Analysis of Keratinocytic Exosomes from Diabetic and Nondiabetic Mice by Charge Detection Mass Spectrometry

Abstract

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. 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.

Figure 1.

(a, b) Mass versus charge CDMS measurements for nondiabetic (N1) and diabetic (D1) wound-edge keratinocyte-derived exosome samples. (c) N1 (red trace) and D1 (black trace) mass spectra generated upon integrating the ion signal across the charge dimension using 0.35 MDa bins. Red (N1) and black (D1) dashed lines mark the average mass. The gray line denotes the expected mass range of exosomes based on assuming a spherical geometry, a 30 nm minimum exosome diameter, and an average density of 1.17 g/mL.

Figure 2.

(Top) EM images of both diabetic (D1) and nondiabetic (N1) exosome-enriched samples. (Bottom) Size distribution (shown in diameter) determined by analyzing 1012 particles across the EM images recorded for three separate diabetic and nondiabetic samples. Note: the particle diameter scale is in 4 nm increments, and deformed or clearly damaged particles as well as those clearly too small to be exosomes (below ~10 nm) were not included in this analysis.

Figure 3.

Two-dimensional mass versus charge plot showing subpopulations obtained from Gaussian fits to the experimental data for the first CDMS measurement of nondiabetic and diabetic Exo_κ samples. See the text for details. This model finds evidence for 15 and 13 subpopulations with abundances over 1% for nondiabetic and diabetic samples, respectively. Each point represents the mass and charge measured for a single particle and is assigned to a subpopulation (indicated by a color). Subfamily assignment is based on the highest probability of each particle belonging to a specific subfamily. Visually, this assignment leads to boundaries that are artificially strict when in reality the subpopulations overlap. The top and left side traces show the integrated raw data for the mass and charge dimensions, respectively, and the corresponding sums of the Gaussian curves as black lines for these dimensions. The determined fits for each subpopulation are also shown and delineated using the same color scheme. The percentage of each subpopulation is also indicated. The dashed vertical line provides an estimate of the delineation between those particles having masses in the range that is expected for exosomes and those particles that are too small to be exosomes. To the right of both the N1 and D1 mass versus charge plots is the integrated mass distribution of the N1 and D1 Exo_κ at specified charge ranges (described above, horizontal dashed lines visually show these regions) corresponding to cross sections of the densely populated S14 subpopulation. The S9, S10, S11, S15, S16, S17, and S18 subpopulation mass distributions are also included. The data were normalized to 1 and treated as a composite data set. Mass spectra generated upon integrating the ion signal across the charge dimension used 0.45 MDa bins.

Figure 4.

Plot of CDMS-derived diameters of nondiabetic (N1, left) and diabetic (D1, right) samples of each subpopulation (shown in Figure 3) using a bin size of 0.5 nm. Particle diameters from CDMS were determined by assuming a spherical geometry and a density of 1.17 g/mL.

Figure 5.

(Left) Representative coimmunofluorescence of heterogeneous nuclear ribonucleoproteins (hnRNP) Q (red) counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) in the wound-edge tissue of nondiabetic (m+/db) and diabetic (db/db) mice. The hnRNP Q expression in db/db wound-edge keratinocytes was significantly lower than that in m+/db. The white dashed line indicates the dermal–epidermal junction with the epidermal edge labeled epi. The white arrowhead indicates the leading edge of the epidermis. Granulation tissue is labeled gt. (Right) Quantification of the hnRNP Q intensity, normalized with DAPI intensity in wound-edge tissue, was plotted graphically. Each dot corresponds to one quantified region of interest (ROI), except for the blue and red dots that correspond to the mean value for one of three mice. At least three ROIs are plotted per mouse. Solid black lines indicate the mean ± standard error of the mean for all three mice in each group and were analyzed by a two-tailed unpaired Student’s t-test to determine a P value of 0.001.

Scheme 1.

Possible Additional Exosome Geometries