Peptide selection for the quantification of P‐III‐NP in human serum by mass spectrometry. (5th March 2018) - Moncrieffe, Danielle
Abstract : Rationale: Procollagen III amino‐terminal propeptide (P‐III‐NP) is currently monitored in human doping control as a biomarker for growth hormone administration and also in clinical diagnostics using immunoassays. Drawbacks to this approach have been highlighted and research is ongoing to develop a mass spectrometric method to complement these methods. However, a lack of traceable reference material, the presence of post‐translational modifications (PTMs), and small blood concentration complicate the development of targeted analytical methods for P‐III‐NP quantification. Methods: Tryptic digest products of P‐III‐NP were assessed by liquid chromatography/mass spectrometry (LC/MS). In silico digestion was used to predict P‐III‐NP peptides for MS analysis; however, these excluded PTMs. With a priori knowledge of PTMs, we associated experimental P‐III‐NP peptides with those derived by in silico digestion. Synthesized P‐III‐NP peptides, h T1 (human) and T5 (human/bovine), were used to develop sensitive micro‐ and nano‐flow LC/MS methods to analyse P‐III‐NP originating from human serum semi‐quantitatively. Results: P‐III‐NP peptides, T1 and T5, were identified using high‐resolution accurate MS (HRAMS). PTMs modified the mass of observed peptides. N‐terminal pyroglutamation ( p E) in T1 and several hydroxylated prolines ( h P) in T5 (G‐X‐ h P motif) were observed. With PTM, h T1 and T5 were observed in a digest of immuno‐captured P‐III‐NP by LC/MS. Using a semi‐quantitative approach, h P‐III‐NP at basal concentrations of 2 ng/mL (50 pmol) could be estimated from a 200‐μL sample volume. Conclusions: Consideration of PTMs is needed to identify P‐III‐NP peptides produced by digestion with trypsin. The information presented here now gives the most appropriate peptide sequences for synthesizing suitable reference materials required for quantification of human P‐III‐NP in blood and evidences methodology that is sufficiently sensitive to develop a quantitative method.