MSACL 2025 Abstract

Optimisation of a LC-MS/MS Method for Quantitation of Human Serum Albumin (HSA) and Vitamin D Binding Protein (VDBP) with Identification of Three VDBP Isoforms

Shauna O’Brien (1), Paweenuch Teerasumran (1), Carl Jenkinson (1,2)
(1) MRC Laboratory of Medical Sciences, London, United Kingdom (2) Imperial College London, London, United Kingdom

Shauna O'Brien, PhD (Presenter) MRC-Laboratory of Medical Sciences

Presenter Bio: Shauna O'Brien received a PhD in Organic and Analytic Chemistry from University of Galway. She is currently an MRC Postdoctoral Researcher at the MRC-Laboratory of Medical Sciences in London, where she develops sample preparation and LC-MS/MS methods for the quantitation of the Vitamin D metabolome and Vitamin D Binding Protein for biomarker discovery.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION
Three common polymorphisms of VDBP are encoded by one of three codominant alleles of the VDBP gene. These isoforms are termed GC1f, GC1S and GC2, with each having a differing affinity of vitamin D metabolite binding, for example 25(OH)D3 and 1,25(OH)2D3, Gc1f > Gc1s > Gc2. In practice, individuals with the Gc1S or Gc2 isoforms tend to have higher circulating vitamin D levels compared to individuals with the Gc1f isoform. Vitamin D metabolites also bind to Human Serum Albumin (HSA), though with a lower affinity than VDBP. Routine clinical measurements of circulating vitamin D metabolites only measure 25(OH)D, regardless of its bioavailability which is dependent on VDBP and HSA levels. This is particularly evident in population and human health studies, such as pregnancy, ethnicity and early life stages. The concentration of both VDBP and HSA, alongside the distinction between different VDBP isoforms, could have implications on the clinical interpretation of measured VDBP levels, alongside 25OHD3 concentrations in human serum that are currently under explored.

OBJECTIVES
This work aimed to develop a “bottom-up” proteomics LC-MS/MS method for measurement of circulating VDBP and HSA levels in human serum, as well as VDBP isoform identification.

METHODS
The LC-MS/MS method has been developed and optimised using synthesised peptide standards of VDBP and HSA tryptic digest peptides; VLEPTLK and ELPEHTVK for the quantification of VDBP, LPEATPTELAK, LPDATPK and LPDATPTELAK for VDBP isoform identification, and THLPEVFLSK for quality control, while LVNEVTEFAK is used to quantify HSA. Each metabolite was measured by multiple reaction monitoring following optimization of MS parameters. LC-MS/MS method development was performed on a Waters ACQUITY UPLC coupled to a Waters Xevo TQ-XS, using a HSS T3 column with a method run time of 5 min. The denaturation, alkylation, reduction and digestion method has been optimised using 2,2,2-trifluoroethanol, dithiothreitol, iodoacetamide and TPCK-treated trypsin, respectfully.

RESULTS
The method development process focused on maximising MS sensitivity and digestion efficiency to ensure complete protein digestion, peptide recovery and accurate quantitation. Digestion factors including reagent concentrations and digestion time have been optimised using pooled human serum spiked with VDBP and HSA. The optimal trypsin concentration for peptide recovery for both 10 µL and 20 µL serum sample volumes was 4 mg/mL. Increasing digestion time beyond 15 minutes had a negative impact on peptide recovery, presumably due to over-digestion. The optimised assay achieves a linear range of 10 – 800 µg/mL VDBP and 1 – 80 mg/mL HSA using 20 µL serum.

CONCLUSIONS
A UPLC-MS/MS assay has been developed to quantify HSA and VDBP, and identify VDBP isoforms in serum samples, achieving optimal digestion and sample preparation parameters. The developed assay will be fully validated and employed alongside multi metabolite vitamin D metabolite assays across human health cohort studies in future work.