Biology subjects

Olsen, J. V.

Publications and source records attributed to Olsen, J. V..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages

ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues. Glutamate- and aspartate-linked ADPr (Glu/Asp-ADPr) is chemically unstable during sample preparation for conventional mass spectrometry (MS)-based proteomics workflows, limiting its detection. Here, we systematically assessed the stability of ADPr linkages using synthetic peptides and confirmed that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, or through hydrolysis by wildtype Af1521. We established an acidic enrichment workflow encompassing an Af1521 mutant, robustly preserving Glu/Asp-ADPr enabling their site-specific and systems-wide MS analysis. Applying this strategy to cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp-ADPr and >200 Cys-ADPr sites. Our analysis uncovered that Glu/Asp-ADPr marks distinct cytoplasmic protein networks enriched in immune functions, contrasting with Ser-ADPr typically observed on nuclear and chromatin-associated proteins. Quantitative profiling revealed reproducible ADPr patterns specific to cell type and treatment. Notably, PARP10 promoted Glu/Asp modification of ubiquitin, highlighting crosstalk between ADPr and ubiquitin signaling. Across interferon treatments, we identified a conserved network of antiviral PARPs and associated cofactors extensively modified on Glu/Asp residues, emphasizing residue-specific ADPr as a regulator of innate immune signaling. Together, our work establishes an MS-based proteomics workflow for identification of Glu/Asp-ADPr, provides a resource of site-specific modification events, and reveals residue-specific ADPr dynamics in immune signaling.

cell biology↗

bioRxiv2025

Benchmarking enrichment and depletion methods for quantitative plasma proteomics in different plasma types and the correlation to clinical routine assays.

Plasma proteomics based on mass spectrometry has great potential for biomarker discovery. Plasma is challenging for mass spectrometry due to high dynamic range in protein abundance. Several workflows have been developed to overcome this, and in this study, we compare prominent workflows using platelet-poor-(PPP), platelet-rich plasma (PRP) and serum (SER). Our results show that depletion workflows including Top14 depletion and acid precipitation allow quantification of very different proteomes than methods based on enrichments of extracellular vesicles such as bead-based enrichment or ultracentrifugation. Enrichment methods are superior in terms of proteome depth and quantitative performance but may be less robust in large cohorts. There is a very high correlation between PPP and PRP samples with all methods and less to SER samples - especially with enrichment workflows. The correlation of 10 protein measurements, performed by clinical routine processes on a Cobas system, showed heterogeneous results. Low abundant proteins with biological dynamics within a healthy cohort, including c-reactive protein and lipoprotein(a), correlated very well to proteomic workflows while others, including albumin and transferrin, correlated poorly. In conclusion, the workflow for plasma proteomics should be aligned with the aim of the analysis and the setup of the sample collection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/686186v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@10b62a7org.highwire.dtl.DTLVardef@79d4f1org.highwire.dtl.DTLVardef@8b21a3org.highwire.dtl.DTLVardef@4d85d9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

biochemistry↗

bioRxiv2025

Single-cell proteomics of pancreatic islet cells reveals type 1 diabetes and donor-specific features

Type 1 diabetes mellitus (T1DM) is the most common severe chronic disease in children and adolescents and requires life-long exogenous insulin treatment. Investigating key differences between the pancreas of healthy and T1DM patients is key to help uncover new treatments. Here we performed a single-cell proteomic (SCP) analysis of purified pancreatic islets from several healthy and T1DM donors at a depth of > 4000 protein quantified. We employed tools designed for single-cell mRNA sequencing (scRNAseq) for cell type annotation highlighting discrepancies between mRNA and protein markers. Subsequently, we defined a subset of markers useful for future proteomics analyses of pancreatic islets. This marker list was employed to refine the annotations of our combined dataset, which we exploited with a machine learning approach to successfully transfer annotations into a new islet dataset. Lastly, we showcase the applicability and value of SCP in a clinical context by providing novel insights into cell type-specific differences between healthy and T1DM islets as well as between healthy islets of different donors. Our study is the first SCP analysis of pancreatic islets from multiple donors and T1DM islets, performed at this level of analytical depth. This provides methodological and biological insight as well as a reference dataset for future SCP studies and for any researcher interested in T1DM and in pancreatic islet physiology.

