.

References

1. Tsuprykov, O, Chen, X, Hocher, CF, Skoblo, R, Lianghong, Y, Hocher, B. Why should we measure free 25(OH) vitamin D? J Steroid Biochem Mol Biol 2018;180:87–104. https://doi.org/10.1016/j.jsbmb.2017.11.014.Search in Google Scholar PubMed

2. Chun, RF, Peercy, BE, Orwoll, ES, Nielson, CM, Adams, JS, Hewison, M. Vitamin D and DBP: the free hormone hypothesis revisited. J Steroid Biochem Mol Biol 2014;144:132–7. https://doi.org/10.1016/j.jsbmb.2013.09.012.Search in Google Scholar PubMed PubMed Central

3. Bouillon, R, Schuit, F, Antonio, L, Rastinejad, F. Vitamin D binding protein: a historic overview. Front Endocrinol 2020;10:1–21. https://doi.org/10.3389/fendo.2019.00910.Search in Google Scholar PubMed PubMed Central

4. Henderson, CM, Lutsey, PL, Misialek, JR, Laha, TJ, Selvin, E, Eckfeldt, JH, et al.. Measurement by a novel LC-MS/MS methodology reveals similar serum concentrations of vitamin D-binding protein in blacks and whites. Clin Chem 2016;62:179–87. https://doi.org/10.1373/clinchem.2015.244541.Search in Google Scholar PubMed PubMed Central

5. Kilpatrick, LE, Boggs, ASP, Davis, WC, Long, SE, Yen, JH, Phinney, KW. Assessing a method and reference material for quantification of vitamin D binding protein during pregnancy. Clin Mass Spectrom 2020;16:11–7. https://doi.org/10.1016/j.clinms.2020.01.002.Search in Google Scholar PubMed PubMed Central

6. Kilpatrick, LE, Phinney, KW. Quantification of total vitamin-D-binding protein and the glycosylated isoforms by liquid chromatography-isotope dilution mass spectrometry. J Proteome Res 2017;16:4185–95. https://doi.org/10.1021/acs.jproteome.7b00560.Search in Google Scholar PubMed

7. Hoofnagle, AN, Eckfeldt, JH, Lutsey, PL. Vitamin D-binding protein concentrations quantified by mass spectrometry. N Engl J Med 2015;373:1480–2. https://doi.org/10.1056/nejmc1502602.Search in Google Scholar PubMed PubMed Central

8. May, W, Parris, R, Beck, C, Fassett, J, Greenberg, R, Guenther, F, et al.. Definitions of terms and modes used at NIST for value-assignment of reference materials for chemical measurements. Gaithersburg, MD: U.S. Government Printing Office; 2000:1–12 pp.Search in Google Scholar

9. Camara, J, Gonzalez, CA, Choquette, SJ. National institute of standards & technology certificate of analysis standard reference material 1950 metabolites in frozen human plasma. Gaithersburg, MD: NIST Office of Reference Materials; 2019:1–15 pp.Search in Google Scholar

10. Russell, A, Gonzalez, CA, Choquette, SJ. National institute of standards & technology certificate of analysis standard reference material 1949 frozen human prenatal serum. Gaithersburg, MD: NIST Office of Reference Materials; 2021:1–8 pp.Search in Google Scholar

11. Hatse, S, Lambrechts, D, Verstuyf, A, Smeets, A, Brouwers, B, Vandorpe, T, et al.. Vitamin D status at breast cancer diagnosis: correlation with tumor characteristics, disease outcome, and genetic determinants of vitamin D insufficiency. Carcinogenesis 2012;33:1319–26. https://doi.org/10.1093/carcin/bgs187.Search in Google Scholar PubMed

12. Magrane, M, Consortium, U. UniProt knowledgebase: a hub of integrated protein data. J Biol Databases Curation 2011;2011:bar009. https://doi.org/10.1093/database/bar009.Search in Google Scholar PubMed PubMed Central

13. Hoofnagle, AN, Whiteaker, JR, Carr, SA, Kuhn, E, Liu, T, Massoni, SA, et al.. Recommendations for the generation, quantification, storage, and handling of peptides used for mass spectrometry-based assays. Clin Chem 2016;62:48–69. https://doi.org/10.1373/clinchem.2015.250563.Search in Google Scholar PubMed PubMed Central

14. Lowenthal, MS, Yen, J, Bunk, DM, Phinney, KW. Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS. Anal Bioanal Chem 2010;397:511–9. https://doi.org/10.1007/s00216-010-3616-9.Search in Google Scholar PubMed

15. Taylor, BN, Kuyatt, CE. NIST guidelines for evaluating and expressing the uncertainty of NIST measurement results. Gaithersburg, MD: U.S. Department of Commerce; 1994. Available from: https://www.nist.gov/pml/nist-technical-note-1297.10.6028/NIST.TN.1297Search in Google Scholar

16. Working Group 1 of the Joint Committee for Guides in Metrology. Evaluation of measurement data — guide to the expression of uncertainty in measurement (ISO GUM 1995 with minor corrections). JCGM 100:2008 2008; 1–120. Available from: http://www.bipm.org/utils/common/documents/jcgm/JCGM_100_2008_E.pdf.Search in Google Scholar

