Evaluation of Digestion Protocols for the Quantification of Vitamin D Binding Protein by IDMS

摘要

1) Molar concentrations of the tryptic peptides vary significantly between different protocols. Relative concentrations of peptides also vary between purified and plasma protein. 2) Heat appears to aid in denaturing the protein, especially when present in plasma, and results in improved coefficients of variation (% CV) for peptide concentrations. 3) For some peptides, digests performed with guanidine and RapiGest result in lower concentrations and have higher % CV. 4) TFE results in higher concentrations of peptides unique for each isoform in digests of plasma. 5) Peptides TSALSAK, VLEPTLK, and ELPEHTVK (with heat denaturing, except in guanidine) have relatively low % CV between all digest conditions for both protein that has been purified or present in plasma and appear to be promising candidates for reproducible, accurate quantification of VDBP. 6) Reproducibility of the method (preparation of digests and labeled mixes) is generally good with <10 % CV. 7) Peptides specific for each of the 3 major isoforms may also be glycosylated and will be the focus of future work.