biochemistry↗

bioRxiv2025

The proteomics and phosphoproteomics landscape of melanoma under T cell attack

Understanding how tumor cells interact with tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) is crucial for identifying targetable immune checkpoints and predictive biomarkers for immunotherapy. While transcriptional responses have been characterized, protein-level changes remain largely unexplored. Here, we used a system reproducing the interaction of TILs with cancer cells occurring in the TME, by co-culturing patient-derived cancer cells with matched autologous TILs at sub-lethal ratios. Using this system, we profiled the early response that cancer (melanoma) cells and TILs activate following autologous T cell attack. To distinguish melanoma from TIL proteomes, we applied stable isotope labeling by amino acids in cell culture (SILAC) combined with Orbitrap Astral-based data-independent acquisition (DIA) mass spectrometry, enabling cell type-specific profiling of protein and phosphorylation dynamics without FACS sorting. This approach also captured the global newly synthesized proteome of the mixed cultures. Our analyses resolved interferon-{gamma}-dependent proteome changes occurring in melanoma cells, identified the cytotoxic and regulatory T-cell molecule (CRTAM) as a selective marker of reactive cytotoxic T lymphocytes, and revealed tumor-intrinsic kinase activation signatures. Among these, multiple DNA damage response-associated kinases were activated during immune attack, suggesting potential therapeutic vulnerabilities. Overall, this framework enables proteomic dissection of tumor-immune interactions and provides a resource for guiding biomarker discovery and therapeutic strategies to improve immunotherapy outcomes.

cancer biology↗

bioRxiv2025

Improved peptide search for identification of SUMO and sequence-based modifications, in MaxSBM

Post-translational modifications (PTMs), such as SUMOylation and ubiquitination, regulate key cellular processes by covalently attaching to lysine residues. While mass spectrometry allows site-specific identification of PTMs, most existing search engines are optimized for small, non-fragmenting modifications and struggle to detect large, fragmenting protein-based modifiers. We refer to these as Sequence-Based Modifiers (SBMs). To overcome this limitation, we developed an SBM-specific search strategy within MaxQuant that accounts for the fragmentation behavior of SBMs during peptide identification. Using publicly available datasets, we validated our approach for SUMO2/3. Our analysis identified distinct diagnostic features and characteristic mass shifts associated with SBM fragmentation, referred to in this study as d-ions (diagnostic ions) and p-ions. By leveraging these features, our method improved the identification of SUMOylated peptides from human cell lines by 13%, SUMOylation sites in mouse embryonic cells by 18%, and in mouse adipocytes by 25%. Our search method improved spectral annotation of SBMs by up to 9% increase in the median Andromeda score. Taken together, we highlight the potential of our SBM search to enhance the discovery of protein-based modifications. HighlightsO_LIDevelopment of a MaxQuant module tailored for identifying Sequence-Based Modifiers (SBMs), including SUMO2/3 C_LIO_LIIncorporation of SBM-specific fragmentation patterns into search algorithms C_LIO_LIEnhanced biological discovery through improved PTM identification from mass spectrometry datasets C_LI In BriefHere, we introduce MaxSBM, an optimized framework for interpreting complex sequence-based modifiers (SBMs), particularly SUMO, within MaxQuant. Our approach incorporates SBM-specific d- and p-ion series into peptide scoring and annotation. By extending the theoretical spectral space to include fragments bearing partial SUMO (or other SBM) peptide remnants, MaxSBM provides a more comprehensive spectral annotation which enhances peptide scoring, resulting in increased identification rates at a higher confidence. Beyond methodological refinement, we validated MaxSBM via reanalysis of several physiological SUMO datasets, ultimately unlocking new insights via mapping of previously obscured modification sites.

bioinformatics↗

bioRxiv2025

Higher-Throughput Proteome Profiling Enabled by Parallelized Pre-Accumulation and Optimized Ion Processing in the Orbitrap Astral Zoom Mass Spectrometer

High-throughput proteomics is critical for understanding biological processes, enabling large-scale studies such as biomarker discovery and systems biology. However, current mass spectrometry technologies face limitations in speed, sensitivity, and scalability for analyzing large sample cohorts. The Thermo Scientific Orbitrap Astral Zoom mass spectrometer (MS) was developed to address these limitations by improving acquisition speed, ion utilization, and spectral processing, which are all essential for advancing proteome depth in high-throughput proteomics. The Orbitrap Astral Zoom MS achieves ultra-fast MS/MS scan rates of up to 270 Hz with enhanced ion utilization through pre-accumulation, enabling the identification of [~]100,000 unique peptides and >8,400 proteins in a single 300 samples-per-day (SPD) analysis of human cell lysate. The optimized system reduces analysis time by 40%, achieves near-complete proteome coverage (>12,000 proteins) in 2.7 hours, and enables ultra-high-throughput workflows, identifying >7,000 proteins in a 500 SPD method with exceptional reproducibility (Pairwise Pearson correlations >0.99). These advancements establish the Orbitrap Astral Zoom MS as a new benchmark in proteomics, significantly enhancing speed, sensitivity, and scalability, paving the way for routine large-scale proteome studies with applications in clinical research and systems biology. TeaserHigh-Speed Human Proteome Analysis using the Orbitrap Astral Zoom Mass Spectrometer

