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A4485 - Proteomic Analysis of Rat Alveolar Macrophage-Derived Microvesicles Using Tandem Mass Tag Isobaric Labeling
Author Block: D. Claar1, S. H. Wettlaufer1, V. Basrur2, M. Peters-Golden1; 1Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI, United States, 2Pathology, University of Michigan, Ann Arbor, MI, United States.
Introduction: Extracellular vesicles (EVs) are increasingly recognized as critical mediators of cell-cell signaling, containing a wide array of cargo including DNA, RNA, lipids, and proteins. Microvesicles (MVs) are a heterogeneous subtype of EV generated by outward budding from the plasma membrane. Our lab has previously shown that MVs derived from alveolar macrophages (AMs) play an important immunomodulatory role in the alveolar space through their uptake by and actions in alveolar epithelial cells. We sought to better understand the global potential for AM-derived MV transfer as a means of cellular crosstalk by defining the proteome of these MVs, which has not been previously reported.
Methods: Conditioned medium was collected from 24-h cultures of NR8383 cells, a spontaneously immortalized rat alveolar macrophage cell line, in F12K medium with 2% EV-free serum. MVs were isolated from the conditioned medium by ultracentrifugation. Vesicular fractions were pooled into three biological replicates to obtain the quantity of protein necessary for mass spectrometry (MS) analysis. Replicates underwent isobaric labeling with tandem mass tags followed by nano ultra-high pressure liquid chromatography with coupled multinotch MS3 analysis on an Orbitrap Fusion mass spectrometer. Protein identification was performed using Proteome Discoverer. The DAVID Bioinformatic Database was used for gene ontology analysis.
Results: We identified 3035 MV proteins with high confidence (identification based on ≥2 peptides with a false discovery rate ≤1%) across all three biological replicates. Gene ontology analysis demonstrated marked enrichment of proteins associated with the cell membrane, RNA binding, extracellular vesicles, mitochondria, and microtubules, consistent with previous MV literature. CD11c, a surface marker found in higher abundance on AMs relative to other macrophage subtypes, was also identified. Multiple proteins implicated in microvesicle biogenesis were found including ARF6, Cdc42, RhoA, and ROCK. Further consistent with the biological origins of MVs, 1037 proteins localizing to the cytoplasm and 752 proteins localizing to the cell membrane were identified, the latter representing a large pool of potential surface markers for targeted isolation of AM MVs in complex fluids such as bronchoalveolar lavage.
Conclusions: We report the first characterization of the AM-derived MV proteome. This work represents an important first step in better understanding the broad range of possible effector mechanisms of these vectors of intercellular signaling in the alveolar space as well as candidate surface markers for improved isolation in complex biologic fluids. Funding for this work was provided by the NIH R01, R01 HL125555.