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N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry.

Li X, Jiang J, Zhao X, Wang J, Han H, Zhao Y, Peng B, Zhong R, Ying W, Qian X - PLoS ONE (2013)

Bottom Line: Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches.The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study.Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, China ; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.

ABSTRACT
Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

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Related in: MedlinePlus

Bioinformatics analysis of identified N-glycoproteins.A) Cellular component annotation of identified N-glycoproteins. B) Biological functions of differentially expressed N-glycoproteins.
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pone-0081921-g003: Bioinformatics analysis of identified N-glycoproteins.A) Cellular component annotation of identified N-glycoproteins. B) Biological functions of differentially expressed N-glycoproteins.

Mentions: To determine what subset of proteins was enriched in the secretome, we performed a prediction analysis of the protein cellular localization using Gene Ontology (GO). Approximately 65 % of the glycoproteins identified were categorized into the extracellular space and plasma membrane in the GO subcellular localization annotation (Figure 3A) (Table S3). Although 27 % of the glycoproteins have a GO subcellular localization term of cytoplasm, 75 % of these proteins were predicted to contain signal peptides. Sixty-four of these cytoplasm-localized proteins contained signal peptides, as determined by Signal P 4.0, and forty-six of them were predicted to be localized to the exosome by comparing the data against an exosome database[25]. Taken together, 90% of the identified proteins were predicted to be secreted. Moreover, the enrichment scores for the clusters of glycoproteins and secretory proteins were 195.2 and 50.2, respectively, as compared to the human proteome background by DAVID analysis (p-values: 2.52×10-204 and 2.42×10-63; Benjamini: 1.26×10-201 and 3.03×10-61) (Table S4).


N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry.

Li X, Jiang J, Zhao X, Wang J, Han H, Zhao Y, Peng B, Zhong R, Ying W, Qian X - PLoS ONE (2013)

Bioinformatics analysis of identified N-glycoproteins.A) Cellular component annotation of identified N-glycoproteins. B) Biological functions of differentially expressed N-glycoproteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852754&req=5

pone-0081921-g003: Bioinformatics analysis of identified N-glycoproteins.A) Cellular component annotation of identified N-glycoproteins. B) Biological functions of differentially expressed N-glycoproteins.
Mentions: To determine what subset of proteins was enriched in the secretome, we performed a prediction analysis of the protein cellular localization using Gene Ontology (GO). Approximately 65 % of the glycoproteins identified were categorized into the extracellular space and plasma membrane in the GO subcellular localization annotation (Figure 3A) (Table S3). Although 27 % of the glycoproteins have a GO subcellular localization term of cytoplasm, 75 % of these proteins were predicted to contain signal peptides. Sixty-four of these cytoplasm-localized proteins contained signal peptides, as determined by Signal P 4.0, and forty-six of them were predicted to be localized to the exosome by comparing the data against an exosome database[25]. Taken together, 90% of the identified proteins were predicted to be secreted. Moreover, the enrichment scores for the clusters of glycoproteins and secretory proteins were 195.2 and 50.2, respectively, as compared to the human proteome background by DAVID analysis (p-values: 2.52×10-204 and 2.42×10-63; Benjamini: 1.26×10-201 and 3.03×10-61) (Table S4).

Bottom Line: Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches.The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study.Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, China ; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.

ABSTRACT
Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

Show MeSH
Related in: MedlinePlus