Quantification of Total Vitamin D-Binding Protein and Three Common Isoforms with Liquid Chromatography-Isotope Dilution Mass Spectrometry
Lisa E. Kilpatrick, Karen W. Phinney
Vitamin D-binding protein (VDBP), a transporter of 25-hydroxyvitamin D [25(OH)D] metabolites, has additional roles in the immune system and bone development. Recent investigations have studied the relationship between VDBP isoforms, having variations in amino acids, and various diseases. Quantification is typically performed using antibodies which may be prone to bias; therefore, the development of an accurate method for quantification of VDBP isoforms will allow further investigation into the physiological importance of this protein. In this work, liquid chromatography coupled with isotope dilution mass spectrometry (LC-IDMS) was evaluated for quantification of total VDBP and the three common isoforms, GC-1f, GC-1s, and GC-2. Because the O-linked glycosylation site(s) is near the amino acid variations, only unglycosylated isoform concentrations were measured. Protocols using either guanidine, urea, RapiGest, trifluoroethanol, or Tris buffer were also evaluated for optimal tryptic digestion. Differences in peptide release were detected between purified and plasma VDBP, however three peptides shared between the isoforms (ELPEHTVK, TSALSAK, and VLEPTLK) were found to have the good reproducibility between most digestion protocols for both protein sources. The isoform- specific peptides LPDATPK, LPDATPTELAK, and LPEATPTELAK, had optimal release when TFE was used for plasma digestions. The total protein concentration calculated from the average of the three shared peptides resulted in > 97 % accuracy compared to the concentration determined by amino acid analysis. Relative amounts of glycosylation were also calculated from the ratio of the isoform-specific peptide(s) to the total protein concentration and were found to be
and Phinney, K.
Quantification of Total Vitamin D-Binding Protein and Three Common Isoforms with Liquid Chromatography-Isotope Dilution Mass Spectrometry, Journal of Proteome Research, [online], https://doi.org/10.1021/acs.jproteome.7b00560
(Accessed July 5, 2022)