Ireland, J. | bioRxiv Science

bioRxiv2025

Quantitative Substrate Kinetics Screening: One-Pot LC/MS Based Approach to Map Protease Specificity

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naive protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/680748v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1496d11org.highwire.dtl.DTLVardef@1979fc1org.highwire.dtl.DTLVardef@17967e8org.highwire.dtl.DTLVardef@194b741_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Defined microenvironments trigger in vitro gastrulation in human pluripotent stem cells

Embryogenesis is orchestrated through local morphogen gradients and endometrial constraints that give rise to the three germ layers in a well-defined assembly. In vitro models of embryogenesis have been demonstrated by treating pluripotent stem cells in adherent or suspension culture with soluble morphogens and small molecules, which leads to tri-lineage differentiation. However, treatment with exogenous agents override the subtle spatiotemporal changes observed in vivo that ultimately underly the human body plan. Here we demonstrate how microconfinement of pluripotent stem cells on hydrogel substrates catalyses gastrulation-like events without the need for supplements. Within six hours of initial seeding, cells at the boundary show elevated cytoskeletal tension and yes-associated protein (YAP) activity, which leads to changes in cell and nuclear morphology, epithelial to mesenchymal transition, and emergence of defined patterns of primitive streak containing SRY-Box Transcription Factor 17 (SOX17)+ T/BRACHYURY+ cells. Immunofluorescence staining, transcript analysis, and the use of pharmacological modulators reveal a role for mechanotransduction-coupled non-canonical wingless-type (WNT) signalling in promoting epithelial to mesenchymal transition and multilayered organization within the colonies. These microscale gastruloids were removed from the substrate and encapsulated in 3D hydrogels, where biomaterials properties correspond to maintenance and spatial positioning of the primitive streak. Together, this approach demonstrates how materials alone can nurture embryonic gastrulation, thereby providing an in vitro model of early development.

bioengineering↗