Influence of carboxylation on osteocalcin detection by mass spectrometry

Abstract

Rationale: Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein extracts, and is predominantly detected by non-MS immunological methods. Here, we analyze bovine osteocalcin and its post-translational modifications to determine why a protein of this size goes undetected.

Methods: Osteocalcin was purified from cow bone using well-established methods. Intact osteocalcin or trypsin-digested osteocalcin were separated using an Agilent 1200 series high-performance liquid chromatography (HPLC) system and analyzed using a ThermoScientific LTQ-Orbitrap XL after fragmentation with higher-energy collision dissociation. Data were analyzed using Mascot or Prosight Lite.

Results: Our results support previous findings that the cow osteocalcin has up to three carboxylations using both intact osteocalcin and digested forms. Using Mascot, we were able to detect osteocalcin peptides, but no fragments that localized the carboxylations. Full annotation using Prosight Lite of the intact (three carboxylations), N-terminal peptide (one carboxylation), and middle peptide (two carboxylations) showed complete fragmentation was present, but complete neutral loss was observed.

Conclusions: Osteocalcin carboxylation, and its associated neutral losses, makes high-throughput detection of this protein challenging; however, alternative fragmentation or limited purification can overcome these challenges. Copyright © 2016 John Wiley & Sons, Ltd.

Figure 1

A) Intact Bos taurus OC showing between 0 and 3 carboxylations. Most are triply-carboxylated. B) Fragmentation of m/z 1171.3408 showing as much as three CO₂ losses. Light gray box = hydroxylation; dark gray box = carboxylation.

Figure 2

A) Spectrum of the N-terminal tryptic peptide showing single carboxylation with carbamidomethyl N-terminus (1-Carboxy+CAM) and uncarboxylated (0-Carboxy+CAM) forms. B) Fragmentation pattern of m/z 1149.05 in (A). No carboxylated y-ion peaks are present; all detected peaks show CO₂ loss except y2, which does not overlap the carboxylation. Light gray box = hydroxylation; dark gray box = carboxylation; empty gray box = carbamidomethylation of the N-terminus. Gray fragment markers indicate fragments detected by Mascot.

Figure 3

A) The middle peptide (REVCELNPDCDELADHIGFQEAYR) showing 0–2 carboxylations. B) Fragmentation of m/z 1009.09 in (A) showing two CO₂ losses for all b-series ions. No y-series ions overlapping the carboxylation were detected. Dark gray box = carboxylation; gray C = carbamidomethylation of cysteine. Gray fragment markers indicate fragments detected by Mascot.