biochemistry↗

bioRxiv2025

Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells

BackgroundGlucocorticoids (GCs) are corticosteroid hormones that are commonly used for treating systemic inflammatory diseases and acute infections. Immunosuppressive effects of GCs have been studied in many cell types, particularly macrophages and T cells. Despite the importance and abundance of neutrophils in the human immune system, GC responses remain understudied in neutrophils. ResultsHere, we performed quantitative mass-spectrometry (MS)-based proteomics of neutrophil-like cells (NLCs) differentiated from human HL-60 promyelocyte cells. Proteome and flow cytometry analysis showed that NLCs share features of neutrophils. Quantitative proteomics and phosphoproteomics of NLCs treated with two synthetic GC compounds, the clinical drugs dexamethasone (DEX) and prednisolone (PRED), identified higher numbers of significantly regulated proteins and phosphosites compared to parental HL-60 cells. GC treatments triggered abnormal neutrophil activation and aging by promoting toll-like receptor (TLR) signaling and CXCR4 serine phosphorylation. We also identified RIPOR2 as a novel target protein of GC which stimulates Rho GTPase signaling networks and upregulates actin cytoskeletal proteins. ConclusionsOur results not only reveal unconventional regulatory mechanism of GCs in the human immune system but also provide valuable resources for discovering novel GC-responsive protein targets.

cell biology↗

bioRxiv2025

Meteorin resolves nociceptive hypersensitivity by reducing connexin-mediated coupling in satellite glial cells

Neuropathic pain, a persistent condition arising from injury to the nervous system, involves complex interactions between neurons and non-neuronal cells, including satellite glial cells (SGCs) in the dorsal root ganglia (DRG). In this study, we examined the glial-targeting effects of meteorin, a neurotrophic protein with gliogenic properties, using mouse models of neuropathic and inflammatory pain. Systemic meteorin administration reversed mechanical hypersensitivity across diverse neuropathic and inflammatory pain models, with therapeutic effects persisting beyond the treatment period. We identified SGCs as the principal site of meteorin expression and action in the DRG, where it selectively activated SGCs and altered their functional state. Proteomic profiling revealed meteorin-mediated downregulation of gap junction proteins in SGCs, particularly connexin 43, which was corroborated by immunohistochemical analyses. Functional assessments demonstrated that meteorin treatment normalized injury-induced increases in intercellular coupling between SGCs, establishing a mechanistic link between glial network modulation and pain resolution. These findings identify meteorin as a regulator of SGC communication through connexin-dependent mechanisms. The sustained therapeutic effects and multi-model efficacy highlight meteorin as a potential intervention for neuropathic pain while advancing our understanding of SGC plasticity in sensory processing.

neuroscience↗

bioRxiv2025

A Solid-Phase Extraction Capture (SPEC) workflow in nanoliter volumes for fast, robust and ultrasensitive proteomics

Sample preparation remains a critical bottleneck in mass spectrometry (MS)-based proteomics, particularly for limited sample amounts where surface adsorption and dilution cause substantial losses. Here, we present Solid-Phase Extraction Capture (SPEC), a workflow that confines protein processing to nanoliter volumes within ion-exchange or C18 matrix inside a pipette tip. This achieves near-complete proteolysis within 5 minutes instead of hours and maintains full compatibility with strong detergents without cleanup steps, enabling effective lysis of challenging samples. From 200 ng FFPE tissue, SPEC achieves proteome depth and reproducibility exceeding conventional bulk protocols using 100 {micro}g, critical when sample is irreplaceable. The modular two-tip configuration enables on-tip chemical modifications for mTRAQ labeling and fractionation, while integration with enrichment workflows yields 2-fold improved glycopeptide identifications from plasma and 3-fold enhanced ubiquitin remnant identification at low amounts. SPEC enables nanoPhos for cell-type resolved tissue phosphoproteomics and provides a universal platform for proteomics sample preparation.

