With the increased realization of the effect of oxygen (O 2 ) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O 2 concentrations and establish biomimetic O 2 gradients to study and manipulate cellular behavior. Nonetheless, O 2 gradients present in 3D engineered platforms result in diverse cell behavior across the O 2 gradient, making it difficult to identify and study O 2 sensitive signaling pathways. Using a layer-by-layer assembled O 2 -controllable hydrogel, the authors precisely control O 2 concentrations and study uniform cell behavior in discretized O 2 gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O 2 concentrations. Using RNA sequencing, it is found that time-dependent regulation of cyclic adenosine monophosphate signaling enables cell survival and clustering in the high stress microenvironments. Various extracellular matrix modulators orchestrate hypoxia-driven endothelial cell clustering. Finally, clustering is facilitated by regulators of cell-cell interactions, mainly vascular cell adhesion molecule 1. Taken together, novel regulators of hypoxic cluster-based vasculogenesis are identified, and evidence for the utility of a unique platform is provided to study dynamic cellular responses to 3D hypoxic environments, with broad applicability in development, regeneration, and disease.

The efficiency of shotgun proteomic analysis is dependent on the reproducibility of the peptide cleavage process during sample preparation. To generate rapid and useful metrics for peptide cleavage efficiency, as in enzymatic or chemical cleavage, SPACEPro was developed to evaluate efficiency and reproducibility of protein cleavage in peptide samples following data-dependent analysis by mass spectrometry. SPACEPro analyzes samples at the peptide-spectrum match (PSM), peptide, and protein levels to provide a comprehensive representation of the overall sample processing to peptides. All output is provided in human-readable text and JSON files that can be further processed to assess the cleavage efficiency on proteins within the sample. SPACEPro provides a snapshot of the protein cleavage efficiency through very minimal effort so that the user is informed of the quality of the sample processing efficiency and can accordingly develop protocols to improve the initial sample preparation for subsequent analyses.