Highly Efficient Exosome Isolation and Protein Analysis by an Integrated Nanomaterial-Based Platform

Abstract

Exosomes play important roles in mediating intercellular communication and regulating a variety of biological processes, but clear understanding of their functions and biogenesis has not been achieved, due to the high technical difficulties involved in analysis of small vesicular structures that contain a high proportion of membrane structures. Herein, we designed a novel approach to integrate two nanomaterials carrying varied surface properties, the hydrophilic, macroporous graphene foam (GF) and the amphiphilic periodic mesoporous organosilica (PMO), for efficient exosome isolation from human serum and effective protein profiling. The high specific surface area of GF, after modification with the antibody against the exosomal protein marker, CD63, allowed highly specific isolation of exosomes from complex biological samples with high recovery. Since the organic solvent, methanol, turned out to be the most effective lysis solution for releasing the exosomal proteins, the amphiphilic PMO was employed to rapidly recover the exosomal proteins, including the highly hydrophobic membrane proteins. The fine pores of PMO also acted as the nanoreactors to accelerate protein digestion that produced peptides subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 334 proteins with 111 membrane proteins [31% of these contained >2 transmembrane domains (TMD)] were identified using the integrated GF/PMO platform. In contrast, with the commercial exosome isolation kit and the in-solution protein digestion method, only 151 proteins were found, with 28 being membrane proteins (only one contained three TMDs). Our results support that the integrated GF/PMO platform is of great value to facilitate the comprehensive characterization of exosomal proteins for better understanding of their functions and for identification of more exosome-based disease markers.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

“…[103]). Such methods include tangential flow filtration and variations thereon [21,[104][105][106][107][108][109][110], field-flow fractionation (FFF) [111], asymmetric flow field-flow fractionation (AFFF, A4F, or AF4) [112][113][114], field-free viscoelastic flow [115], alternating current electrophoretics [116,117], acoustics [118], variations on size exclusion chromatography (SEC) [100,[119][120][121], ion exchange chromatography [122][123][124], microfiltration [125], fluorescenceactivated sorting [126,127] (especially for larger EVs including large apoptotic bodies [128] and large oncosomes [129]), deterministic lateral displacement (DLD) arrays [130], novel immunoisolation or other affinity isolation technologies [131][132][133][134][135][136][137][138], including lipid affinity [139], novel precipitation/combination techniques [140][141][142], hydrostatic filtration dialysis [143], ...…”

Progress in exosome associated tumor markers and their detection methods

“…Currently, antibodies have been combined with some new functional nanomaterials and a series of new immunoaffinity isolation techniques have been developed. Apart from magnetic and latex beads, the most commonly used immobilization tools for antibody coating [84] include [78] highly porous monolithic silica microtips [85], graphene foam [86], superparamagnetic nanoparticles [87] and temperature-responsive magnetic nanoparticle [88] to isolate exosomes. It is reported that IAC is the most effective method, and this study shows that the specific marker in exosome isolated by IAC is more than 2-fold higher than that of UC and gradient centrifugation [89].…”

Mesenchymal Stem Cell Derived Exosomes: a Nano Platform for Therapeutics and Drug Delivery in Combating COVID-19

“…Freeze drying formulations are one of the most reliable methodologies for highly unstable molecules like proteins, nucleic acids, etc. This enhances their practicality by increasing shelf life, lowering storage demands, reducing costs by curtailing cold chain maintenance during transportation [82,83]. However, clumping of exosomes and degradation of their cargo might be a problem during the freezedrying process.…”