The study of extracellular vesicles (EVs) and the subclass of particles known as exosomes has been rapidly expanding, in part due to their roles in communication between various cell types. Wound edge tissue from mice was harvested immediately and five days after wounding from the same mice then processed using differential centrifugation followed by density gradient ultracentrifugation to produce four EV populations with defined densities. Proteomic experiments indicated that all fractions were found to contain the exosome marker proteins CD9 and CD81. From these fractions the density range 1.10 – 1.14 g/mL was chosen for further analyses due to previous reports that this density range is depleted for high- and low-density lipoproteins, microvesicles and protein aggregates. The selected fractions displayed intact vesicles when subjected to transmission electron microscopy (TEM) that yielded similarly broad populations at day 0 and day 5. Charge Detection Mass Spectrometry (CDMS) provides mass and charge information for particles greater than 10 megadaltons.  CDMS data reveals the day 5 EV fraction contains a similar population to the day 0 EV fraction centered at 26 MDa and 370 charges; however, CDMS was able to resolve a new, smaller EV population centered at 18 MDa and 250 charges not present in the day 0 sample. Combining a density-based isolation with CDMS allows for the particle masses to be converted to diameters and directly compared to TEM.  The population found across day 0 and day 5 was centered around 41 nm in diameter, and the five days post-wounding population was centered around 35 nm in diameter. Proteomic measurements found 197 proteins enriched more than threefold across all day 5 densities including CD177 and CD209d, as well as 194 proteins enriched more than twofold across all day 0 densities including Aldoa. The data suggests that the population found across day 0 and day 5 is enriched in energy transport and metabolic proteins, and the new population seen at day 5 is enriched in mRNA processing proteins and ribosomal proteins for RNA translation.<br/>.

CDMS of 1.10 – 1.14 g*mL<sup>-1</sup> murine wound-edge tissue exosomes: Observation of 13 MDa particles at five days post-wounding

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CDMS of 1.10 – 1.14 g*mL-1 murine wound-edge tissue exosomes: Observation of 13 MDa particles at five days post-wounding

CDMS of 1.10 – 1.14 g*mL^-1 murine wound-edge tissue exosomes: Observation of 13 MDa particles at five days post-wounding