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Facile preparation of salivary extracellular vesicles for cancer proteomics.

Sun Y, Xia Z, Shang Z, Sun K, Niu X, Qian L, Fan LY, Cao CX, Xiao H - Sci Rep (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, Laboratory of Analytical Biochemistry and Bioseparation, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f4: 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.

Mentions: LC-MS/MS based shotgun proteomic approach was applied for the proteome analysis of SEVs. All experiments (both conventional centrifugation method and ACCF method) were carried out in triplicate. For each SEVs sample obtained by ACCF method, we identified 128, 138 and 107 proteins, respectively. For each SEVs sample prepared by conventional centrifugation method, we discovered 87, 76 and 72 proteins, respectively. To extract the most reliable data, we used the overlapped results of 3 experiments. Finally, we consistently obtained 95 SEVs proteins for ACCF method and 56 SEVs proteins for conventional centrifugation method (Fig. 4). The number of identified SEVs proteins was increased more than 70% through using ACCF method (Fig. 4A). Further analysis revealed that 42 proteins were shared by both methods, which include SEVs marker protein Integrin beta-234. The ACCF method was able to discover 53 unique proteins from SEVs, while 14 proteins were unique to conventional centrifugation method. It could be observed from Fig. 2C that the use of ACCF approach for the removal of amylase led to find more number of low molecule weight proteins (<20‚ÄČkDa) than conventional centrifugation method. These results were consistent with the SDS-PAGE analysis of both methods (Fig. 2B).


Facile preparation of salivary extracellular vesicles for cancer proteomics.

Sun Y, Xia Z, Shang Z, Sun K, Niu X, Qian L, Fan LY, Cao CX, Xiao H - Sci Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835767&req=5

f4: 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.
Mentions: LC-MS/MS based shotgun proteomic approach was applied for the proteome analysis of SEVs. All experiments (both conventional centrifugation method and ACCF method) were carried out in triplicate. For each SEVs sample obtained by ACCF method, we identified 128, 138 and 107 proteins, respectively. For each SEVs sample prepared by conventional centrifugation method, we discovered 87, 76 and 72 proteins, respectively. To extract the most reliable data, we used the overlapped results of 3 experiments. Finally, we consistently obtained 95 SEVs proteins for ACCF method and 56 SEVs proteins for conventional centrifugation method (Fig. 4). The number of identified SEVs proteins was increased more than 70% through using ACCF method (Fig. 4A). Further analysis revealed that 42 proteins were shared by both methods, which include SEVs marker protein Integrin beta-234. The ACCF method was able to discover 53 unique proteins from SEVs, while 14 proteins were unique to conventional centrifugation method. It could be observed from Fig. 2C that the use of ACCF approach for the removal of amylase led to find more number of low molecule weight proteins (<20‚ÄČkDa) than conventional centrifugation method. These results were consistent with the SDS-PAGE analysis of both methods (Fig. 2B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, Laboratory of Analytical Biochemistry and Bioseparation, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.

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.

No MeSH data available.


Related in: MedlinePlus