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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

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.
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f2: 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.

Mentions: In order to evaluate purification efficiency of the ACCF system, 1D SDS-PAGE was used to separate salivary and SEVs proteins, as shown in Fig. 2A. Comparing the protein bands obtained from SEVs isolated by ACCF (lane b) and saliva (lane c), notable difference appeared between 49 kDa and 62 kDa, because this range was mostly salivary amylase33. In lane (c), amylase was a wide and strong band while other protein bands were masked in the same region. In addition, lane (b) displayed more refined protein bands than lane (c), which suggests that high content amylase create interference in the separation and detection of other low abundant proteins.


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)

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

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

f2: 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.
Mentions: In order to evaluate purification efficiency of the ACCF system, 1D SDS-PAGE was used to separate salivary and SEVs proteins, as shown in Fig. 2A. Comparing the protein bands obtained from SEVs isolated by ACCF (lane b) and saliva (lane c), notable difference appeared between 49 kDa and 62 kDa, because this range was mostly salivary amylase33. In lane (c), amylase was a wide and strong band while other protein bands were masked in the same region. In addition, lane (b) displayed more refined protein bands than lane (c), which suggests that high content amylase create interference in the separation and detection of other low abundant proteins.

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