Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

Abstract

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

Keywords: extracellular vesicles; precipitation kits; proteomic; serum; size exclusion; ultracentrifugation.

Figure 1

Schematic representation of the EV separation design. Each serum was diluted (the 100 µL loaded for each test contains 80 µL of undiluted serum), cleared and subsequently divided in four aliquots processed by four different techniques, ultracentrifugation (UC), INV (Total Exosome Purification kit, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), GAG (Exo-GAG precipitation solution, Nasabiotech, A Coruña, Spain) and SEC (size exclusion chromatography). The results presented in this study were obtained from four independent biological replicates.

Figure 2

Western blot analysis of human EV-enriched serum preparations by using different procedures (UC, ultracentrifugation, SEC3, fractions 2–4 from SEC, SEC8 fractions 7–9 from SEC, INV, Total isolation solution from Invitrogen, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, GAG, Exo-GAG precipitation solution from Nasabiotech, A Coruña, Spain). * In the case of the sample INV, it was not possible to assay the sample against IgG, due to overload of protein, as shown in the small picture of the gel at the right corner.

Figure 3

Differences in the proteomic profile enrichment achieved by each technique. (A) Unsupervised heatmap of normalized intensity values for each technique and identified protein. The color intensity correlates with the value of normalized abundance. (B) Proteins significatively enriched for each technique (sense of the comparison y axe vs. x axe) according to ANOVA and Tukey’s post hoc analysis (significance is considered for p value < 0.05, n = 4). The color intensity correlates with the number of molecules enriched.

Figure 4

(A) The upper graph shows the number of proteins annotated in UNIPROT as harboring glycosylation sites (in black) within the total number of proteins detected for each technique (at least two peptides should be detected in 3 biological replicates). (B) The graph shows the number of proteins with glycosylated sites within the number of enriched or underrepresented proteins for each technique compared with the rest or between SEC3 and INV.

Figure 5

Summary of the cell component analysis associated with the proteins overrepresented in the preparations obtained with different isolation techniques.