Facile preparation of salivary extracellular vesicles for cancer proteomics

Abstract

Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

Figure 1. Schematic diagram for EVs isolation from human saliva.

Figure 2. SDS-PAGE of salivary proteins and SEVs proteins.

(A) 1D SDS-PAGE of salivary protein. (a) Protein ladder; (b) 1.5 μg of salivary proteins prepared by ACCF method; (c) 1.5 μg of primordial salivary proteins. (B) 1D SDS-PAGE of SEVs proteins. (a) Protein ladder; (b) 1.0 μg of salivary proteins; (c) 1.0 μg of EVs’ proteins prepared by ACCF method; (d) 1.0 μg of EVs’ proteins prepared by conventional centrifugation method. (C) Molecular weight distribution of SEVs proteins prepared by conventional centrifugation method and ACCF method.

Figure 3

Particle size and distribution of the SEVs obtained by primordial saliva (A) and purified saliva sample using ACCF system (B).

Figure 4. Venn diagram of identified proteins.

(A) Overlap of SEVs proteins prepared through ACCF method and conventional method; (B) Overlap of SEVs proteins extracted from the saliva of lung cancer patients and healthy subjects.

Figure 5

Total ion chromatogram of SEVs’ peptides from primordial saliva (A) and ACCF purified saliva (B).

Figure 6. Gene Ontology analysis of candidate SEVs’ protein biomarkers for lung cancer.

Figure 7. IPA analysis of the candidate SEVs’ protein biomarkers for lung cancer.

Number of lung cancer specific proteins involve in cancer related network (A) and cellular movement related network (B). Gray symbols are SEVs proteins identified in this study.