Osteocalcin (OC) is an important protein in the extracellular matrix of bone that controls hydroxyapatite crystal properties. It also plays a role in whole-bone mechanics because of its interaction with both hydroxyapatite and bone collagen I. OC is of particular interest in human and model organism studies due to its roles within bone and as a hormone promoting insulin production. However, OC is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein extracts. Researchers predominantly use non-MS immunological methods to detect OC. Cleland et al. (2016) sought to determine why OC goes undetected.1
The investigators used bovine OC isolated from Bos taurus bone. They prepared samples for analysis as OC peptides and intact OCs. They also prepared a sample with bovine collagen I and OC in a 100:1 ratio, to reflect a similar ratio of collagen to OC in bone and demonstrate the effect of collagen I on OC detection by MS. Cleland et al. separated all three OC preparations for 75 minutes at increasing gradients and then characterized the eluted peptides in positive mode on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). They fragmented the three most abundant peaks using higher energy collision dissociation (HCD). To identify OC peaks, Cleland et al. converted raw files to peak lists and searched against Swiss-Prot and a decoy database using Mascot 2.3. Finally, the investigators performed in silico digestion of B. taurus OC. They extracted averaged MS/MS spectra corresponding to these peaks.
For intact OC, Cleland et al. detected proteoforms with up to three carboxylations. They also identified chromium-adducted forms with up to three carboxylations. They found fully carboxylated B. taurus OC with hydroxyproline. They also found doubly, singly and uncarboxylated proteoforms. The chromium-adducted forms showed more consistent carboxylation levels, which the investigators suggest indicates that the chromium stabilizes the OC carboxylation, and that some of the carboxylation variation detected is the result of decarboxylation during or before their analysis. Further, they found extensive overalkylation of the cysteine-free N-terminal peptide and the dicysteine middle OC peptide. The investigators suggest that this makes an added N-terminal carbamidomethylation (CAM) modification in Mascot critical to detect these peptides.
Cleland et al. note the following hindrances to OC detection by MS:
Gamma carboxylation of one or more glutamic acids
OC acidity: gamma carboxylation of glutamic acid results in neutral loss after fragmentation
In positive ionization mode, acidic peptides show reduced ion signals compared to relatively basic ones, such as those from collagen I. In the samples prepared with bovine collagen I, the investigators could not detect OC.
In conclusion, Cleland et al. recommend that researchers do not use HCD fragmentation. They should instead consider alternative fragmentation methodology and/or additional search strategies.
1. Cleland, T.P. (2016) “Influence of carboxylation on osteocalcin detection by mass spectrometry,” Rapid Communications in Mass Spectrometry, 30(19) (pp. 2109–2115), doi: 10.1002/rcm.7692.
Post Author: Miriam Pollak. Miriam a Nutritionist specialising in women’s health and works from her Bondi Beach clinic. She is also currently completing her Masters by research in nutrition.
Prior to this, Miriam majored in neuroscience as an undergraduate before completing a post graduate degree in science communication. She spent over a decade working in science communication and medical research, collaborating with some of the best oncologists and researchers in Australia and the U.S.