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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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Capture and enrichment of azide-tagged sialoglycoproteins. A, Detection of sialylated glycoprotein in cell lysates. N2 and ML2 cells were metabolically labeled with 20 μm ManNAz for 24 h and conjugated with 25% (v/v) biotin-alkyne. Cells were lysed and total protein was extracted as described under “Experimental Procedures.” Top panel: Protein was resolved by SDS-PAGE and visualized by incubation with streptavidin-IR 800. Reactive bands indicate sialylated glycoproteins. The same blot was probed with anti-β-actin to verify equal protein loading. The relative expression of sialylated glycoproteins after normalization with the β-actin is shown at the bottom of each lane. B, Enrichment of azide-tagged sialoglycoproteins by affinity chromatography. Azide-tagged and biotin-conjugated sialoglycoproteins from the total lysate of N2 and ML2 cells were captured by streptavidin beads, separated by SDS-PAGE and visualized by reaction with streptavidin-IR 800. Shown are the results from 20 μg of postclick cell lysate (Input), 5% of the flow through material that did not bind to the beads (Flow-Through), 5% of the eluted material from 2 mg of protein that bound to the beads (Eluent). In, input; FT, flow through; E, eluent. Shown is a representative of four experimental replicates.
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Figure 3: Capture and enrichment of azide-tagged sialoglycoproteins. A, Detection of sialylated glycoprotein in cell lysates. N2 and ML2 cells were metabolically labeled with 20 μm ManNAz for 24 h and conjugated with 25% (v/v) biotin-alkyne. Cells were lysed and total protein was extracted as described under “Experimental Procedures.” Top panel: Protein was resolved by SDS-PAGE and visualized by incubation with streptavidin-IR 800. Reactive bands indicate sialylated glycoproteins. The same blot was probed with anti-β-actin to verify equal protein loading. The relative expression of sialylated glycoproteins after normalization with the β-actin is shown at the bottom of each lane. B, Enrichment of azide-tagged sialoglycoproteins by affinity chromatography. Azide-tagged and biotin-conjugated sialoglycoproteins from the total lysate of N2 and ML2 cells were captured by streptavidin beads, separated by SDS-PAGE and visualized by reaction with streptavidin-IR 800. Shown are the results from 20 μg of postclick cell lysate (Input), 5% of the flow through material that did not bind to the beads (Flow-Through), 5% of the eluted material from 2 mg of protein that bound to the beads (Eluent). In, input; FT, flow through; E, eluent. Shown is a representative of four experimental replicates.

Mentions: Having demonstrated efficient labeling of cell surface glycoconjugates, we continued with a general characterization of whole-cell lysates of Biotin-labeled glycoconjugates via SDS-PAGE separation and visualization by reaction with streptavidin-IR800. Expression of β-actin was used for normalization. Overall sialylation, as judged by streptavidin staining, was restricted to extracts from the ManNAz labeled cells (Fig. 3A). In addition, there appeared to be greater sialylated glycoproteins in the ML2 cell line when compared with the N2 cell line. We also observed a major protein band that migrated just under 150 kDa and reacted with streptavidin. These observations were consistent with the prior immunofluorescence analyses in demonstrating a specific uptake and labeling of the ManNAz treated cells.


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)

Capture and enrichment of azide-tagged sialoglycoproteins. A, Detection of sialylated glycoprotein in cell lysates. N2 and ML2 cells were metabolically labeled with 20 μm ManNAz for 24 h and conjugated with 25% (v/v) biotin-alkyne. Cells were lysed and total protein was extracted as described under “Experimental Procedures.” Top panel: Protein was resolved by SDS-PAGE and visualized by incubation with streptavidin-IR 800. Reactive bands indicate sialylated glycoproteins. The same blot was probed with anti-β-actin to verify equal protein loading. The relative expression of sialylated glycoproteins after normalization with the β-actin is shown at the bottom of each lane. B, Enrichment of azide-tagged sialoglycoproteins by affinity chromatography. Azide-tagged and biotin-conjugated sialoglycoproteins from the total lysate of N2 and ML2 cells were captured by streptavidin beads, separated by SDS-PAGE and visualized by reaction with streptavidin-IR 800. Shown are the results from 20 μg of postclick cell lysate (Input), 5% of the flow through material that did not bind to the beads (Flow-Through), 5% of the eluted material from 2 mg of protein that bound to the beads (Eluent). In, input; FT, flow through; E, eluent. Shown is a representative of four experimental replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Capture and enrichment of azide-tagged sialoglycoproteins. A, Detection of sialylated glycoprotein in cell lysates. N2 and ML2 cells were metabolically labeled with 20 μm ManNAz for 24 h and conjugated with 25% (v/v) biotin-alkyne. Cells were lysed and total protein was extracted as described under “Experimental Procedures.” Top panel: Protein was resolved by SDS-PAGE and visualized by incubation with streptavidin-IR 800. Reactive bands indicate sialylated glycoproteins. The same blot was probed with anti-β-actin to verify equal protein loading. The relative expression of sialylated glycoproteins after normalization with the β-actin is shown at the bottom of each lane. B, Enrichment of azide-tagged sialoglycoproteins by affinity chromatography. Azide-tagged and biotin-conjugated sialoglycoproteins from the total lysate of N2 and ML2 cells were captured by streptavidin beads, separated by SDS-PAGE and visualized by reaction with streptavidin-IR 800. Shown are the results from 20 μg of postclick cell lysate (Input), 5% of the flow through material that did not bind to the beads (Flow-Through), 5% of the eluted material from 2 mg of protein that bound to the beads (Eluent). In, input; FT, flow through; E, eluent. Shown is a representative of four experimental replicates.
Mentions: Having demonstrated efficient labeling of cell surface glycoconjugates, we continued with a general characterization of whole-cell lysates of Biotin-labeled glycoconjugates via SDS-PAGE separation and visualization by reaction with streptavidin-IR800. Expression of β-actin was used for normalization. Overall sialylation, as judged by streptavidin staining, was restricted to extracts from the ManNAz labeled cells (Fig. 3A). In addition, there appeared to be greater sialylated glycoproteins in the ML2 cell line when compared with the N2 cell line. We also observed a major protein band that migrated just under 150 kDa and reacted with streptavidin. These observations were consistent with the prior immunofluorescence analyses in demonstrating a specific uptake and labeling of the ManNAz treated cells.

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