Integrated Proteomics and Single-Cell Mass Cytometry Analysis Dissects the Immune Landscape of Ankylosing Spondylitis.

Mass cytometry is a powerful single-cell technology widely adopted to depict immune cell heterogeneity in different contexts. However, this method is only capable of examining several dozens of proteins simultaneously and requires a prior knowledge of the markers to be analyzed. Here we propose that the integration of mass cytometry with shot-gun proteomics may serve as a valuable tool to achieve an in-depth understanding of the immune system. By implementing such a strategy, we investigated the immune landscape of ankylosing spondylitis (AS), a chronic inflammatory arthritis with unclear etiology. The proteome alteration in peripheral blood mononuclear cells (PBMCs) was investigated by quantitative proteomics, and then mass cytometry analysis was conducted to decipher the immunome by considering the signaling molecules identified with differential expression by proteomics. As a result, we identified a wide spectrum of proteins dysregulated in AS, e.g., upregulation of glycolytic enzymes, downregulation of lipid transporters, and dysregulation of chemokine signaling molecules involved in proinflammatory cytokine production and leucocyte migration. Moreover, the single-cell analysis showed the upregulation of chemokine signaling regulators in subclusters of both innate and adaptive immune cells in AS. In addition, correlation analysis unveiled the interplay among Phenograph-identified subclusters of monocytes, CD4+ T cells, and CD8+ T cells. Taken together, our findings demonstrated that the integration of mass spectrometry-based proteomics and single-cell mass cytometry may serve as a useful tool to reveal clinically relevant information regarding useful targets and cellular phenotypes that could be further exploited to develop novel therapeutic strategies.