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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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Experimental workflow. (1) Metabolic labeling of cells with the mannose analog, peracetylated azido-mannose (AC4ManNAz). (2) Chemoselective conjugation of azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry. (3) Lysis of labeled cells. (4) Affinity purification using streptavidin (SAv) resins. (5) Elution of captured sialoglycoproteins. (6) SDS-PAGE separation of sialoglycoproteins. (7) Isolation of gel slice and subsequent digestion and release of peptides. (8) Analysis of peptides by LC-MS/MS. (9) Bioinformatic analysis.
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Figure 1: Experimental workflow. (1) Metabolic labeling of cells with the mannose analog, peracetylated azido-mannose (AC4ManNAz). (2) Chemoselective conjugation of azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry. (3) Lysis of labeled cells. (4) Affinity purification using streptavidin (SAv) resins. (5) Elution of captured sialoglycoproteins. (6) SDS-PAGE separation of sialoglycoproteins. (7) Isolation of gel slice and subsequent digestion and release of peptides. (8) Analysis of peptides by LC-MS/MS. (9) Bioinformatic analysis.

Mentions: Our strategy for interrogation of cell surface sialoglycoproteins using selective chemical tagging followed by high-affinity enrichment and GeLC-MS/MS analysis is summarized in Fig. 1. The method consists of several steps: (1) metabolic labeling of N2 and ML2 cells with the azide-containing mannose analog, peracetylated azido-mannose (AC4ManNAz); (2) chemoselective conjugation of azide sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry in live cells; (3) affinity enrichment of the labeled cell-surface sialylated proteins by streptavidin capture; and (4) separation by one-dimensional gel electrophoresis and identification by LC-MS/MS. When compared with other published approaches, the theoretical advantage of our approach is the targeted selectivity for sialyl glycosylated proteins on the cell surface.


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)

Experimental workflow. (1) Metabolic labeling of cells with the mannose analog, peracetylated azido-mannose (AC4ManNAz). (2) Chemoselective conjugation of azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry. (3) Lysis of labeled cells. (4) Affinity purification using streptavidin (SAv) resins. (5) Elution of captured sialoglycoproteins. (6) SDS-PAGE separation of sialoglycoproteins. (7) Isolation of gel slice and subsequent digestion and release of peptides. (8) Analysis of peptides by LC-MS/MS. (9) Bioinformatic analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental workflow. (1) Metabolic labeling of cells with the mannose analog, peracetylated azido-mannose (AC4ManNAz). (2) Chemoselective conjugation of azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry. (3) Lysis of labeled cells. (4) Affinity purification using streptavidin (SAv) resins. (5) Elution of captured sialoglycoproteins. (6) SDS-PAGE separation of sialoglycoproteins. (7) Isolation of gel slice and subsequent digestion and release of peptides. (8) Analysis of peptides by LC-MS/MS. (9) Bioinformatic analysis.
Mentions: Our strategy for interrogation of cell surface sialoglycoproteins using selective chemical tagging followed by high-affinity enrichment and GeLC-MS/MS analysis is summarized in Fig. 1. The method consists of several steps: (1) metabolic labeling of N2 and ML2 cells with the azide-containing mannose analog, peracetylated azido-mannose (AC4ManNAz); (2) chemoselective conjugation of azide sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry in live cells; (3) affinity enrichment of the labeled cell-surface sialylated proteins by streptavidin capture; and (4) separation by one-dimensional gel electrophoresis and identification by LC-MS/MS. When compared with other published approaches, the theoretical advantage of our approach is the targeted selectivity for sialyl glycosylated proteins on the cell surface.

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