Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Consecutive digestion of archaeological skeletal proteomes with multiple proteases

ABSTRACT: Ancient skeletal proteomes are increasingly utilised for phylogenetic and evolutionary analysis. These proteomes are, however, often small and with low sequence coverage. We expand on previous observations which have shown that parallel digestion of Pleistocene skeletal proteomes increases proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The sequential utilisation of several proteases is a promising avenue for the study of highly degraded ancient proteomes for phylogenetic purposes.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Equus Caballus (horse) Bos Taurus (bovine)

TISSUE(S): Bone

SUBMITTER: Zandra Fagernäs

LAB HEAD: Frido Welker

PROVIDER: PXD047069 | Pride | 2024-03-06

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part3

Project description:Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in monoclonal antibody. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C-terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of non-collagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a non-reduced monoclonal antibody.

2020-10-19 | PXD021703 | Pride

A Pleistocene hominin enamel proteome from Homo antecessor

Project description:Hominin relationships and their dispersal throughout Eurasia during the Early and Middle Pleistocene are highly debated. The relationships between Early and Middle Pleistocene hominins, like Homo antecessor and Homo erectus, and hominin species that dominate the Late Pleistocene fossil record, like Homo sapiens, Neanderthals and Denisovans, are unclear. Here, we obtain enamel proteomes from Homo antecessor (Atapuerca, Spain) and Homo erectus (Dmanisi, Georgia), two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal, and divergence. We recover endogenous ancient proteomes from the Atapuerca and Dmanisi hominins. In addition, our proteomic data indicates that the sampled Homo antecessor molar derives from a male hominin through amelogenin protein sexing. Our data includes preserved in vivo phosphorylation and enamel proteome proteolytic digestion.

2020-04-02 | PXD014342 | Pride

Multi-protease analysis of Pleistocene bone proteomes

Project description:Palaeoproteomics of skeletal tissues has the potential to provide new insights into the evolutionary relationships between globally distributed hominin fossils across the Pleistocene. The use of alternative or additional proteases has the potential to increase both proteome size and protein. However, the potential of alternative proteases beside trypsin remains unexplored systematically in palaeoproteomic studies. Here, we present a comparison of the bone proteome size and protein sequence recovery of six different proteases from four Pleistocene bone specimens. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also note that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics.

2020-10-13 | PXD018264 | Pride

The TNH2 hominin enamel proteome

Project description:Here we analyse two enamel protein extractions from a Middle Pleistocene hominin from Laos, Asia. The proteome is analyzed for potential phylogenetic information and characterized in terms of its molecular damage and in vivo (serine) phosphorylation.

2022-04-06 | PXD018721 | Pride

Middle Pleistocene protein sequences from the rhinoceros genus Stephanorhinus and the phylogeny of extant and extinct Middle/Late Pleistocene Rhinocerotidae.

Project description:Characterization of Middle Pleistocene rhinoceros proteins and the phylogenetic relationships between extinct and extanct rhinoceros was investigated by obtaining ancient protein data for two extinct rhinoceros genera (Coelodonta antiquitatis and Stephanorhinus sp.).

2017-02-02 | PXD005534 | Pride

ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part2

Project description:Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of digestion solution. In this study we characterized ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestions are carried out at acidic pH (1.5) for relatively short (2 hr) time periods. To elucidate the potential of ProAlanase in proteomic applications, we conducted a series of investigations comprising digestion of proline-rich proteins, PTM analysis, de novo protein sequencing and disulfide bond mapping. The results demonstrated that digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain and improves non-collagenous bone protein identification. Notably, cleavage also occurs at the C-terminus of hydroxyproline, facilitating efficient digestion of bone collagen. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in monoclonal antibodies, as well as achieving nearly complete database-independent sequence reconstruction of endogenous protein by de novo sequencing.

2020-10-19 | PXD021191 | Pride

ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping

Project description:Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of digestion solution. In this study we characterized ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestions are carried out at acidic pH (1.5) for relatively short (2 hr) time periods. To elucidate the potential of ProAlanase in proteomic applications, we conducted a series of investigations comprising digestion of proline-rich proteins, PTM analysis, de novo protein sequencing and disulfide bond mapping. The results demonstrated that digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain and improves non-collagenous bone protein identification. Notably, cleavage also occurs at the C-terminus of hydroxyproline, facilitating efficient digestion of bone collagen. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in monoclonal antibodies, as well as achieving nearly complete database-independent sequence reconstruction of endogenous protein by de novo sequencing.

2020-10-19 | PXD019039 | Pride

Palaeoproteomic analysis of Pleistocene cave hyenas from east Asia

Project description:Spotted hyena (Crocuta crocuta) is the only extant species of the genus Crocuta, which once occupied a much wider range during the Pliocene and Pleistocene. However, its origin and evolutionary history is somewhat contentious due to discordances being found between morphological, nuclear, and mitochondrial data. Due to the limited molecular data from east Asian Crocuta, and the difficulty of extracting ancient DNA from this area, here we present proteomic analysis of cave hyenas from three locations in northern China. This marks the first proteomic data generated from cave hyenas, adding new molecular data to the east Asian populations. Phylogenetic analysis based on these protein sequences reveals two different groups of cave hyenas in east Asia, one of which could not be distinguished from modern spotted hyenas from northern Africa, tentatively the result of previously suggested gene flow between these lineages. With developments of instrumentation and analytical methods, proteomics holds promising potential for the phylogenetic reconstruction of ancient fauna previously thought to be unreachable using ancient DNA.

2020-10-19 | PXD020530 | Pride

Ostrich eggshell peptides survival into the Miocene (6-9 Ma)

Project description:The oldest authenticated peptide sequences to date were reported in 2016 from 3.8 Ma old ostrich eggshell (OES) from the site of Laetoli, Tanzania (Demarchi et al., 2016). Here we demonstrate survival of the same sequences in 6.5-9Ma OES recovered from a palaeosteppe setting in northwestern China. The eggshell is thicker than those observed in extant species and consistent with the Liushu Struthio sp. ootaxon. These findings push the preservation of ancient proteins back to the Miocene and highlight their potential for paleontology, paleoecology and evolutionary biology.

2022-12-29 | PXD035872 | Pride