Jason L. Pittman

Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the Cβ incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post‐translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimers disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to cn•+58 and zℓ−n‐57 fragment ions (n, position of Asp; ℓ, total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the Cα—Cβ backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top‐down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the α-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the α-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c·, and they also show c·-1 peaks in the deuterated peptides only. The presence of the c·-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H· and −CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.

Biological rationale for the intramedullary canal as a source of autograft material.

Bone harvested by intramedullary reaming offers a minimally invasive alternative to harvesting bone from the iliac crest, which has long been considered the gold standard for autogenous bone grafting. The biologic potential of intramedullary reaming material has been studied both in vitro and in vivo. The material provides osteogenic, osteoinductive, and osteoconductive properties that are comparable to the material harvested from the iliac crest. In addition to the ability to obtain a large volume of bone, the graft harvested by the Reamer-Irrigator-Aspirator has been shown to be rich in growth factors, including BMP-2, TGF-beta1, IGF-I, FGFa, and PDGFbb.

BMPR1A antagonist differentially affects cartilage and bone formation during fracture healing

A soluble form of BMP receptor type 1A (mBMPR1A‐mFC) acts as an antagonist to endogenous BMPR1A and has been shown to increase bone mass in mice. The goal of this study was to examine the effects of mBMPR1A‐mFC on secondary fracture healing. Treatment consisted of 10 mg/kg intraperitoneal injections of mBMPR1A‐mFC twice weekly in male C57BL/6 mice. Treatment beginning at 1, 14, and 21 days post‐fracture assessed receptor function during endochondral bone formation, at the onset of secondary bone formation, and during coupled remodeling, respectively. Control animals received saline injections. mBMPR1A‐mFC treatment initiated on day 1 delayed cartilage maturation in the callus and resulted in large regions of fibrous tissue. Treatment initiated on day 1 also increased the amount of mineralized tissue and up‐regulated many bone‐associated genes (p = 0.002) but retarded periosteal bony bridging and impaired strength and toughness at day 35 (p < 0.035). Delaying the onset of treatment to day 14 or 21 partially mitigated these effects and produced evidence of accelerated coupled remodeling. These results indicate that inhibition of the BMPR1A‐mediated signaling has negative effects on secondary fracture healing that are differentially manifested at different stages of healing and within different cell populations. These effects are most pronounced during the endochondral period and appear to be mediated by selective inhibition of BMPRIA signaling within the periosteum.

Erratum: “BMPR1A Antagonist Differentially Affects Cartilage and Bone Formation During Fracture Healing” [J Orthop Res. Vol 34, 2096–2105 (2016)]

Erratum: “BMPR1A Antagonist Differentially Affects Cartilage and Bone Formation During Fracture Healing” [J Orthop Res. Vol 34, 2096–2105 (2016)] Elise F. Morgan, Jason Pittman, Anthony DeGiacomo, Daniel Cusher, Chantal M. J. de Bakker, Keri A. Mroszczyk, Mark W. Grinstaff, Louis C. Gerstenfeld Department of Mechanical Engineering, Boston University, Boston 02215 MA , Department of Orthopaedic Surgery, Boston University School of Medicine, Boston 02118 MA, Department of Biomedical Engineering, Boston University, Boston 02215 MA , Department of Chemistry, Boston University, Boston 02215 MA Received 22 July 2015; accepted 10 March 2016 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.23677