Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exo κ-GFP ) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo κ-GFP were also characterized. Unlike skin exosome, wound-edge Exo κ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by woundmacrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exo κ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo κ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP κ ) were designed with 94.3% encapsulation *

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle’s mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.

Here, we show that hypoxia drives especially long-lasting epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) primarily through a positive-feedback histone methylation-MAPK signaling axis. We find that neoplastic PDAC cells preferentially undergo EMT in hypoxic tumor regions in multiple model systems and that hypoxia drives a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, can last for weeks. We further demonstrate that hypoxia reduces histone demethylase KDM2A activity, suppresses PP2 family phosphatase expression, and activates MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors play only a supporting role. This mechanism can be antagonized in vivo by combinations of MAPK inhibitors that may be effective in multi-drug therapies designed to target EMT.

Unresolved inflammation compromises diabetic wound healing. Recently, we reported that inadequate RNA packaging in murine wound-edge keratinocyte-originated exosomes (Exo κ ) leads to persistent inflammation [Zhou, X.et al.ACS Nano 2020, 14(10), 12732−12748]. Herein, we use charge detection mass spectrometry (CDMS) to analyze intact Exo κ isolated from a 5 day old wound-edge tissue of diabetic mice and a heterozygous nondiabetic littermate control group. In CDMS, the charge (z) and mass-to-charge ratio (m/z) of individual exosome particles are measured simultaneously, enabling the direct analysis of masses in the 1−200 MDa range anticipated for exosomes. These measurements reveal a broad mass range for Exo κ from ∼10 to >100 MDa. The m and z values for these exosomes appear to fall into families (subpopulations); a statistical modeling analysis partially resolves ∼10−20 Exo κ subpopulations. Complementary proteomics, immunofluorescence, and electron microscopy studies support the CDMS results that Exo κ from diabetic and nondiabetic mice vary substantially. Subpopulations having high z (>650) and high m (>44 MDa) are more abundant in nondiabetic animals. We propose that these high m and z particles may arise from differences in cargo packaging. The veracity of this idea is discussed in light of other recent CDMS results involving genome packaging in vaccines, as well as exosome imaging experiments. Characterization of intact exosome particles based on the physical properties of m and z provides a new means of investigating wound healing and suggests that CDMS may be useful for other pathologies.

Peak broadening in ion mobility (IM) is a relatively predictable process and abnormally broad peaks can be indicative of the presence of unresolved species. Here, we introduce a new ion mobility peak fitting (IM_FIT) software package for automated and systematic determination of traveling wave ion mobility (TWIM) unresolved species. To identify IM unresolved species, the IM_FIT software generates a trend line by plotting ions' mobility peak widths as a function of their arrival times. Utilizing user-defined thresholds, IM_FIT allows for automated and rapid detection of ions that deviate from the peak width trend line. To demonstrate the advantages of IM_FIT for automated detection of IM unresolved species, IM-mass spectrometry (IM-MS) data from a sample mixture containing polypropylene glycol and multiple peptides were analyzed. A total of 14 out of the 34 observed singly-charged IM peaks above 5% relative abundance (i.e., signal-to-noise ratios above ∼200) were tagged as potentially co-eluting ions by IM_FIT. Subsequently, the 14 IM peaks tagged as potentially unresolved (presumably, peaks corresponding to co-eluting compounds), were further analyzed by automated IM deconvolution (AIMD), liquid chromatography-IM-MS (LC-IM-MS), and/or ultra-high resolution mass spectrometry. Using the aforementioned techniques, more than 85% of the tagged IM peaks (12 out of 14) were confirmed to contain co-eluting ions. As an additional new finding, IM_FIT facilitated the discovery of an unexpected sequence-scrambled y-type fragment ion.