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 ∼ 120,000 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 well-preserved 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. The data are available via ProteomeXchange with identifier PXD001827.

Fig. 1.

Cranial bone sample from B. latifrons (13. 009a) with inside surface shown. Presence of material that is consistent with meninges connective tissue is shown. Structure that may be the site of a former blood vessel is found in the upper right of the bottom image (boxed area).

Fig. 2.

Relative quantification of COL1A1 peptide deamidation using extracted ion chromatogram of the ion envelopes. Area under the curve was used to calculate percentage of peptide with glutamine deamidation. Insets: MS signal for the right and left peak showing the COL1A1 deamidated form of DGEAGAQ*GPPGPAGPAGER (top) and the parent COL1A1 peptide DGEAGAQGPPGPAGPAGER (bottom).

Fig. 3.

Occurrence plot from open mass modification search using Protein Prospector Batch-Tag. The Y-axis is limited to <100 peptide spectra matches to improve visualization of lower abundant mass modifications. Mass additions consistent with galactosylation and glucosylgalactosylation of hydroxylysine are highlighted.

Fig. 4.

Spectra assignments for glucosylgalactosyl-hydroxylysine containing peptides are reported in (A) COL5A1 peptide GEK††GESGPSGAAGPPGPK (K753) with oxidation of P15 and (B) COL1A2 peptide GFPGTPGLPGFK††GIR (K175) with oxidation of P3, P6, and P9. †† denotes site of modification (Δ 340.10 Da). For y and b ion identifications; † denotes the loss of 162.05 Da (Hex), †† denotes the loss of 324.11 Da (2 Hex), * denotes the loss of NH₃, and ^o denotes the loss of H₂O. Assigning these hexose groups as Hyl O-linked galactose and glucosylgalactose is consistent with the well-characterized modification of fibrillar collagens (–79). Consistent with this assignment, all modifications are found in the triple helix regions.

Fig. 5.

Glycosylated hydroxylysine residues identified within several collagen proteins. Line lengths mirror the primary structure organization of the represented proteins. Colored circles represent significant modifications (expectation value ≤ 0.05), while grayscale circles represent modifications with an e-value > 0.05 and are included only for sites where a significant glycan modification was identified in at least one of the two datasets. PTMs are either oriented upwards or downwards, indicating whether a specific glycosylation pattern has been identified in the present B. latifrons dataset (up) or in a recent proteomics report on woolly mammoth (M. primigenius) femur proteomics by Cappellini et al. (29) (down). The boxed COL1A2 and COL5A1 glycosylation sites represent the peptides shown in Fig. 4(A) and 4(B). * denotes that this specific site, K753, is found in a different isoform of the α1-chain of type V collagen (Accession # K7QCV6) than the one used for the figure (Accession # G3MZI7). The placement of this site at residue #1491 was determined by homology searching.