The discovery of objective non-invasive biomarkers that may allow to diagnose Systemic Sclerosis- associated interstitial lung disease ( SSc-ILD) at an early stage and to identify patients with Raynaud’s Phenomenon evolving into SSc would be highly valuable for prompt initiation of SSc-disease- modifying therapy. We propose to utilize novel Aptamer-Based Proteomics for the analysis of the protein cargo of Serum Exosomes isolated from patients with recent-onset diffuse SSc with and without SSc-ILD and from patients with Primary Raynaud’s Phenomenon and patients with Raynaud’s Phenomenon with positive serum antinuclear autoantibodies (ANA). The aptamer proteomic approach measures simultaneously 1,305 proteins and offers an extraordinarily high level of sensitivity (from femtomolar to micromolar). Exosomes, microvesicles secreted by all cells, contain a broad assortment of macromolecules, including proteins. The exosome cargo reflects their cell of origin and, most importantly, the pathologic status of the cells. We propose to test the following hypothesis: “The unbiased proteomic analysis of serum exosomes employing a highly sensitive aptamer-based proteomic assay will identify proteins that may serve as specific biomarkers for the early diagnosis of SSc-ILD and for the identification of patients with Raynaud’s Phenomenon at high risk of evolving into SSc.” We will pursue the following Specific Aims: Specific Aim 1: Identify employing aptamer-based proteomics differentially expressed proteins in serum exosomes from patients with recent onset SSc-ILD compared with exosomes from SSc patients without SSc-ILD. Specific Aim 2: Identify employing aptamer-based proteomics differentially expressed proteins in serum exosomes from patients with Primary Raynaud’s Phenomenon compared with exosomes from patients with Raynaud’s Phenomenon at high risk of evolving into SSc. Specific Aim 3: Examine whether the most differentially increased proteins identified in Aims 1 and 2 induce in vitro a profibrotic phenotype in normal human lung fibroblasts or endothelial to mesenchymal transition (EndMT) in normal human lung microvascular endothelial cells. To accomplish these Aims, exosomes will be isolated from serum from: 1; patients with diffuse SSc without SSc-ILD, 2; patients with diffuse SSc and recent onset SSc-ILD, 3; patients with Primary Raynaud’s Phenomenon, and 4; patients with Raynaud’s Phenomenon harboring serum ANA without other clinical evidence of SSc. The novel aptamer-based proteomic analysis should allow the identification of proteins that may serve as valuable biomarkers for the early diagnosis of SSc-ILD and of Raynaud’s Phenomenon at high risk of evolving into SSc. The proposed studies may also uncover novel proteins involved in the molecular pathogenesis of SSc tissue fibrosis and vasculopathy.