<p> Musculoskeletal injuries are one of the leading causes of human disability. Tissue engineers aim to restore damaged musculoskeletal tissues by creating scaffolds that promote cellular adhesion, proliferation, and eventual differentiation into functional tissue. It is known that the extracellular matrix (ECM) regulates cellular behavior and is often used as a basis for biological scaffolds; however, current scaffolds often mimic the ECM of adult, homeostatic tissue and frequently lead to poor tissue restoration. What is rarely taken into consideration is that the ECM undergoes extensive remodeling during development to facilitate growth.</p> <p>In the musculoskeletal system, myogenic progenitors (<em>Pax3</em>+) and connective tissue cells (<em>Prx1</em>+) proliferate and differentiate into muscle, tendon, cartilage, and conjoining interfaces (<em>e.g.</em> myotendinous junction), while depositing and remodeling the ECM. As tissues mature, cells continue to refine ECM networks to withstand the functional demands to facilitate movement. The ECM composition and architecture of adult musculoskeletal tissues have been studied individually and are thought to be distinct; however, there has yet to be a comprehensive comparative analysis of the ECM in adult muscle, tendon, and the myotendinous junction (MTJ) in a single study. Additionally, how the matrisome of adult musculoskeletal system compares to the ECM dynamics during forelimb development, remain largely unknown due to lack of techniques to analyze embryonic matrisome composition and synthesis. </p> <p>To address these research gaps, we (1) used quantitative proteomics to map the matrisome composition in the mature murine MTJ, relative to the tendon and muscle; (2) adapted tissue fractionation and biorthogonal non-canonical amino acid tagging techniques to embryonic tissues as a method to quantify the global and nascent embryonic matrisome; and (3) subsequently used these techniques to establish a baseline of ECM dynamics during forelimb morphogenesis (embryonic day, E11.5-E14.5) and growth (postnatal day, P3 and P35). We hypothesized that proteomic evaluation of ECM composition and synthesis in developing and adolescent limbs would resolve differences between embryonic and growing tissues. Indeed, we saw significant differences in global and nascent matrisome composition between embryonic and adolescent forelimbs. Notably, the relative abundance and ratios of collagens associated with type I fibrillogenesis (I, III, and V) were significantly different as a function of development embryogenesis and across the adult muscle, MTJ, and tendon.</p> <p>Type I collagen fibrils are critical for tissue architecture and function. Using genetic mouse models, the regulatory roles of COL5A1 in the initiation of type I collagen fibrillogenesis, and organization of subsequent fibrils, have been well characterized in tendons and ligaments; however, is it unknown which cell types contribute COL5A1 to the ECM in the forelimb. To identify the functional contribution of COL5A1 by myogenic or connective tissue cell populations, we generated conditional (cre-flox) knock-out mouse models to inactivate <em>Col5a1</em> using <em>Pax3</em>- or <em>Prx1</em>-drivers, respectively. Haploinsufficiency of <em>COL5A1</em> in humans is associated classical Ehlers-Danlos syndrome, characterized by skin fragility and join instability; similar, albeit more severe, phenotypes were present in <em>Prx1Cre/+;Col5a1fl/fl</em> mutants, but not in <em>Pax3Cre/+;Col5a1fl/fl</em> mutants or controls. Interestingly, THBS4+ and COL22A1+ networks at the MTJ were morphologically affected in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs. Additional work needs to be conducted to characterize the systematic phenotypes observed in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs.</p> <p>Together, our results indicate that there are distinct, complex ECM dynamics, originating from distinct cell-types, that drive musculoskeletal morphogenesis in the forelimb. Further, the tools developed here will serve as a foundation for quantitative proteomic analyses of the matrisome composition in embryonic tissues. Collectively, this work provides a baseline of ECM protein dynamics during musculoskeletal morphogenesis, a helpful guide for tissue engineers in designing scaffolds to promote restoration of damaged tissues, with enhanced integration into the host tissue.</p>

<div>The use of computational approaches to understand kinase substrate preference has been a powerful tool in the search to develop artificial peptide probes to monitor kinase activity, however, most of these efforts focus on a small portion of the human kinome. The use of high throughput techniques to identify known kinase substrates plays an important role in development of sensitive protein kinase activity assays.</div><div>The KINATEST-ID pipeline is an example of a computational tool that uses known kinase substrate sequence information to identify kinase substrate preference. This approach was used to design three artificial substrates for ABL, JAK2 and SRC family kinases. These biosensors were used to design ELISA and lanthanide-based assays to monitor in vitro kinase activity. The KINATEST-ID pipeline relies on a high number of reported kinase substrates to predict artificial substrate sequences, however, not all kinases have the sufficient number of known substrates to make an accurate prediction. </div><div>The adaptation of kinase assay linked with phosphoproteomics technique was used to increase the number of known FLT3 kinase variant substrate sequences. Subsequently, a set of data formatting tools were developed to curate the mass spectrometry data to become compatible with a command line version of the KINATEST-ID pipeline modules. This approach was used to design seven pan-FLT3 artificial substrate (FAStides) sequences. The pair of FAStides that were deemed the most sensitive toward FLT3 kinase phosphorylation were assayed in increasing concentrations of clinically relevant tyrosine kinase inhibitors. </div><div>To improve the automation of the mass spectrometry data analysis and formatting for use with the KINATEST-ID pipeline, a streamlined process was developed within a bioinformatic platform, GalaxyP. The data formatting tools used to process the FLT3 mass spectrometry data were converted into compatible versions to execute within the GalaxyP framework. This process was used to design four BTK artificial substrates (BAStide) to monitor kinase activity. Additionally, one of the BAStide sequences was designed in the lanthanide chelating motif to develop an antibody-free activity assay for BTK. </div><div>Lastly, a multicolored time resolved lanthanide assay was designed by labeling SYK artificial substrate and a SRC family artificial substrate to measure the activity of both kinases in the same kinase reaction. This highlighted the functionality of lanthanide-based time resolved assays for potential multiplexing assay development. </div><div><br></div>