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

Abstract

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of '-omics' research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol

Keywords: MCF-10A; MCF-7; MDA-MB-231; Vn96; cell-conditioned media; exosomes; extracellular vesicle.

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.

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.