109 4 Proteomic signature of macrophages cultured on collagen type 1 recorded over a m/z-range from 350-1500. One full spectrum was followed by a DIA scan from m/z 400-700, followed by a full spectrum and a DIA scan from m/z 700-1000. For protein identification and quantification, a library was created with Skyline (MacCoss Lab, University of Washington, USA, version 19.1.0.193) based on the DDA data. The Proteome Discoverer results were imported in Skyline and the peptide library was generated using the following parameters: transition settings: precursor charges: 2, 3, 4, 5; fragment ion charges: 1, 2; ion types: p (precursor), b, y; ion match tolerance: 0.02 Da; minimum number of product ions: 4; m/z-range: 350-2000; the DIA acquisition method file was uploaded to determine the DIA isolation scheme; RT tolerance: 5 minutes. The generated library consisted of 6.310 proteins with at least one unique peptide for each protein and 74.654 unique peptides, covering 37% of the theoretical mouse proteome. The LC-DIA-MS files were imported and filtered with a dotp-threshold of 0.85 (best match). Only proteins with at least one unique peptide were kept. The Skyline results were exported for further data analysis in R and Python. This included missing values imputations, median normalization and log2 transformation of the protein intensities. For feature selection, partial least squares discriminant analysis and statistical analysis were performed. Extracellular flux analysis The glycolytic rate was measured using an XFe96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, California). Alveolar macrophages were seeded into Seahorse XF96 cell culture microplates that were coated with either fibrous or globular collagen or left uncoated (control) and incubated for 72 hours. To measure extracellular acidification and oxygen consumption rates, cells were equilibrated for 1 hour in the absence of CO2 at 37 °C in XF base media (Agilent Technologies, Santa Clara, California). The manufacturer’s protocol for the XF Glycolysis Stress Test was used to assess the glycolytic rate (Agilent Technologies). After equilibration, macrophages were stimulated with sequential injections of glucose (20 mM), oligomycin (1 µM), and 2-deoxy-D-glucose (50 mM). Glycolytic function is represented as glycolysis, glycolytic capacity and glycolytic reserve. Glycolysis was calculated as the difference between the maximal rate before oligomycin addition and the last rate measurement before glucose addition. Glycolytic capacity was calculated as the difference between the maximal rate after oligomycin addition and the last rate measurement before glucose addition. The glycolytic reserve was calculated as the difference between glycolytic capacity and glycolysis. Measurements were normalized to the protein amount in each well using a microplate BCA protein assay.
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