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Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ - Mol. Cell Proteomics (2011)

Bottom Line: A selective enrichment of sialoglycoproteins was confirmed.When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach.Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

View Article: PubMed Central - PubMed

Affiliation: Leroy T. Canoles Cancer Research Center, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

ABSTRACT
Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

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Characterization of Integrin β1 and CDCP1 Expression. A, Western blot analysis of cell-surface glycoprotein enriched fractions. N2 and ML2 cells were metabolically labeled with ManNAz or ManNAc, conjugated with biotin-alkyne and isolated via streptavidin. The purified eluates from each group were separated by SDS-PAGE, and immunoblotted for selected proteins as described. Streptavidin (SAv) was analyzed to normalize for loading. B, Western blot analysis of expression of the indicated proteins in total lysates of N2 and ML2 cells. Anti-GAPDH was included as a loading control. (C, D) Characterization of the glycosylation status of integrin β1 and CDCP1. Total cell lysates (20 μg) of N2 and ML2 cells were subjected to digestions with PNGase F (C) or neuraminidase (D) as described and then subjected to SDS-PAGE. The separated proteins were analyzed by Western blot analysis as described. Analysis of endogenous GAPDH or β actin was included as a loading control.
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Figure 8: Characterization of Integrin β1 and CDCP1 Expression. A, Western blot analysis of cell-surface glycoprotein enriched fractions. N2 and ML2 cells were metabolically labeled with ManNAz or ManNAc, conjugated with biotin-alkyne and isolated via streptavidin. The purified eluates from each group were separated by SDS-PAGE, and immunoblotted for selected proteins as described. Streptavidin (SAv) was analyzed to normalize for loading. B, Western blot analysis of expression of the indicated proteins in total lysates of N2 and ML2 cells. Anti-GAPDH was included as a loading control. (C, D) Characterization of the glycosylation status of integrin β1 and CDCP1. Total cell lysates (20 μg) of N2 and ML2 cells were subjected to digestions with PNGase F (C) or neuraminidase (D) as described and then subjected to SDS-PAGE. The separated proteins were analyzed by Western blot analysis as described. Analysis of endogenous GAPDH or β actin was included as a loading control.

Mentions: We next employed orthoganol approaches to examine two candidate proteins as a verification of the ability of the described methodology to target sialoglycoproteins that are differentially expressed across our cell model. We targeted Integrin β1 and CDCP1 for further analysis and would propose that such verification be a routine component of our work-flow. Integrin β1 (CD29) was identified in both N2 and ML2 cells, where as CDCP1 was uniquely identified in the metastatic ML2 cell line. We first examined the cell surface glycoprotein enriched eluents derived from the streptavidin-based purification shown in Fig. 3B. An equal volume of surface enriched fractions from N2 and ML2 cells and their respective controls was resolved by SDS-PAGE and probed with antibodies to the indicated proteins. When analyzed for Integrin β1, a band with an apparent molecular weight of 130 kDa was found in N2 and ML2 ManNAz-treated cells at similar abundance (Fig. 8A, top panel), consistent with the observed LC-MS/MS results. Immunoblotting for CDCP1 revealed the 135 kDa full-length form as well as a 70 kDa truncated species (truncated species not shown). The CDCP1 levels were ∼fourfold greater on the surface of ML2 compared with N2 cells (Fig. 8A, middle panel). The ManNAc-labeled control cells exposed to the same enrichment procedure had undetectable levels of the selected proteins, underscoring the efficiency of our surface glycoproteomic strategy. The consistent amounts of SAv across all samples served as a control for normalization of the data. This result provides verification of the relative expression of these proteins.


Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ - Mol. Cell Proteomics (2011)

Characterization of Integrin β1 and CDCP1 Expression. A, Western blot analysis of cell-surface glycoprotein enriched fractions. N2 and ML2 cells were metabolically labeled with ManNAz or ManNAc, conjugated with biotin-alkyne and isolated via streptavidin. The purified eluates from each group were separated by SDS-PAGE, and immunoblotted for selected proteins as described. Streptavidin (SAv) was analyzed to normalize for loading. B, Western blot analysis of expression of the indicated proteins in total lysates of N2 and ML2 cells. Anti-GAPDH was included as a loading control. (C, D) Characterization of the glycosylation status of integrin β1 and CDCP1. Total cell lysates (20 μg) of N2 and ML2 cells were subjected to digestions with PNGase F (C) or neuraminidase (D) as described and then subjected to SDS-PAGE. The separated proteins were analyzed by Western blot analysis as described. Analysis of endogenous GAPDH or β actin was included as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108840&req=5

Figure 8: Characterization of Integrin β1 and CDCP1 Expression. A, Western blot analysis of cell-surface glycoprotein enriched fractions. N2 and ML2 cells were metabolically labeled with ManNAz or ManNAc, conjugated with biotin-alkyne and isolated via streptavidin. The purified eluates from each group were separated by SDS-PAGE, and immunoblotted for selected proteins as described. Streptavidin (SAv) was analyzed to normalize for loading. B, Western blot analysis of expression of the indicated proteins in total lysates of N2 and ML2 cells. Anti-GAPDH was included as a loading control. (C, D) Characterization of the glycosylation status of integrin β1 and CDCP1. Total cell lysates (20 μg) of N2 and ML2 cells were subjected to digestions with PNGase F (C) or neuraminidase (D) as described and then subjected to SDS-PAGE. The separated proteins were analyzed by Western blot analysis as described. Analysis of endogenous GAPDH or β actin was included as a loading control.
Mentions: We next employed orthoganol approaches to examine two candidate proteins as a verification of the ability of the described methodology to target sialoglycoproteins that are differentially expressed across our cell model. We targeted Integrin β1 and CDCP1 for further analysis and would propose that such verification be a routine component of our work-flow. Integrin β1 (CD29) was identified in both N2 and ML2 cells, where as CDCP1 was uniquely identified in the metastatic ML2 cell line. We first examined the cell surface glycoprotein enriched eluents derived from the streptavidin-based purification shown in Fig. 3B. An equal volume of surface enriched fractions from N2 and ML2 cells and their respective controls was resolved by SDS-PAGE and probed with antibodies to the indicated proteins. When analyzed for Integrin β1, a band with an apparent molecular weight of 130 kDa was found in N2 and ML2 ManNAz-treated cells at similar abundance (Fig. 8A, top panel), consistent with the observed LC-MS/MS results. Immunoblotting for CDCP1 revealed the 135 kDa full-length form as well as a 70 kDa truncated species (truncated species not shown). The CDCP1 levels were ∼fourfold greater on the surface of ML2 compared with N2 cells (Fig. 8A, middle panel). The ManNAc-labeled control cells exposed to the same enrichment procedure had undetectable levels of the selected proteins, underscoring the efficiency of our surface glycoproteomic strategy. The consistent amounts of SAv across all samples served as a control for normalization of the data. This result provides verification of the relative expression of these proteins.

Bottom Line: A selective enrichment of sialoglycoproteins was confirmed.When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach.Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

View Article: PubMed Central - PubMed

Affiliation: Leroy T. Canoles Cancer Research Center, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

ABSTRACT
Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

Show MeSH
Related in: MedlinePlus