systems biology↗

bioRxiv2025

Composition variations in archaeological human bone proteomes

Sampling strategies within the field of skeletal palaeoproteomics are often based on specimen availability. Knowledge of bone biology might assist in improving sample selection strategies and minimise unnecessary sampling of precious (hominin) material. We study ten bone sample locations across four bone elements, for a total of 10 adult, archaeological human skeletons. We compare bone proteome composition and modification for skeletal elements formed through endochondral and intramembranous ossification, as well as cortical-trabecular bone pairs of three skeletal locations. We observe minimal differences in bones formed through the two ossification processes, outside of the exclusive presence of cartilage-related proteins in endochondral bone samples. We observe higher protein concentrations, a larger number of protein groups and peptides, and lower rates of deamidation in cortical bone compared to trabecular bone proteomes, this indicates that cortical bone provides a better preservation environment compared to trabecular bone. Throughout our analysis, the petrous bone stands out, with the largest and most complex proteomes recovered for all studied individuals. Formed through endochondral ossification, the petrous bone undergoes minimal turnover during life. Our observations indicate that the petrous bone is the ideal source of ancient protein sequence information.

evolutionary biology↗

bioRxiv2025

Identification and removal of contamination in palaeoproteomic analysis of dental enamel

Analysis of archaeological and palaeontological dental enamel allows for the study of taxonomy, phylogenetic relationships, and genetic sex of Pleistocene fauna and hominin remains. However, high-quality data is required for the study of degraded, chemically modified ancient proteomes. Protein contamination may hinder acquisition of such data, due to the abundance and better preservation of modern contaminating proteins. Here, we artificially contaminate a Pleistocene woolly rhinoceros tooth in order to investigate decontamination of archaeological dental enamel. We find that although the contaminating proteins are not identified, likely due to a lack of denaturation and digestion steps in archaeological dental enamel protein extraction protocols, contamination leads to significant loss of endogenous proteomic information. We thereafter compare five published decontamination methods, and find that a simple water or bleach wash prior to demineralization is most efficient at removing protein contamination. The water wash does not affect the endogenous proteome, while a bleach wash may lead to a loss of shorter intercrystalline peptides. We therefore conclude that decontamination is necessary in the study of ancient dental enamel, to ensure maximal retrieval of endogenous proteomic information, and can easily be achieved by incorporating a water or bleach wash step into the protein extraction protocol.

evolutionary biology↗

bioRxiv2025

New methods on the block: Taxonomic identification of archaeological bones in resin-embedded sediments through palaeoproteomics

The integration of biomolecular studies of past organisms with geoarchaeological studies can significantly improve our understanding of the relative chronology and context of archaeologically (in)visible behaviours. However, the complexity and sedimentological heterogeneity of archaeological deposits at a microscopic scale is often not taken into consideration in biomolecular studies. Here, we investigate the preservation and retrieval of palaeoproteomic data from bone fragments embedded in Pleistocene resin-impregnated sediment blocks. We show that resin impregnation has minimal effect on skeletal protein taxonomic identifications in modern skeletal material, but observe an increase in oxidation-related post-translational modifications. We then successfully retrieve proteins from resin-impregnated blocks from the Palaeolithic sites of Bacho Kiro Cave, La Ferrassie and Quincay. The taxonomic identifications of minute bones encased in resin are in line with previous analyses of the faunal communities of these sites, with a diversity of taxa (Bos sp./Bison sp., Equus sp., Ursus sp., and Caprinae) observed at a microscale in Bacho Kiro. This differs from results from La Ferrassie where most of the samples are identified as a single taxon (Bos sp./Bison sp.) across different areas of the site. The block from Quincay only provided taxonomic identification of two out of eleven bone-derived samples, likely due to diagenesis. Our work indicates that palaeoproteomes can be retrieved from bone fragments at a microstratigraphic resolution, enabling the detailed study of faunal community composition at a scale that more closely matches that of past human occupations. Significance StatementResin-embedded sediment blocks are widely used in archaeology and soil sciences to reconstruct past environments and human behavior, but their potential for biomolecular analysis is underexplored. Here, we demonstrate that ancient proteins can be successfully retrieved from bone fragments embedded in resin-impregnated sediment blocks from Pleistocene archaeological sites. Our findings show that resin impregnation has a minimal impact on protein recovery and that palaeoproteomics enables taxonomic identification of Pleistocene bone fragments at a microstratigraphic scale. This approach allows for reconstructing past faunal communities with unprecedented detail, improving our understanding of ancient ecosystems and the environmental contexts of early hominin occupations.

