Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Abstract

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ϳ120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in wellpreserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1-4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4 -7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6,8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more pre...

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

“…For samples in which analysis of a separate demineralization fraction is not feasible, alternative methods that do not fractionate demineralization, or do not demineralize (e.g., Cleland & Vashishth, 2015; Hill et al, 2015), should be considered to avoid loss of identified protein diversity.…”

“…Because the extant chicken bones used for these analyses were not subject to the diagenetic factors that can cause chemical alterations in fossil proteins (e.g., deamidation, carboxymethylation of lysine, loss of hydroxylations to proline (Cleland et al, 2016; Cleland, Schroeter & Schweitzer, 2015; Hill et al, 2015)), it is unclear whether the disparity of efficiencies between protocols observed here would be similar if conducted on a fossil sample. Indeed, given the chemical differences in depositional environments experienced by fossils from different localities, it is possible that similar comparison analyses of 10 different fossil specimens would yield 10 different results.…”

“…In either case, for studies specifically seeking NCPs in low protein samples (e.g., osteonectin in fossils) it is imperative that any demineralization fractions be analyzed rather than discarded, regardless of comparatively minute amounts of extract. Conversely, extraction protocols that do not include a separate demineralization stage (e.g., Cleland & Vashishth, 2015; Hill et al, 2015) may be particularly well suited for the recovery of NCPs from fossils, and should be further explored.…”

Analysis of the Preserved Amino Acid Bias in Peptide Profiles of Iron Age Teeth from a Tropical Environment Enable Sexing of Individuals Using Amelogenin MRM

“…Methods for the extraction of dentin and enamel proteins has been adapted from the groups of Jàgr, Hill,and Capellini . Briefly, teeth were cut lengthwise with a diamond blade.…”

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry

“…Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has become the favorite technique to routinely identify posttranslational modifications (PTMs) of proteins of biological interest; however, a number of practical issues remain to be scrutinized for the analyses of highly modified large proteins such as collagens. 49–51 Indeed, although collagen IV is very abundant, a surprising underrepresentation of sequence coverage has been observed in proteomics inventories of highly enriched BM preparations. 52–56 This may be explained by the inherent properties of collagen IV such as repetitive nature of primary sequence, high insolubility, cross-linking, and resistance to cleavage due to extensive PTM, which could lead to low peptide abundance, poor primary sequence coverage, and poor identification of the location of PTMs.…”