17. Ravnsborg, T, Olsen, DT, Thysen, AH, Christiansen, M, Houen, G, Hojrup, P. The glycosylation and characterization of the candidate Gc macrophage activating factor. Biochim Biophys Acta 2010;1804:909–17. https://doi.org/10.1016/j.bbapap.2009.12.022.Search in Google Scholar PubMed

18. Borges, CR, Jarvis, JW, Oran, PE, Nelson, RW. Population studies of vitamin D binding protein microheterogeneity by mass spectrometry lead to characterization of its genotype-dependent O-glycosylation patterns. J Proteome Res 2008;7:4143–53. https://doi.org/10.1021/pr8002936.Search in Google Scholar PubMed

19. Borges, CR, Jarvis, JW, Oran, PE, Rogers, SP, Nelson, RW. Population studies of intact vitamin D binding protein by affinity capture ESI-TOF-MS. J Biomol Tech JBT 2008;19:167–76.Search in Google Scholar

20. Rehder, DS, Nelson, RW, Borges, CR. Glycosylation status of vitamin D binding protein in cancer patients. Protein Sci 2009;18:2036–42. https://doi.org/10.1002/pro.214.Search in Google Scholar PubMed PubMed Central

21. Yu, L, Vizel, A, Huff, MB, Young, M, Remmele, RLJr, He, B. Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development. J Pharmaceut Biomed Anal 2006;42:455–63. https://doi.org/10.1016/j.jpba.2006.05.008.Search in Google Scholar PubMed

22. CLSI. Liquid chromatography-mass spectrometry methods; approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.Search in Google Scholar

23. U.S. Department of Health and Human Services Food and Drug Administration. Bioanalytical method validation guidance for industry: food and drug administration; 2018. Available from: https://www.fda.gov/media/70858/download.Search in Google Scholar

24. Shuford, CM, Sederoff, RR, Chiang, VL, Muddiman, DC. Peptide production and decay rates affect the quantitative accuracy of protein cleavage isotope dilution mass spectrometry (PC-IDMS). Mol Cell Proteomics 2012;11:814–23. https://doi.org/10.1074/mcp.o112.017145.Search in Google Scholar PubMed PubMed Central

25. Grangeon, A, Clermont, V, Barama, A, Gaudette, F, Turgeon, J, Michaud, V. Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics. J Pharmaceut Biomed Anal 2019;173:96–107. https://doi.org/10.1016/j.jpba.2019.05.006.Search in Google Scholar PubMed

26. Trenchevska, O, Nelson, RW, Nedelkov, D. Mass spectrometric immunoassays for discovery, screening and quantification of clinically relevant proteoforms. Bioanalysis 2016;8:1623–33. https://doi.org/10.4155/bio-2016-0060.Search in Google Scholar PubMed PubMed Central

27. Zhou, H, Hoek, M, Yi, P, Rohm, RJ, Mahsut, A, Brown, P, et al.. Rapid detection and quantification of apolipoprotein L1 genetic variants and total levels in plasma by ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom : RCM 2013;27:2639–47. https://doi.org/10.1002/rcm.6734.Search in Google Scholar PubMed

28. Vilà-Rico, M, Colomé-Calls, N, Martín-Castel, L, Gay, M, Azorín, S, Vilaseca, M, et al.. Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS. J Proteonomics 2015;127:234–46. https://doi.org/10.1016/j.jprot.2015.04.016.Search in Google Scholar PubMed

29. Little, RR, Rohlfing, CL, Hanson, S, Connolly, S, Higgins, T, Weykamp, CW, et al.. Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods. Clin Chem 2008;54:1277–82. https://doi.org/10.1373/clinchem.2008.103580.Search in Google Scholar PubMed

30. Shuford, CM, Walters, JJ, Holland, PM, Sreenivasan, U, Askari, N, Ray, K, et al.. Absolute protein quantification by mass spectrometry: not as simple as advertised. Anal Chem 2017;89:7406–15. https://doi.org/10.1021/acs.analchem.7b00858.Search in Google Scholar PubMed

31. van den Broek, I, Romijn, FP, Smit, NP, van der Laarse, A, Drijfhout, JW, van der Burgt, YE, et al.. Quantifying protein measurands by peptide measurements: where do errors arise? J Proteome Res 2015;14:928–42. https://doi.org/10.1021/pr5011179.Search in Google Scholar PubMed

32. Kritmetapak, K, Pongchaiyakul, C. Parathyroid hormone measurement in chronic kidney disease: from basics to clinical implications. Internet J Nephrol 2019;2019:5496710. https://doi.org/10.1155/2019/5496710.Search in Google Scholar PubMed PubMed Central

33. Smit, MA, van Kinschot, CMJ, van der Linden, J, van Noord, C, Kos, S. Clinical guidelines and PTH measurement: does assay generation matter? Endocr Rev 2019;40:1468–80. https://doi.org/10.1210/er.2018-00220.Search in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2022-0642).

