Quantitative proteomic analysis of trypsin‐treated extracellular vesicles to identify the real‐vesicular proteins

Abstract

Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer and released into the extracellular milieu by most of cells. Although various EV isolation methods have been established, most of the current methods isolate EVs with contaminated non-vesicular proteins. By applying the label-free quantitative proteomic analyses of human colon cancer cell SW480derived EVs, we identified trypsin-sensitive and trypsin-resistant vesicular proteins. Further systems biology and protein-protein interaction network analyses based on their cellular localization, we classified the trypsin-sensitive and trypsin-resistant vesicular proteins into two subgroups: 363 candidate real-vesicular proteins and 151 contaminated non-vesicular proteins. Moreover, the protein interaction network analyses showed that candidate real-vesicular proteins are mainly derived from plasma membrane (46.8%), cytosol (36.6%), cytoskeleton (8.0%) and extracellular region (2.5%). On the other hand, most of the contaminated non-vesicular proteins derived from nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, ribosomal protein complexes and T-complex proteins were classified as the contaminated nonvesicular proteins. Taken together, our trypsin-digested proteomic approach on EVs is an important advance to identify the real-vesicular proteins that could help to understand EV biogenesis and protein cargo-sorting mechanism during EV release, to identify more reliable EV diagnostic marker proteins, and to decode pathophysiological roles of EVs.

Bovine milk extracellular vesicles induce the proliferation and differentiation of osteoblasts and promote osteogenesis in rats

“…This result is consistent with the previous proteomic analysis of bovine milk EVs identifying the lactoferrin and β-lactoglobulin as one of the major proteins of milk EV proteome (Benmoussa et al, 2019;Sanwlani et al, 2020;Yang et al, 2017). Extracellular soluble proteins can be attached to the surface of EVs and co-isolated with EVs (Choi et al, 2020).…”

Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes

“…The molecular composition of EVs is a key focus in the field, as they carry a wide variety of functional proteins (Choi et al., 2020; Hoshino et al., 2020). To identify the specific proteomic features of MSC‐derived apoVs, we prepared proteins of apoVs and parental MSCs, and performed LC‐MS/MS analysis.…”

“…For the induction of apoptosis, MSCs were washed with PBS and then treated with STS, which showed significant  of  morphological alteration and positive TUNEL staining (Supplementary Figure 1(C)). We isolated apoVs from apoptotic MSCs using the optimized gradient centrifugation protocol (Liu et al, 2020) (Supplementary Figure 2(A The molecular composition of EVs is a key focus in the field, as they carry a wide variety of functional proteins (Choi et al, 2020;Hoshino et al, 2020). To identify the specific proteomic features of MSC-derived apoVs, we prepared proteins of apoVs and parental MSCs, and performed LC-MS/MS analysis.…”

Extracellular vesicles in Inflammatory Skin Disorders: from Pathophysiology to Treatment

“…Thus, several techniques were developed to improve our understanding of EV contents. A recent study used a trypsin-digested proteomics approach to classify trypsin-sensitive and trypsin-resistant vesicular proteins, and to systematically study the actual-vesicular proteins, which helped to identify reliable functional proteins, and revealed their pathophysiological roles, an important aspect in the use of EVs as liquid biopsies 242 . In addition, new techniques such as exosome-templated nanoplasmonics 243 and nanopatterned microchips 244 were recently developed to accurately and sensitively monitor the molecular profiles of EVs, which would facilitate the development of liquid biopsies.…”