Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry

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, hT1 (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 (pE) in T1 and several hydroxylated prolines (hP) in T5 (G-X-hP motif) were observed. With PTM, hT1 and T5 were observed in a digest of immuno-captured P-III-NP by LC/MS. Using a semi-quantitative approach, hP-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.