Quantitative Proteomic Profiling of MSCs and EVs

Liquid chromatography mass spectrometry (LC-MS/MS) proteomic analysis was performed as previously described [22 (link), 23 (link)]. MSC and EV pellets were solubilized and lysed, and protein samples denatured by incubation at 85°C for 10min. Aliquots were resolubilized in reducing sample buffer and samples electrophoresed in 4–20% TGX Ready gels at 200V for 30min. Gel sections were digested with trypsin [23 (link)], and peptides extracted and transferred onto a 35cmx100μm PicoFrit column 9 (NewObjective), self-packed with Agilent Poroshell 120S 2.7μm EC-C18 stationary phase, using a Dionex UltiMate 3000 RSLC LC system (Thermo-Fisher Scientific). Peptides were separated and eluting peptides analyzed using a QExactive mass spectrometer (Thermo-Fisher Scientific). Label-free peptide MS1 intensity-based methods were used to identify differentially expressed proteins between MSCs and EVs. Data quality was assessed using MaxQuant 1.5.1) software [24 (link)] and reversed protein sequences appended to the database for estimating protein identification false discovery rates (FDRs). Protein group intensities of each sample were log₂ transformed, normalized, and modeled using a Gaussian-linked generalized linear model. Data was normalized by protein loading, and differential p-values FDR-corrected using the Benjamini-Hochberg-Yekutieli procedure [25 (link)].