Biology subjects

Fort, K.

Publications and source records attributed to Fort, K..

7 recordsLinked to original sources

bioRxiv2026

Genomic signatures of selection in the mound-building mouse Mus spicilegus

The steppe mouse Mus spicilegus is distinguished by several unusual phenotypes: animals of this species build complex mounds from brush and dirt, and this behavior as well as the pace of development is regulated by season of birth. The genetic and evolutionary basis of species-defining traits in Mus spicilegus is unknown. To discover genomic clues to the mechanisms at play, we took a molecular-evolution approach. Sequence-based tests of protein-coding exons, and of brain-active cis-regulatory regions, revealed a striking excess of evidence for evolutionary acceleration in the M. spicilegus genome relative to those of closely related species. Transcriptional analyses also revealed unique expression programs in the M. spicilegus brain, which could be traced in part to cis-regulatory sequence variation. Hit loci from our molecular-evolution tests tended to be active in the cerebral cortices and hypothalamus and have annotated functions in neuronal development, neural plasticity, or endocrine signaling. In many cases, their orthologs in other animal systems had been implicated in neoteny/the pace of maturation, unusual building behaviors, and autism. Together, our data indicate that M. spicilegus has undergone divergence in brain-active genes to an extent far more than its relatives. We interpret the affected genes under a model in which they mediate the seasonal capacity by M. spicilegus for protracted maturation and mound-building. Thus our work provides an evolutionary interpretation of, and catalog of candidate determinants for, shifts in motivated behavior and development in this intriguing mouse species.

evolutionary biology↗

bioRxiv2025

High-throughput screening reveals mechanisms of environmental control of germination in a fungal thermophile

Thermothelomyces thermophilus is a filamentous fungus isolated from self-heating compost. Unlike most of the fungal kingdom, this species exhibits a growth optimum at 45{degrees}C and is intolerant of temperatures below 30{degrees}C. To investigate genetic contributors to temperature-dependent fitness in this system, we implemented a large-scale insertional mutagenesis approach. We generated thousands of T. thermophilus mutants and cultured them at temperature extremes in standard medium. Phenotyping-by-sequencing identified dozens of disrupted loci representing candidate determinants of thermophilic life history, including several annotated in metal transport. We then validated a subset of screen hits with a directed, single-gene knockout paradigm. The results revealed a temperature-dependent regulatory logic for germination, the developmental decision by which a fungal spore initiates growth. Surprisingly, most mutants germinated far better at 50{degrees}C than the wild-type in standard medium and showed markedly slower germination at lower temperatures, consistent with altered germination regulation rather than enhanced intrinsic heat tolerance. We hypothesized that T. thermophilus has evolved sophisticated regulatory machinery to block germination at high temperature unless environmental conditions are favorable. As a proof of concept, we surveyed media conditions and established that elevated zinc dampened germination of wild-type T. thermophilus at 50{degrees}C but promoted it at lower temperature; mutation experiments made clear that such sensitivity was mediated in part by the zinc transporter zip. We interpret these results under a model in which T. thermophilus integrates temperature and nutrient availability to control the transition from spore dormancy to vegetative growth, a developmental decision that shapes fitness outcomes across temperatures. SignificanceFungal thermophiles thrive at temperatures that represent the upper limits of eukaryotic life. The regulatory and developmental mechanisms that shape their temperature-dependent fitness remain poorly understood. In this work, we elucidate how Thermothelomyces thermophilus integrates temperature cues with other environmental inputs during germination, a key life-cycle stage for dispersal. Our findings highlight germination regulation as an important contributor to fitness at elevated temperatures in a thermophilic eukaryote. These insights are of basic biological interest and provide a foundation for rational strategies to modulate temperature-dependent performance in industrial strains, with applications for high-temperature bioprocessing.

microbiology↗

bioRxiv2025

Increased EThcD efficiency on the hybrid Orbitrap Excedion Pro Mass Analyzer extends the depth in identification and sequence coverage of HLA class I immunopeptidomes

Gaining a complete and unbiased understanding of the non-tryptic peptide repertoire presented by HLA-I complexes by LC-MS/MS is indispensable for therapy design for cancer, autoimmunity and infectious diseases. A serious concern in HLA peptide analysis is that the routinely used, collision-based fragmentation methods (CID/HCD) do not always render sufficiently informative MS2 spectra, whereby gaps in the fragmentation sequence coverage prevent unambiguous assignments. EThcD can be utilized to generate complementary ion series, i.e. b/y ions and c/z ions, resulting in richer, more informative MS2 spectra, thereby filling in the gaps. Here, we present data generated on a novel hybrid Orbitrap mass spectrometer, facilitating fast and efficient hybrid fragmentation due to the implementation of EThcD in the ion routing multipole. We hypothesized that this would enable more comprehensive and less error-prone analysis of immunopeptidomes at minimal costs in duty-cycle. First, we optimized ETD/EThcD methods using an elastase-digested cell lysate, as this contains peptides of similar length and charge distributions to immunopeptides. Next, we compared HCD and EThcD on immunopeptidomes originating from three cell lines with distinct HLA-I complexes that present peptides with varying physicochemical properties. We demonstrate that the new instrument not only enables efficient and fast ETD reactions, but when combined with collision-based supplemental activation, i.e. EThcD, also consistently increases the sequence coverage and identification of peptide sequences, otherwise missed by using solely HCD. We reveal several of the biochemical properties that make HLA peptides preferably identifiable by EThcD, with internal Arg residues being one of the most dominant determinants. Finally, we demonstrate the power of EThcD for the identification and localization of HLA peptides harboring post-translational modifications, focusing here on HLA Arg mono-/di-methylation. We foresee that this new instrument with efficient EThcD capabilities enhances not only immunopeptidomics analysis, but also analysis of peptides harboring post-translational modifications and de novo sequencing.

biochemistry↗

bioRxiv2023

Characterisation of an Omnitrap-Orbitrap platform equipped with IRMPD, UVPD and ExD for the analysis of peptides and proteins

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions, that can be used to perform resonant based collision induced dissociation, several forms of electron associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation on a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the interrogation of polypeptides.

systems biology↗

bioRxiv2022

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

systems biology↗

bioRxiv2021

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.

molecular biology↗

bioRxiv2019

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 unique peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 minute LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

biochemistry↗