evolutionary biology↗

bioRxiv2025

Organoid-based modeling of platinum resistance identifies KRT17 as both a response mediator and biomarker for targeted therapy in ovarian cancer

Variable platinum responses drive high mortality in high-grade serous ovarian cancer (HGSOC), but to date, there is no molecular approach to define resistance levels for clinical decision-making. Here, we developed the organoid drug resistance assay (ODR-test) with patient-derived organoids from our ovarian cancer biobank and found that all HGSOC patients develop molecular resistance under exposure to carboplatin, although with varying clinical implications. Sustained phenotypic reprogramming and cellular plasticity under carboplatin pressure emerged as a conserved mechanism irrespective of the basal resistance level. Transcriptional and proteomic analyses revealed changes in cell adhesion and differentiation in post-platinum lines as adaptive responses that drive the increase in resistance. We identified Keratin 17 (KRT17) as a mediator of developing platinum resistance and validated its function by CRISPR/Cas9 and overexpression. Additionally, we found that KRT17 expression status (K-score) is a significant negative prognostic histopathological biomarker in a large cohort (N=384) of advanced HGSOC patients. In organoids, increased KRT17 levels enhanced sensitivity to PI3K/Akt inhibitors Alpelisib and Afuresertib, highlighting the potential of KRT17 as a stratification biomarker for targeted therapies.

cancer biology↗

bioRxiv2025

Enhancing tandem MS sensitivity and peptide identification via ion pre-accumulation in an Orbitrap mass spectrometer

High-throughput mass spectrometry-based proteomics has gained increasing interest for both academic and industrial applications. As implementation of faster gradients has facilitated higher sample throughput, mass spectrometers must adapt to shorter analysis times by enhancing scanning speed and sensitivity. For Orbitrap mass spectrometers, faster scan rates are constrained by the need for sufficient ion accumulation time, particularly given limitations on duty cycle at high repetition rate, and transient length, which determines analyzer sensitivity and resolving power. In this context, implementing alternative ion scheduling and better ion signal-processing strategies are needed to unleash the speed of these instruments. Here, we introduce a new scanning strategy termed pre-accumulation, which enables the storage of ions in the bent flatapole in parallel to the operation of the C-trap/IRM, leading to a significant improvement in ion beam utilization and enabling for the first time scanning speeds of >70 Hz on hybrid Orbitrap instruments. The combination of pre-accumulation and increased scan speeds notably enhances peptide and protein group identifications for short LC gradients and improves sensitivity for high-throughput applications. These benefits were further amplified when coupled with the full mass range phase-constrained spectrum deconvolution method ({Phi}SDM), especially for fast, lower-resolving Orbitrap measurements used with short LC gradients. Overall, we demonstrate that pre-accumulation of ions in the bent flatapole offers distinct advantages, particularly for conditions with reduced signal input. Since no hardware changes are required, this approach is highly attractive for Orbitrap mass spectrometers operated with fast MS/MS acquisition methods.

biochemistry↗

bioRxiv2024

Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows

This study presents a comparative analysis of three LysC homologues from Achromobacter lyticus, Pseudomonas aeruginosa, and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture (PAC) workflow with HeLa cell lysate, we assessed the enzymes cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three homologues exhbihited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with its superior peptide identification, digestion efficiency, and protein coverage, especially at short digestion times. Combination of A. lyticus LysC and trypsin demonstrated that importance of LysC for signifancanlty minimizing missed cleavage rates in tryptic digests. This study underscores A. lyticus LysCs potential as an optimal choice for enhancing mass spectrometry-based proteomics.

biochemistry↗

bioRxiv2024

Phosphoproteomics highlights complex resource management upon inflammatory stimulation of fibroblasts

