Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5–12.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered in vivo will enable future studies that seek to characterize the murine proteome during growth, disease and repair.

Perlecan, or heparan sulfate proteoglycan (Hspg2), is an extracellular matrix (ECM) protein localized to the pericellular matrix (PCM). When Hspg2 is knocked down, the phenotype mimics the skeletal defects observed in human Schwartz‐Jampel syndrome. We previously demonstrated, using atomic force microscopy, that perlecan knockdown significantly decreased the stiffness of the ECM and chondrocytes in developing cartilage; however, it is not clear what changes occur in ECM structure and organization to cause this decrease. To address this question, our goal is to develop computational models that incorporate geometries and material properties that will enable us to analyze the mechanical response of cells in situ. Therefore, we created 3D physiologically‐derived geometries that can generate repeatable simulations of cartilage mechanics. In parallel, we are using mass spectrometry (LC‐MS/MS) and transmission electron microscopy (TEM) to define how cartilage composition and organization changes as a function of development and perlecan knockdown.Multi‐cellular geometries from embryonic day (E)16.5 and postnatal day (P)3 murine cartilage were imaged in 3D using confocal microscopy. Image stacks were processed using MATLAB to create multicellular geometries for finite element analysis using ANSYS (Fig 1A,B). Geometries based on confocal images and isolated, single cell models were compressed 5% and cells and ECM strain were compared (Fig 1C,D). Our simulations indicated that cells had similar strains at both time points, even though cell and ECM stiffness at P3 was significantly higher than at E16.5. In addition, the ECM at P3 took on more strain than at E16.5. The isolated, single cell geometries underestimated both cell and ECM strain and were not able to capture the similarity in cell strain observed with physiologically‐derived geometries.A limitation of the current model is that cells and ECM were treated as homogenous materials, whereas native cartilage is a biphasic material comprised of type II collagen fibrils embedded in an amorphous proteoglycan matrix. To identify how ECM composition and organization changes, cartilage from distal humeri or femurs were dissected from E16.5 and P3 wildtype, heterozygous, and homozygous Hspg2 mice for analysis via LC‐MS/MS or TEM. Proteins were extracted with guanidine‐HCl and processed for LC‐MS/MS. Relative increases in the abundance of ECM and associated proteins, including collagen types II & IX and aggrecan highlighted expected developmental changes between E16.5 and P3. Multiple proteins were significantly more abundant in homozygous vs. wildtype P3 mice, including collagens type II & IX, fibronectin and matrilin‐3. Surprisingly, TEM studies indicate that collagen fibril density and diameter decreased between wildtype and homozygous mice, suggesting that perlecan knockdown leads to a decrease in cross‐linking and increased ECM extractability. We aim to integrate these changes in material properties with our simulations to generate a computational model that can describe and predict the mechanics of developing cartilage.Support or Funding InformationThis work was supported by the National Institutes of Health [R21 AR069248, R01 AR071359 and DP2 AT009833 to S.C.].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.