Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

The Influence of Proteoforms: Assessing the Accuracy of Total VDBP Quantification by Proteolysis and LC-MS/MS

Published

Author(s)

Lisa Kilpatrick, Roger Bouillon, Clay Davis, Clark Henderson, Andrew N. Hoofnagle, Steven Pauwels, Dirk Vanderschueren, Etienne Waelkens, Hans Wildiers, James H. Yen, Karen W. Phinney

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.
Citation
Clinical Chemistry and Laboratory Medicine
Volume
61
Issue
1

Keywords

LC-MS/MS, proteoform, quantification, reference value, vitamin D-binding protein

Citation

Kilpatrick, L. , Bouillon, R. , Davis, C. , Henderson, C. , Hoofnagle, A. , Pauwels, S. , Vanderschueren, D. , Waelkens, E. , Wildiers, H. , Yen, J. and Phinney, K. (2022), The Influence of Proteoforms: Assessing the Accuracy of Total VDBP Quantification by Proteolysis and LC-MS/MS, Clinical Chemistry and Laboratory Medicine, [online], https://doi.org/10.1515/cclm-2022-0642, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=931885 (Accessed June 18, 2024)

Issues

If you have any questions about this publication or are having problems accessing it, please contact reflib@nist.gov.

Created October 24, 2022, Updated March 8, 2023