The stimulation of cells by inflammatory mediators gives rise to intricate signaling cascades, inducing specific biological functions. The role of kinase activation for the establishment of specific inflammatory functions has only scarcely been established. A time-course analysis of human fetal fibroblasts stimulated with Interleukin-1{beta} (IL-1{beta}) and/or Dexamethasone (Dex) was conducted using mass spectrometry-based proteomics and phosphoproteomics in conjunction with lysolipid and oxylipin profiling. The IL-1{beta} induced proteome alterations indicated metabolic, transcriptional, and translational activation, including inflammatory marker proteins such as NOS1, THBS1, and STING1. The induction of mitochondrial proteins and the formation of numerous lysolipids indicated an increase in beta-oxidation. In addition to the NF-{kappa}B and STAT signaling pathways, which are characteristic of inflammatory activation, the MAP and AKT kinase signaling pathways were found to be strongly induced. Six hours after treatment, the observed signaling events exhibited a notable decline, nearly returning to their initial states after 24 hours. It is noteworthy that nearly all of these signaling activities were also observed in cells treated with Dex alone. Additionally, the proteome exhibited transient alterations, which included proteins otherwise characteristic of an inflammatory response, such as MMP3 and NFKB2. Activation of the kinase PIKFYVE was apparently specific for dexamethasone but constituted a minority of the total phosphorylation events. Only after 24 hours was the induction of proteins characteristic of glucocorticoids, such as TSC22D3 and MAOA, observed. The analysis of the effects of dexamethasone on the background of established inflammatory signaling verified the known inhibitory functions and resulted in the expression of anti-inflammatory proteins and oxylipins, but hardly affected the signaling events involving MAP and AKT kinases. In conclusion, this data demonstrates that a majority of inflammation-associated signaling events in fibroblasts needs to be attributed to resource and stress management rather than the establishment of specific inflammatory effector functions.

biochemistry↗

bioRxiv2024

Cleaning the Dead: Optimized decontamination enhances palaeoproteomic analyses of a Pleistocene hominin tooth from Khudji, Tajikistan

The study of ancient proteins preserved in a range of archaeological, cultural heritage, and palaeontological materials is increasingly contributing to our understanding of human evolution and archaeological research questions. Many of the specimens studied have been excavated and stored for a significant duration prior to their proteomic analysis. Human handling and storage environments therefore provide ample opportunities for protein contamination onto and into specimens of interest to palaeoproteomic studies. As such, modern protein contamination limits access to endogenous proteomes. Here, we compare five approaches of bone protein decontamination applied to a Pleistocene Equus sp. bone fragment contaminated with a modern dog salivary proteome. We find that all tested methods reduce the protein contamination, but with different efficiencies. We find that a brief bleach wash is the most effective approach in removing modern protein contamination, and that no additional damage is caused to the endogenous proteome by this treatment. Next, we apply this approach to a hominin tooth found at Khudji, a Late Pleistocene archaeological site in Tajikistan. We demonstrate that a brief bleach wash removes almost all human skin protein contamination while retaining the endogenous hominin dentine proteome. Subsequent phylogenetic analysis of the Khudji dentine proteome allowed determination that the specimen is likely not a Denisovan, but still leaves ambiguity between an assignment to either modern humans or Neanderthals.

evolutionary biology↗

bioRxiv2024

A 20+ Ma old enamel proteome from Canada's High Arctic reveals diversification of Rhinocerotidae in the middle Eocene-Oligocene

In the past decade, ancient protein sequences have emerged as a valuable source of data for deep-time phylogenetic inference. Still, the recovery of protein sequences providing novel phylogenetic insights does not exceed 3.7 Ma (Pliocene). Here, we push this boundary back to 21-24 Ma (early Miocene), by retrieving enamel protein sequences of an early-diverging rhinocerotid (Epiaceratherium sp. - CMNF-59632) from the Canadian High Arctic. We recover partial sequences of seven enamel proteins (AHSG, ALB, AMBN, AMELX, AMTN, ENAM, MMP20) and over 1000 peptide-spectrum matches, spanning over at least 251 amino acids. Authentic endogeneity of these sequences is supported by indicators of protein damage, including several spontaneous and irreversible post-translational modifications accumulated during prolonged diagenesis and reaching near-complete occupancy at many sites. Bayesian tip-dating, across 15 extant and extinct perissodactyl taxa, places the divergence time of CMNF-59632 in the middle Eocene-Oligocene, and identifies a later divergence time for Elasmotheriinae in the Oligocene. The finding weakens alternative models suggesting a deep basal split between Elasmotheriinae and Rhinocerotinae. This divergence time of CMNF-59632 coincides with a phase of high diversification of rhinocerotids, and supports a Eurasian origin of this clade in the late Eocene or Oligocene. The findings are consistent with previous hypotheses on the origin of the enigmatic fauna of the Haughton crater, which, in spite of their considerable degree of endemism, also display similarity to distant Eurasian faunas. Our findings demonstrate the potential of palaeoproteomics in obtaining phylogenetic information from a specimen that is ten times older than any sample from which endogenous DNA has been obtained.

paleontology↗