Charge Detection Mass Spectrometry Measurements of Exosomes and other Extracellular Particles Enriched from Bovine Milk

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.