Biologically and diagenetically derived peptide modifications in moa collagens

Abstract

The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa.

Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus canadensis

“…Two of the backbone cleavages are directly adjacent to positions that may be deamidated, leading us to propose deamidation as an additional mechanism for diagenetic backbone cleavage beyond the previously hypothesized proline oxidation reactions. 54 The presence of these diagenetically cleaved peptides, together with noncleaved versions of B. canadensis peptides, provides further support for this process. We were also able to detect several biologically derived PTMs (e.g., acetylation, methylation) that, through future research, may result in a better understanding of how these proteins evolve.…”

“…We were also able to detect several biologically derived PTMs (e.g., acetylation, methylation) that, through future research, may result in a better understanding of how these proteins evolve. Both acetylation and methylation have been shown to persist in early diagenesis, 54 a critical time for preserving proteins and their modifications into deep time.…”

Peptide sequences from the first Castoroides ohioensis skull and the utility of old museum collections for palaeoproteomics

“…and (iii) collagen I alpha 1 GjVSVPGPMGPSGPR. These truncations and the CML-modified peptides are located at different positions than those from previously detected from non-mammalian collagen I sequences [10], supporting their diagenetic, nonenzymatic origin. These truncations, which would result in many collagen sequences of variable, unpredictable lengths, may be a source of the smearing frequently observed in palaeoproteomics SDS-PAGE gels [12].…”

Bone protein “extractomics”: comparing the efficiency of bone protein extractions ofGallus gallusin tandem mass spectrometry, with an eye towards paleoproteomics

“…Primary among these challenges is the inherent uncertainty surrounding the nature of the fossils themselves, which have undergone processes that are impossible to directly measure or observe, but which profoundly affect the chemistry of organic matter remaining within them. These include, but are not limited to, uncertainty in the rates at which proteins may degrade over geologic time, and the role that any given environmental factor plays in protein preservation and/or degradation (Hedges, 2002; Nielsen-Marsh et al, 2000; Nielsen-Marsh & Hedges, 2000), uncertainty regarding diagenetic modifications that might alter the chemical structure of ancient molecules (e.g., AGEs) (Cleland, Schroeter & Schweitzer, 2015; Nielsen-Marsh et al, 2005; Van Klinken & Hedges, 1995), and finally, uncertainty over what fluctuations in the biological, thermal, and hydrological conditions a fossil may have been subjected to over thousands to millions of years, and how these may affect their molecular composition (Schroeter & Cleland, 2016; Smith et al, 2003). However, although molecular studies on fossil remains will always be laden with unknown (and unknowable) variables that must be studied indirectly, the nascent field of molecular paleontology is also affected by unexplored gaps in our knowledge which must be addressed and resolved for the field to continue to progress.…”

“…A variety of reagents have been employed to remove bone mineral and increase access to remaining organic fractions, such as hydrochloric acid (HCl) (Buckley, 2013; Buckley, 2015; Buckley et al, 2009; Buckley et al, 2010; Buckley, Larkin & Collins, 2011; Buckley & Wadsworth, 2014; Cleland, Schroeter & Schweitzer, 2015; Cleland, Voegele & Schweitzer, 2012; Ostrom et al, 2000; Wadsworth & Buckley, 2014; Welker et al, 2015), sodium ethylenediaminetetraacetic acid (EDTA) (Buckley et al, 2008; Cappellini et al, 2012; Cleland, Voegele & Schweitzer, 2012; Humpula et al, 2007; Nielsen-Marsh et al, 2002; Nielsen-Marsh et al, 2005; Ostrom et al, 2006; Schweitzer et al, 2007; Schweitzer et al, 2009), and ammonium EDTA (Ostrom et al, 2000). Reagents used to solubilize bone proteins for subsequent mass spectrometry (MS) and immunological applications are equally diverse, and have included (among others) ammonium bicarbonate (ABC) (Buckley et al, 2009; Buckley et al, 2010; Buckley, Larkin & Collins, 2011; Cappellini et al, 2012; Cleland, Schroeter & Schweitzer, 2015; Cleland, Voegele & Schweitzer, 2012), and guanidine hydrochloride (GuHCl) (Buckley, 2015; Buckley & Wadsworth, 2014; Cleland, Voegele & Schweitzer, 2012; Schweitzer et al, 2007; Schweitzer et al, 2009; Wadsworth & Buckley, 2014). However, these methods have not been consistently employed across fossil studies, which may hamper direct comparisons of preserved protein content between fossil specimens, as these reagents have been shown to vary in efficacy when used to extract bone proteins for enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Cleland, Voegele & Schweitzer, 2012).…”