Peptide selection for the quantification of P‐III‐NP in human
serum by mass spectrometry
Mark C. Parkin
David A. Cowan
Drug Control Centre, King's Forensics,
Department of Analytical, Environmental and
Forensic Sciences, King's College London,
D. A. Cowan, Drug Control Centre, King's
Forensics, Department of Analytical,
Environmental and Forensic Sciences, King's
College London, London, UK.
Partnership for Clean Competition, Grant/
Award Number: 306508 R314
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
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.
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.
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
Type III collagen is the second most abundant collagen (varying in
abundance with age) in humans; it is typically found with type I
collagen and is present in tissues with elastic properties such as
skin, blood vessels and various internal organs.
Type III collagen is
formed from a precursor pro‐collagen unit in the cell, which includes
amino‐ and carboxyl‐terminal extension propeptides (denoted P‐III‐NP
and P‐III‐CP, respectively).
P‐III‐NP is a clinical biomarker used to monitor chronic active
hepatitis, liver fibrosis and cirrhosis.
Elevated levels of P‐III‐NP have
been associated with hormonally induced growth,
one of two biomarkers used to detect rhGH (recombinant human growth
hormone) doping in sport;
the other being insulin‐like growth factor I
(IGF‐I). These compounds have little diurnal variation and are largely
unaffected by exercise or sex, but respond to hGH administration.
Also, body mass index and race contribute little to the variability of
For humans, blood P‐III‐NP concentrations are inversely
proportional to age, with maximum concentrations being observed
during pubertal years. Sex however affects P‐III‐NP blood concentrations,
men (average conc. 5.4 ± 2.3 ng/mL) having a slightly larger natural
concentration than women (average conc. 5.1 ± 1.5 ng/mL).
P‐III‐NP consists of three identical 130 amino acid pro α1‐chains,
connected by disulphide cysteine bonds to create a unique pro‐
collagen subunit with three distinct domains, Col 1–3.
domain, Col 1, has a globular structure resulting from its richness in acidic
Received: 1 November 2017 Revised: 3 January 2018 Accepted: 10 January 2018
Rapid Commun Mass Spectrom. 2018;32:535–542. Copyright © 2018 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/rcm 535