Biology subjects

Seale, B.

Publications and source records attributed to Seale, B..

6 recordsLinked to original sources

bioRxiv2026

Reference-Based Library Construction Improves Performance in low-input diaPASEF Workflows

In low input mass spectrometry-based proteomics, Data Independent Acquisition (DIA), is quickly becoming the method of choice for label free quantification. Whether using empirical or in silico spectral libraries, performance is dependent on the library; however, the optimal library construction strategy for low input proteomics remains an open question. To address this, we examine and develop library construction approaches that are compatible with both spectrum-centric and peptide-centric analysis workflows. These approaches leverage a closely related, high-quality sample to improve library quality. First, we validated our approach in bulk sample amounts where we observed that the effects of gas-phase fractionation based library construction is dependent on the software framework, with improvements more pronounced in OpenSWATH compared to DIA-NN. In OpenSWATH, our peptide-centric library reconstruction workflow consistently outperforms a transfer learning strategy, an emerging alternative approach. In DIA-NN, trends are dependent on library source highlighting OpenSWATHs stronger dependence on the search space. In low-input applications, such as single-cell-equivalent injection amounts (100 pg) of HeLa cell digest on a timsTOF SCP, our library construction approach provided more pronounced improvements across both software tools compared to bulk samples. Using a peptide-centric reconstruction approach with the OpenSWATH analysis framework, we detected over 15,000 peptide precursors (2480 protein groups), a 90% improvement over the original library. Furthermore, using a spectrum-centric construction approach, peptide precursor identification rates improved over 6-fold ( [~]1000 to [~]6000). Our strategy provides a practical solution for generating high-quality libraries in low-input applications.

bioinformatics↗

bioRxiv2025

Benchmarking of proximity-dependent biotinylation enzymes across cellular compartments and time windows

Proximity-dependent biotinylation has become a powerful approach for mapping protein interactions and subcellular organization in living cells. Although a growing number of engineered biotin ligases have been introduced, their performance has not been systematically evaluated across diverse cellular contexts. Here, we benchmark ten proximity ligases spanning three bacterial lineages using standardized proteomic workflows across multiple labeling durations, subcellular compartments, and two human cell types. While all enzymes efficiently detect proximal associations, they differ in labeling kinetics, background activity, and spatial specificity. TurboID exhibits the highest overall activity but generates substantial background in standard media. miniTurbo and ultraID support rapid, biotin-dependent labeling with low background, making them better suited for dynamic and time-resolved applications. However, miniTurbo showed aberrant mitochondrial localization with two cytoskeletal baits (VASP and PFN1). Across 15 diverse baits, ultraID consistently provides an excellent combination of specificity, efficiency, and spatial compatibility--including unique recovery of Golgi-resident glycosyltransferases. This study serves as a comparative resource, offering guidance for enzyme selection and experimental design in proximity proteomics.

systems biology↗

bioRxiv2025

Integrated spatial proteomics of human PDAC uncovers an expanded tumour-immune-stroma spectrum with genomic associations

Distinctively, pancreatic ductal adenocarcinoma (PDAC) consists of sparse tumour lesions intertwined with extensive desmoplastic stroma. The complexity of tumour-microenvironment interactions within this desmoplasia poses a challenge for accurate tumour profiling and patient stratification, and characterizes a profoundly chemoresistant tumour. Here we mapped the spatial relationships between tumour, stroma, and immune cell compartments delineating tumour and microenvironment types that expand the classical to basal spectrum of human PDAC. We used imaging mass cytometry to profile the in situ multi-cellular organization of 81 cell types in resected cases with paired whole genome sequencing. Cell types, functions, and pathway activation were distributed as highly reproducible environments in discrete locations throughout these tumours, which we deep-profiled using laser-capture mass spectrometry. We show that the connections between tumour phenotypes, vascularization, immune response, and stromal biophysical state are reinforced by genomic aberrations, altered by treatment, and associated with patient outcome. Predictive machine-learning models showed that spatial single cell data outperformed genomic or clinical features but integrated multi-omics models provide the best prediction of patient survival with compressed models requiring only 10 non-redundant robust molecular measures associated with the phenotypic spectrum of PDAC. Together, these findings define a phenotypic and molecular framework of PDAC that captures tumour-microenvironment co-dependencies and offers a refined basis for patient stratification and therapeutic targeting.

