Optimization of a high-throughput shotgun immunoproteomics pipeline for antigen identification.

Identification of proteins which initiate and/or perpetuate adaptive immune responses has potential to greatly impact pre-clinical and clinical work across numerous fields. To date, however, the methodologies available to identify antigens responsible for driving adaptive immune responses have been plagued by numerous issues which have drastically limited their widespread adoption. Therefore, in this study, we sought to optimize a shotgun immunoproteomics approach to alleviate these persistent issues and create a high-throughput, quantitative methodology for antigen identification. Three individual components of a previously published approach, namely the protein extraction, antigen elution, and LC-MS/MS analysis steps, were optimized in a systematic manner. These studies determined that preparation of protein extracts using a one-step tissue disruption method in immunoprecipitation (IP) buffer, eluting antigens from affinity chromatography columns with 1% trifluoroacetic acid (TFA), and TMT-labeling & multiplexing equal volumes of eluted samples for LC-MS/MS analysis, resulted in quantitative longitudinal antigen identification, with reduced variability between replicates and increased total number of antigens identified. This optimized pipeline provides a multiplexed, highly reproducible, and fully quantitative approach to antigen identification which is broadly applicable to determine the role of antigenic proteins in inciting (i.e., primary antigens) and perpetuating (i.e., secondary antigens) a wide range of diseases. SIGNIFICANCE: Using a systematic, hypothesis-driven approach, we identified potential improvements for three individual steps of a previously published approach for antigen-identification. Optimization of each step created a methodology which resolved many of the persistent issues associated with previous antigen identification approaches. The optimized high-throughput shotgun immunoproteomics approach described herein identifies more than five times as many unique antigens as the previously published method, greatly reduces protocol cost and mass spectrometry time per experiment, minimizes both inter- and intra-experimental variability, and ensures each experiment is fully quantitative. Ultimately, this optimized antigen identification approach has the potential to facilitate novel antigen identification studies, allowing evaluation of the adaptive immune response in a longitudinal manner and encourage innovations in a wide array of fields.

Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation.

Off-the-shelf small diameter vascular grafts are an attractive alternative to eliminate the shortcomings of autologous tissues for vascular grafting. Bovine saphenous vein (SV) extracellular matrix (ECM) scaffolds are potentially ideal small diameter vascular grafts, due to their inherent architecture and signaling molecules capable of driving repopulating cell behavior and regeneration. However, harnessing this potential is predicated on the ability of the scaffold generation technique to maintain the delicate structure, composition, and associated functions of native vascular ECM. Previous de-cellularization methods have been uniformly demonstrated to disrupt the delicate basement membrane components of native vascular ECM. The antigen removal (AR) tissue processing method utilizes the protein chemistry principle of differential solubility to achieve a step-wise removal of antigens with similar physiochemical properties. Briefly, the cellular components of SV are permeabilized and the actomyosin crossbridges are relaxed, followed by lipophilic antigen removal, sarcomeric disassembly, hydrophilic antigen removal, nuclease digestion, and washout. Here, we demonstrate that bovine SV ECM scaffolds generated using the novel AR approach results in the retention of native basement membrane protein structure, composition (e.g., Collagen IV and laminin), and associated cell modulatory function. Presence of basement membrane proteins in AR vascular ECM scaffolds increases the rate of endothelial cell monolayer formation by enhancing cell migration and proliferation. Following monolayer formation, basement membrane proteins promote appropriate formation of adherence junction and apicobasal polarization, increasing the secretion of nitric oxide, and driving repopulating endothelial cells toward a quiescent phenotype. We conclude that the presence of an intact native vascular basement membrane in the AR SV ECM scaffolds modulates human endothelial cell quiescent monolayer formation which is essential for vessel homeostasis.