Exosomes derived from mesenchymal stem cells (MSCs) have been used for cancer treatment, however, an in‐depth analysis of the exosomal proteomes is lacking. In this manuscript, we use the diaPASEF (parallel accumulation serial fragmentation combined with the data‐independent acquisition) method to quantify exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) and rat bone marrow stem cells (BMSCs), resulting in identification of 4200 human proteins and 5362 rat proteins. Comparison of human exosomal proteins and total cellular proteins reveals that some proteins exist in the exosomes exclusively that can be served as potential markers for exosomes. Quantitative proteomic analysis of exosomes from different passages of BMSCs shows that the proteins involved in TGF‐β signaling pathway are regulated in abundance, which could be markers for the therapeutic ability of BMSC exosomes. Collectively, the data presented by this study can be a resource for further study of exosome research.
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