cancer biology↗

bioRxiv2025

Stress Granules Underlie Acute Myeloid Leukemia Stem Cell Survival and Stress Adaptation

The link between cancer maintenance and an ability to sustain continued growth through stresses conferred by the cancer state itself is growing. However, there are significant gaps in our understanding of how this stress is managed, particularly at the level of cancer initiating cells. Here, we identify proteins comprising the dynamic, stress-adaptive ribonucleoprotein complexes known as stress granules (SG) to be enriched among the factors essential for leukemic stem cell (LSC)-driven leukemic propagation. Focusing on core SG nucleator G3BP1, we dissect the role of SGs in human acute myeloid leukemia (AML), their targetability, and the mechanisms they govern to uncover a novel propensity for AML, and in particular LSC-enriched fractions, to prime the expression of SG components, form SGs with greater fidelity and to be reliant on their establishment and continued integrity for LSC maintenance. We further unveil the transcript and protein interactome of G3BP1 in the AML context and show that consolidated control of innate immune signaling, and apoptosis repression is executed through regional binding specificity of G3BP1 to highly structured 3UTRs and cooperation with the RNA helicase UPF1 to mediate transcript decay in SGs. Altogether our findings advance novel fundamental principles of stress adaptation exploited in AML and LSCs that may extend to other cancers and uncover SGs as a novel axis for therapy development.

cancer biology↗

bioRxiv2023

Dynamic extracellular proximal interaction profiling reveals Low-Density Lipoprotein Receptor as a new Epidermal Growth Factor signaling pathway component

Plasma membrane proteins are critical mediators of cell-cell and cell-environment interactions, pivotal in intracellular signal transmission vital for cellular functionality. Proximity-dependent biotinylation approaches such as BioID combined with mass spectrometry have begun illuminating the landscape of proximal protein interactions within intracellular compartments. However, their deployment in studies of the extracellular environment remains scarce. Here, we present extracellular TurboID (ecTurboID), a method designed to profile cell surface interactions in living cells on short timescales. We first report on the careful optimization of experimental and data analysis strategies that enable the capture of extracellular protein interaction information. Leveraging the ecTurboID technique, we unveiled the proximal interactome of multiple plasma membrane proteins, notably the epidermal growth factor receptor (EGFR). This led to identifying the low-density lipoprotein receptor (LDLR) as a newfound extracellular protein associating with EGFR, contingent upon the presence of the EGF ligand. We showed that 15 minutes of EGF stimulation induced LDLR localization to the plasma membrane to associate with proteins involved in EGFR regulation. This modified proximity labelling methodology allows us to dynamically study the associations between plasma membrane proteins in the extracellular environment. One Sentence SummaryWe developed extracellular TurboID (ecTurboID) as a new proximity dependent biotinylation approach that can capture dynamic interactions at the cell surface, identifying Low-Density Lipoprotein Receptor as a new ligand-dependent extracellular partner of Epidermal Growth Factor Receptor.

molecular biology↗

bioRxiv2021

The petrosal and basicranial morphology of Protoceras celer

Protoceratids are an extinct family of endemic North American artiodactyls. The phylogenetic position of protoceratids in relation to camelids and ruminants has been contentious for over a century. The petrosal morphology of basal (Leptotragulus) and derived (Syndyoceras) protoceratids has suggested that protoceratids are closely related to ruminants, whereas a prior description of a disarticulated intermediate protoceratid petrosal (Protoceras celer) indicated that protoceratids were closely related to camelids. This contradictory evidence implied that there were several character reversals within the protoceratid lineage and brought into question the utility of basicranial characters in artiodactyl phylogenetics. Here, we provide descriptions of an additional P. celer petrosal. The descriptions are based on data produced by computed tomography scans, which allowed us to image the petrosal in situ in the skull. Our results indicate that the petrosal morphology of P. celer is similar to that of other protoceratids, implying that, contrary to previous evidence, petrosal morphology is conserved within the Protoceratidae.

paleontology↗