Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods

Figures & data

Figure 1. EV capture, protein extraction and identification workflow.

Figure 2. Verification of EV isolation. Laemmli and TRIzol©-extracted samples were interrogated by Western blot for the expression of the canonical EV markers HSP-70, CD63, Flotillin-1, CD81 and CD9. MCF-7 (7), MCF-10A (10A) and MDA-MB-231 (231) cell EVs were examined. HSP-70 (top), CD63 (middle) and Flotillin-1 (bottom) were detected in the isolated EVs.

Figure 3. Comparison of the Laemmli and TRIzol© protein-extraction methods for the extraction of protein cargo from (A) MCF-10A, (B) MCF-7 and (C) MDA-MB-231 EVs. The unique proteins (number and percentage) from Laemmli are shown in blue, while TRIzol©-extracted proteins are shown in red. The common proteome is shown in the central region of the Venn diagram. Percentages below these numbers indicate the percentage of total proteins identified in each region. The upper percentages in bold show the fraction of proteins isolated from each methodology.

Table 1. List of top 25 proteins from the Exocarta web site that were identified in EVs captured from cell-culture-conditioned media of MCF-10A, MCF-7 and MDA-MB-231 cells.

		MCF-10A		MCF-7		MDA-MB-231
Protein name	Gene	LAE	TRI	LAE	TRI	LAE	TRI
Heat shock cognate 71 kDa protein	HSPA8	–	–	–	–	–	–
CD9 antigen	CD9	–	–	–	–	–	–
Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	+	+	+	+	+	+
Actin, cytoplasmic 1	ACTB	–	–	–	–	–	–
CD63 antigen	CD63	–	–	–	–	–	–
CD81 antigen	CD81	–	–	–	–	–	–
Annexin A2	ANXA2	+	+	+	+	+	+
Alpha-enolase	ENO1	+	+	+	+	+	+
Heat shock protein HSP 90-alpha	HSP90AA1	+	+	+	+	+	+
Elongation factor 1-alpha 1	EEF1A1	+	+	+	+	+	+
Pyruvate kinase	PKM2	+	+	+	+	–	–
14-3-3 protein epsilon	YWHAE	+	+	+	+	+	+
Syntenin-1	SDCBP	+	–	–	–	+	+
Prog. cell death 6-interacting protein	PDCD6IP	+	+	+	–	–	–
Serum albumin	ALB	–	–	–	–	+	-
14-3-3 protein zeta/delta	YWHAZ	+	+	+	+	+	+
Elongation factor 2	EEF2	+	+	+	+	+	+
Actin, cytoplasmic 2	ACTG1	+	+	+	–	+	+
l-lactate dehydrogenase A chain	LDHA	+	+	+	+	+	+
Heat shock protein HSP 90-beta	HSP90AB1	+	+	+	+	+	+
Fructose-bisphosphate aldolase A	ALDOA	+	+	+	+	+	+
Moesin	MSN	+	+	–	–	+	+
Annexin A5	ANXA5	–	–	–	–	–	–
Phosphoglycerate kinase 1	PGK1	+	+	+	+	+	+
Cofilin-1	CFL1	+	+	+	+	+	+

EVs from MCF-7, MDA-MB-231 and MCF-10A were captured using Vn96, and the proteins were extracted with either Laemmli (LAE) or TRIzol© (TRI) extraction methods..

Figure 4. Proteomic analysis of MCF-10A, MCF-7 and MDA-MB-231 EVs revealing sets of common, overlapping and unique proteins from different EV groups. Proteins included in the analysis of each cell type EVs were present using both Laemmli and TRIzol© extraction reagents in both biological replicates.

Figure 5. Principal component analysis (PCA) of Laemmli and TRIzol©-extracted EV proteins from MCF-10A, MCF-7 and MDA-MB-231 cells. The two Laemmli (L) and TRIzol© (T) biological replicates selected from each cell line that were used for all analyses were compared. The total list of extracted proteins within each biological replicate of the extraction method was compiled from the technical replicates.

Figure 6. Unique and overlapping gene ontologies (GOs) from MCF-10A, MCF-7 and MDA-MB-231 EVs. The GO terms were identified using ToppFun from the lists of proteins identified using both Laemmli and TRIzol© extracted EV proteins.