Received: 2022-07-05
Accepted: 2022-09-26
Published Online: 2022-10-24
Published in Print: 2023-01-27

© 2022 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. A new milestone on the road to global standardization of apolipoprotein measurements
  4. Review
  5. Saliva – a new opportunity for fluid biopsy
  6. Opinion Papers
  7. Emerging technology: a definition for laboratory medicine
  8. The next wave of innovation in laboratory automation: systems for auto-verification, quality control and specimen quality assurance
  9. EFLM Paper
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  16. The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS
  17. Total serum vitamin B12 (cobalamin) LC-MS/MS assay as an arbiter of clinically discordant immunoassay results
  18. The preanalytical process in the emergency department, a European survey
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  20. Umbilical cord blood gases: probability of arterial or venous source in acidemia
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  24. Performance of digital morphology analyzer CellaVision DC-1
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  27. Cardiovascular Diseases
  28. Quantitation of cardiac troponin I in cancer patients treated with immune checkpoint inhibitors: a case-control study
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  31. Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: institutional experience performed in all clinical laboratories of a Regional Health Service
  32. Acknowledgment
  33. Acknowledgment
  34. Letters to the Editor
  35. About the estimation of albuminuria based on proteinuria results
  36. Response to “About the estimation of albuminuria based on proteinuria results”
  37. Reply to Abildgaard et al.: lot variation and inter-device differences contribute to poor analytical performance of the DCA vantage™ HbA1c POCT instrument in a true clinical setting
  38. Reply to letter from Mayfield et al. regarding “Lot variation and inter-device differences contribute to poor analytical performance of the DCA Vantage™ HbA1c POCT instrument in a true clinical setting”
  39. High-sensitive cardiac troponin T: are turbulences coming?
  40. Analytical performance evaluation of bioactive adrenomedullin on point-of-care platform
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  42. Neopterin level can be measured by intraocular liquid biopsy
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https://doi.org/10.1515/cclm-2022-0642
Keywords for this article
LC-MS/MS; proteoform; quantification; reference value; vitamin D-binding protein

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. A new milestone on the road to global standardization of apolipoprotein measurements
  4. Review
  5. Saliva – a new opportunity for fluid biopsy
  6. Opinion Papers
  7. Emerging technology: a definition for laboratory medicine
  8. The next wave of innovation in laboratory automation: systems for auto-verification, quality control and specimen quality assurance
  9. EFLM Paper
  10. The new, race-free, Chronic Kidney Disease Epidemiology Consortium (CKD-EPI) equation to estimate glomerular filtration rate: is it applicable in Europe? A position statement by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)
  11. Guidelines and Recommendations
  12. Overcoming challenges regarding reference materials and regulations that influence global standardization of medical laboratory testing results
  13. General Clinical Chemistry and Laboratory Medicine
  14. Quantitative protein mass-spectrometry requires a standardized pre-analytical phase
  15. Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone
  16. The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS
  17. Total serum vitamin B12 (cobalamin) LC-MS/MS assay as an arbiter of clinically discordant immunoassay results
  18. The preanalytical process in the emergency department, a European survey
  19. Proenkephalin A as a marker for glomerular filtration rate in critically ill children: validation against gold standard iohexol GFR measurements
  20. Umbilical cord blood gases: probability of arterial or venous source in acidemia
  21. Reference Values and Biological Variations
  22. Pediatric reference interval verification for 17 specialized immunoassays and cancer markers on the Abbott Alinity i system in the CALIPER cohort of healthy children and adolescents
  23. Hematology and Coagulation
  24. Performance of digital morphology analyzer CellaVision DC-1
  25. Cancer Diagnostics
  26. Managing the impact of inter-method bias of prostate specific antigen assays on biopsy referral: the key to move towards precision health in prostate cancer management
  27. Cardiovascular Diseases
  28. Quantitation of cardiac troponin I in cancer patients treated with immune checkpoint inhibitors: a case-control study
  29. Infectious Diseases
  30. A novel scoring system combining Modified Early Warning Score with biomarkers of monocyte distribution width, white blood cell counts, and neutrophil-to-lymphocyte ratio to improve early sepsis prediction in older adults
  31. Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: institutional experience performed in all clinical laboratories of a Regional Health Service
  32. Acknowledgment
  33. Acknowledgment
  34. Letters to the Editor
  35. About the estimation of albuminuria based on proteinuria results
  36. Response to “About the estimation of albuminuria based on proteinuria results”
  37. Reply to Abildgaard et al.: lot variation and inter-device differences contribute to poor analytical performance of the DCA vantage™ HbA1c POCT instrument in a true clinical setting
  38. Reply to letter from Mayfield et al. regarding “Lot variation and inter-device differences contribute to poor analytical performance of the DCA Vantage™ HbA1c POCT instrument in a true clinical setting”
  39. High-sensitive cardiac troponin T: are turbulences coming?
  40. Analytical performance evaluation of bioactive adrenomedullin on point-of-care platform
  41. Increased PD-L1 surface expression on peripheral blood granulocytes and monocytes after vaccination with SARS-CoV2 mRNA or vector vaccine
  42. Neopterin level can be measured by intraocular liquid biopsy
  43. The stability of pleural fluid pH under slushed ice and room temperature conditions
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