MSACL 2016 US Abstract

Quantification of Vitamin D-Binding Protein Isoforms by MRM

Lisa Kilpatrick (Presenter)
NIST

Authorship: Lisa E. Kilpatrick, Karen W. Phinney
NIST, Gaithersburg, MD

Short Abstract Vitamin D-binding protein (VDBP) is a plasma protein that transports vitamin D metabolites to target tissues. Recent work has shown that quantification of VDBP using immunoassays gives variable results depending on the antibody specificity; therefore, standardization of the methods used for the measurement of this protein is needed. Because there are three common isoforms, GC-1s, GC-1f, and GC-2, with up to two sites of glycosylation on the peptide unique to each isoform, quantification of the protein is challenging. In this work, a method for quantification of the three main VDBP isoforms was investigated using multiple reaction monitoring (MRM).

Long Abstract

Introduction

Vitamin D-binding protein (VDBP) is a plasma protein that transports vitamin D metabolites to target tissues. Recent work has shown that quantification of VDBP using immunoassays gives variable results depending on the antibody specificity(1); therefore, standardization of the methods used for the measurement of this protein is needed. Because there are three common isoforms, GC-1s, GC-1f, and GC-2, with up to two sites of glycosylation on the peptide unique to each isoform, quantification of the protein is challenging. In this work, a method for quantification of the three main VDBP isoforms was investigated using multiple reaction monitoring (MRM).

Methods

Purified VDBP (Athens Research and Technology), pooled human plasma (SRM 1950, NIST), or donor plasma (Bioreclamation) was reduced with TCEP and alkylated with iodoacetamide in the presence of trifluoroethanol. Samples were digested with trypsin overnight. Heavy labeled peptides were gravimetrically spiked into samples and LC-MRM was performed using a triple quadrupole mass spectrometer (Agilent Technologies). Concentrations of the unlabeled peptides in the digests were then determined.

Conclusions

Purified VDBP and synthesized peptides were used to identify MRM transitions for peptides in common to all forms of VDBP and for each of the main isoforms. Two transitions per peptide were selected based on intensity and used for further testing in a sample of pooled human plasma. The preliminary data show that the total VDBP concentration in the pooled sample is 5.3x10^3 pmol/g with 3.7 % CV between samples analyzed on different days. The pooled plasma was also found to contain each of the three common isoforms with concentrations of 8.5x10^2, 1.7x10^3 and 4.7x10^2 pmol/g for GC-1f, GC-1s, and GC-2, respectively. Because the glycosylation sites are present on the isoform-specific peptides, only the concentrations of unglycosylated forms of the isoforms were determined. Quantification of VDBP isoforms was also performed on human plasma from individual donors. This work is the first step toward the development of a VDBP reference material.

References & Acknowledgements:

(1) Henderson CM, Lutsey PL, Misialek JR, Laha TJ, Selvin E, Eckfeldt JH, Hoofnagle AN. Clin Chem. 2015 Oct 9. pii: clinchem.2015.244541.

Financial Disclosure

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

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