期刊
PROTEOMICS
卷 19, 期 8, 页码 -出版社
WILEY
DOI: 10.1002/pmic.201700453
关键词
exosomes; extracellular vesicles; integral membrane proteins; membrane shaving; surfaceome
资金
- La Trobe University, Melbourne, Australia
- Core Research for Evolutional Science and Technology (CREST) from Japan Science and Technology Agency (JST CREST grant) [JPMJCR13M2]
Exosomes are important bidirectional cell-cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X-114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label-free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database-annotated proteins reveals 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 108 RNA-binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface-localized miRNA Let-7a-5p is RNase A-resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.
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