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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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Improved capture of known glycoproteins. Identified proteins were cross-referenced to the UniProt Knowledgebase for known glycoproteins. The percent of total proteins that were matched to known glycoproteins is shown, in comparison to that typically determined by global proteomic approaches.
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Figure 4: Improved capture of known glycoproteins. Identified proteins were cross-referenced to the UniProt Knowledgebase for known glycoproteins. The percent of total proteins that were matched to known glycoproteins is shown, in comparison to that typically determined by global proteomic approaches.

Mentions: As a final assessment of the extent of enrichment of glycoproteins on the cell surface and in the extracellular compartment, we compared our targeted method to a nonselective global proteomic analysis of whole-cell lysates of the same cell model. Whole-cell lysates were prepared from N2 and ML2 cells, directly digested by trypsin and subjected to LC-MS/MS as described in materials and methods. Three nano-LC-MS/MS runs of tryptic digests showed that a significant number of total proteins (310 for N2 and 280 for ML2) could be detected using this simple nonfractionation approach (see supplemental Table 4). When analyzed for protein content, we found that 3.8% of the N2 proteome and 2.9% of the ML2 proteome identified from this global approach were annotated as glycoproteins using the UniProt database. In comparison, our targeted approach yielded 9.3% and 13.5% respectively (Fig. 4). Although the use of the UniProt database to determine if a protein is glycosylated is notoriously under-representative, a fourfold enrichment was revealed. Most striking was that 10 of 12 internal glycoproteins, found to be abundant and identified in the targeted approach, were not identified in the whole-cell proteome approach.


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)

Improved capture of known glycoproteins. Identified proteins were cross-referenced to the UniProt Knowledgebase for known glycoproteins. The percent of total proteins that were matched to known glycoproteins is shown, in comparison to that typically determined by global proteomic approaches.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Improved capture of known glycoproteins. Identified proteins were cross-referenced to the UniProt Knowledgebase for known glycoproteins. The percent of total proteins that were matched to known glycoproteins is shown, in comparison to that typically determined by global proteomic approaches.
Mentions: As a final assessment of the extent of enrichment of glycoproteins on the cell surface and in the extracellular compartment, we compared our targeted method to a nonselective global proteomic analysis of whole-cell lysates of the same cell model. Whole-cell lysates were prepared from N2 and ML2 cells, directly digested by trypsin and subjected to LC-MS/MS as described in materials and methods. Three nano-LC-MS/MS runs of tryptic digests showed that a significant number of total proteins (310 for N2 and 280 for ML2) could be detected using this simple nonfractionation approach (see supplemental Table 4). When analyzed for protein content, we found that 3.8% of the N2 proteome and 2.9% of the ML2 proteome identified from this global approach were annotated as glycoproteins using the UniProt database. In comparison, our targeted approach yielded 9.3% and 13.5% respectively (Fig. 4). Although the use of the UniProt database to determine if a protein is glycosylated is notoriously under-representative, a fourfold enrichment was revealed. Most striking was that 10 of 12 internal glycoproteins, found to be abundant and identified in the targeted approach, were not identified in the whole-cell proteome approach.

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