Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Surfaceome of Exosomes Secreted from the ColorectalCancer Cell Line SW480: Peripheral and Integral MembraneProteins Analyzed by Proteolysis and TX114 (#932)

Rong Xu 1 , David Greening 1 , Maoshan Chen 1 , Alin Ray 1 , Hong Ji 1 , Nobuhiro Takahashi 2 , Richard Simpson 1 2
  1. La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia
  2. Global Innovation Research Organization, Tokyo University of Agriculture and Technology, Tokyo, Japan

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.