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

Autor:	Kilpatrick LE; Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, USA., Bouillon R; Laboratory of Clinical and Experimental Endocrinology, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium., Davis WC; Hollings Marine Laboratory, National Institute of Standards and Technology, Charleston, SC, USA., Henderson CM; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.; Seagen, Inc., Bothell, WA, USA., Hoofnagle AN; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA., Pauwels S; Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium., Vanderschueren D; Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium., Waelkens E; Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium., Wildiers H; Department of General Medical Oncology, University Hospitals Leuven, Leuven, Belgium., Yen JH; Statistical Engineering Division, Information Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA., Phinney KW; Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
Jazyk:	angličtina
Zdroj:	Clinical chemistry and laboratory medicine [Clin Chem Lab Med] 2022 Oct 24; Vol. 61 (1), pp. 78-85. Date of Electronic Publication: 2022 Oct 24 (Print Publication: 2023).
DOI:	10.1515/cclm-2022-0642
Abstrakt:	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 (R 2 ≥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. (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)
Databáze:	MEDLINE
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