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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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Labeling of cell surface sialoglycoproteins. A, Flow cytometry analysis of N2 and ML2 cells treated with AC4ManNAz or ManNAc. Intact cells were biotin-tagged through click reaction, and visualized with streptavidin-FITC. PI negative gated FACS histogram showing three populations of cells: control cells (red), cells treated with ManNAc (gray filled), and cells treated with 40 μm AC4ManNAz (green) for 3 days. B, Confocal microscopy of N2 and ML2 cells for visualization of the fluorescently labeled cell-surface sialoglycoproteins. Cells were treated with 40 μm AC4ManNAz or ManNAc for 3 days and then fixed, conjugated with biotin-alkyne via click reaction and “stained” with streptavidin-FITC (green) and PI (red).
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Figure 2: Labeling of cell surface sialoglycoproteins. A, Flow cytometry analysis of N2 and ML2 cells treated with AC4ManNAz or ManNAc. Intact cells were biotin-tagged through click reaction, and visualized with streptavidin-FITC. PI negative gated FACS histogram showing three populations of cells: control cells (red), cells treated with ManNAc (gray filled), and cells treated with 40 μm AC4ManNAz (green) for 3 days. B, Confocal microscopy of N2 and ML2 cells for visualization of the fluorescently labeled cell-surface sialoglycoproteins. Cells were treated with 40 μm AC4ManNAz or ManNAc for 3 days and then fixed, conjugated with biotin-alkyne via click reaction and “stained” with streptavidin-FITC (green) and PI (red).

Mentions: A critical parameter in the proposed strategy is the efficient labeling and surface expression of the azide-containing sialoglycoproteins. N2 and ML2 cells were subjected to metabolic incorporation of AC4ManNAz, conjugated with biotin with the click reaction, and subsequently stained with streptavidin-FITC to visualize the glycans harboring the azide group. We conducted fluorescence-assisted flow cytometric analysis of nonpermeabilized cells for determining the efficiency of streptavidin-FITC labeling. As quantified by flow cytometry (Fig. 2A), the AC4ManNAz-treated N2 and ML2 cells displayed 80–100 fold greater FITC-specific fluorescence intensity than the control ManNAc-treated and untreated cells. This result demonstrates that the azide moieties were efficiently incorporated into cellular glycans. We next conducted confocal microscopy analysis of streptavidin labeled cells to specifically assess the cellular location of the azide-modified glycans. As seen in Fig. 2B, prominent FITC staining primarily at the cell membrane was observed when the cells were subjected to metabolic incorporation of ManNAz. Consistent with flow cytometric data, confocal microscopic analysis of cells treated with control ManNAc displayed much reduced staining, confirming the labeling via azide-containing glycans is specific. Furthermore, there was no evidence of cell surface labeling for the control groups. We further optimized the azide labeling and click reaction to maximize cell viability/vigor (see supplemental Fig. S1), and determined that 24 h incubation of 20 μm AC4ManNAz and 25% (v/v) biotin-alkyne was optimal.


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)

Labeling of cell surface sialoglycoproteins. A, Flow cytometry analysis of N2 and ML2 cells treated with AC4ManNAz or ManNAc. Intact cells were biotin-tagged through click reaction, and visualized with streptavidin-FITC. PI negative gated FACS histogram showing three populations of cells: control cells (red), cells treated with ManNAc (gray filled), and cells treated with 40 μm AC4ManNAz (green) for 3 days. B, Confocal microscopy of N2 and ML2 cells for visualization of the fluorescently labeled cell-surface sialoglycoproteins. Cells were treated with 40 μm AC4ManNAz or ManNAc for 3 days and then fixed, conjugated with biotin-alkyne via click reaction and “stained” with streptavidin-FITC (green) and PI (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Labeling of cell surface sialoglycoproteins. A, Flow cytometry analysis of N2 and ML2 cells treated with AC4ManNAz or ManNAc. Intact cells were biotin-tagged through click reaction, and visualized with streptavidin-FITC. PI negative gated FACS histogram showing three populations of cells: control cells (red), cells treated with ManNAc (gray filled), and cells treated with 40 μm AC4ManNAz (green) for 3 days. B, Confocal microscopy of N2 and ML2 cells for visualization of the fluorescently labeled cell-surface sialoglycoproteins. Cells were treated with 40 μm AC4ManNAz or ManNAc for 3 days and then fixed, conjugated with biotin-alkyne via click reaction and “stained” with streptavidin-FITC (green) and PI (red).
Mentions: A critical parameter in the proposed strategy is the efficient labeling and surface expression of the azide-containing sialoglycoproteins. N2 and ML2 cells were subjected to metabolic incorporation of AC4ManNAz, conjugated with biotin with the click reaction, and subsequently stained with streptavidin-FITC to visualize the glycans harboring the azide group. We conducted fluorescence-assisted flow cytometric analysis of nonpermeabilized cells for determining the efficiency of streptavidin-FITC labeling. As quantified by flow cytometry (Fig. 2A), the AC4ManNAz-treated N2 and ML2 cells displayed 80–100 fold greater FITC-specific fluorescence intensity than the control ManNAc-treated and untreated cells. This result demonstrates that the azide moieties were efficiently incorporated into cellular glycans. We next conducted confocal microscopy analysis of streptavidin labeled cells to specifically assess the cellular location of the azide-modified glycans. As seen in Fig. 2B, prominent FITC staining primarily at the cell membrane was observed when the cells were subjected to metabolic incorporation of ManNAz. Consistent with flow cytometric data, confocal microscopic analysis of cells treated with control ManNAc displayed much reduced staining, confirming the labeling via azide-containing glycans is specific. Furthermore, there was no evidence of cell surface labeling for the control groups. We further optimized the azide labeling and click reaction to maximize cell viability/vigor (see supplemental Fig. S1), and determined that 24 h incubation of 20 μm AC4ManNAz and 25% (v/v) biotin-alkyne was optimal.

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