Difference gel electrophoresis analysis of Ras‐transformed fibroblast cell‐derived exosomes

Abstract

Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras-transformed NIH3T3 cells. The vesicular nature and size (30-100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2-D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras-transformed NIH3T3 cells. LC-MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen alpha-1 (VI), 14-3-3 isoforms, guanine nucleotide-binding proteins (G proteins), the eukaryotic translation initiation factors elF-3 gamma and elF-5A accumulated (>2-fold) in exosomes upon Ras-induced oncogenic transformation. Significantly, the 10.4-fold increase in v-Ha-Ras p21 protein in exosomes derived from Ras-transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.

Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS

“…HRAS and NRAS have been found in exosomes purified from transformed cells (41,42) and urine (43). HRAS, NRAS, and KRAS were previously detected in exosomes via LC-MS/ MS; however, that study did not describe the peptides identified, and further validation was not provided (44).…”

Extracellular vesicle isolation and characterization: toward clinical application

“…Over the past decade, our laboratory has accumulated exhaustive proteomic profiling data sets for highly purified EV subtypes (exosomes and sMVs) from the human colon cancer cell lines LIM1863 (35, 37, 66), HCT116, SW1222, LIM1215 (102), SW480, SW620 (74), human NIH3T3 fibroblast, and Rastransformed NIH3T3 fibroblast cells (103), and the Madin-Darby canine kidney (MDCK) cell line and an MDCK cell line transformed with oncogenic H-Ras (21D1 cells) (4). These extensive proteomic data sets (RAW files) are publically accessible through PeptideAtlas (104).…”

Microvesicles and Viral Infection

“…(B) Dying or apoptotic cells release shedding microvesicles in the early stages of apoptosis and larger apoptotic bodies at later times that contain nuclear and cytoplasmic remnants of the degrading cell. stress (93), stimulation with cytokines (139), exposure to ATP (17), cell death (61), hypoxia (119), transformation (45), expression of oncogenes (6,27,78), and viral infection (11,88,102,140,162). Thus, in addition to the two defined MVB populations, one destined for a lysosomal degradation and the other for exosomal secretion, it is likely that additional MVB populations produce exosomes with distinct compositions.…”