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

Abstract

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.

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

“…With regard to EVs, a slightly negative zeta-potential suggests that EVs exhibit sub-par stability behaviors in biological colloids and therefore are more likely than lipoproteins to interact with cell surfaces and other particles (Lan et al, 2011 ; Wang and Reed, 2011 ; Kozak et al, 2012 ; Deregibus et al, 2016 ; Vogel et al, 2017 ; Heath et al, 2018 ; Brown et al, 2020 ). EVs have masses in the megadalton range (Brown et al, 2020 ), or about 0.25 pg on average (Stratton et al, 2014 ). Most EVs are identified biochemically by the presence of certain proteins within their membrane (Théry et al, 2018 ).…”

Exosomal Cargo May Hold the Key to Improving Reproductive Outcomes in Dairy Cows

“…In relation to dairy cow reproduction, MS has been utilized to perform thorough and reproducible analyses of bovine plasma, milk, follicular fluid, and uterine flushings [ 101 , 102 , 103 ]. To provide a better understanding of the signaling pathways associated with reproduction, exosomes isolated from milk of dairy cows have also been analyzed using a range of MS strategies in a number of studies [ 16 , 104 , 105 , 106 ]. Additionally, charge detection mass spectrometry (CDMS) and label-free spectral counting have been used successfully to characterize and quantify exosomes from milk and colostrum [ 104 , 105 , 107 ], and both milk and plasma exosomes have undergone qualitative analysis in DDA studies [ 4 , 16 ].…”

“…To provide a better understanding of the signaling pathways associated with reproduction, exosomes isolated from milk of dairy cows have also been analyzed using a range of MS strategies in a number of studies [ 16 , 104 , 105 , 106 ]. Additionally, charge detection mass spectrometry (CDMS) and label-free spectral counting have been used successfully to characterize and quantify exosomes from milk and colostrum [ 104 , 105 , 107 ], and both milk and plasma exosomes have undergone qualitative analysis in DDA studies [ 4 , 16 ]. What is currently lacking in the field is a thorough quantitative analysis of the bovine blood plasma exosomal proteome, which may provide a better systemic snapshot of overall health and pathways associated with fertility, and thus clues to reproductive status in dairy cows.…”

Recent Progress on the Isolation and Detection Methods of Exosomes

“…Based on the characteristics of exosomes particle size and charge, Brown and his colleagues analyzed physical and chemical properties by mass spectrometry, which would be beneficial to the classification of exosomes subtypes. [84] Besides, magnetic nanoparticles were used to separate exosomes from placental cells, while it's tested by enzyme-linked immunosorbent assay. [85] With the increasing maturity of microfluidic technology, the identification and detection of exosomes can be applied through the microprocessing of test channels, and the sensitivity shows obvious advantages.…”