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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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Related in: MedlinePlus

Subcellular location of identified proteins. The cellular location was determined by cross-referencing the annotations from UniProtKB. Each protein was designated with only one subcellular location. The protein classes are shown as percent of total protein on the x axis. This analysis was conducted for both the surface glycoprotein and global methodologies.
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Figure 5: Subcellular location of identified proteins. The cellular location was determined by cross-referencing the annotations from UniProtKB. Each protein was designated with only one subcellular location. The protein classes are shown as percent of total protein on the x axis. This analysis was conducted for both the surface glycoprotein and global methodologies.

Mentions: We next conducted a comparative analysis of the two approaches with specific emphasis on which cellular compartment the proteins were derived. Fig. 5 displays the UniProt-designated subcellular distribution of proteins identified in the global proteomic approach and the surface sialoglycoprotein targeted approach. We observed that about 13.2% of proteins from the whole-cell analysis of N2 and ML2 were classified as being extracellular and membrane-bound. This result is consistent with studies using whole-cell approaches to examine breast cancer cell (33) and ovarian cancer cell proteome (34). In contrast, our cell-surface glycoprotein approach resulted in a proteome for N2 and ML2 with 25.9% and 29.8% of proteins being secreted or cell-surface proteins, respectively. These cell-surface proteins include cluster differentiation markers, cell surface receptors, membrane transport, and cell adhesion proteins, such as CD44, CD166, integrins, anexins, calnexin, etc. In contrast, a small portion of proteins identified using our targeted approach were classified as having endoplasmic reticulum/Golgi location (∼2%). A large portion of proteins were classified as intracellular (>50%), either cytoplasm or nucleus, whereas 18 proteins for N2 and 13 proteins for ML2 remain unclassified. Recently, Sardana et al. conducted a cellular “secretome” analysis in which the conditioned medium of three prostate cancer cell lines (including PC3) was examined. They identified ∼2000 proteins of which 12% were classified as known extracellular proteins (35). Our surface glycoprotein approach observed 32 proteins that were in common with this “secretome” study and that 85% of these were classified as membrane proteins. These findings underscore the value of our targeted strategy to capture and isolate cell-surface glycoproteins.


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)

Subcellular location of identified proteins. The cellular location was determined by cross-referencing the annotations from UniProtKB. Each protein was designated with only one subcellular location. The protein classes are shown as percent of total protein on the x axis. This analysis was conducted for both the surface glycoprotein and global methodologies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Subcellular location of identified proteins. The cellular location was determined by cross-referencing the annotations from UniProtKB. Each protein was designated with only one subcellular location. The protein classes are shown as percent of total protein on the x axis. This analysis was conducted for both the surface glycoprotein and global methodologies.
Mentions: We next conducted a comparative analysis of the two approaches with specific emphasis on which cellular compartment the proteins were derived. Fig. 5 displays the UniProt-designated subcellular distribution of proteins identified in the global proteomic approach and the surface sialoglycoprotein targeted approach. We observed that about 13.2% of proteins from the whole-cell analysis of N2 and ML2 were classified as being extracellular and membrane-bound. This result is consistent with studies using whole-cell approaches to examine breast cancer cell (33) and ovarian cancer cell proteome (34). In contrast, our cell-surface glycoprotein approach resulted in a proteome for N2 and ML2 with 25.9% and 29.8% of proteins being secreted or cell-surface proteins, respectively. These cell-surface proteins include cluster differentiation markers, cell surface receptors, membrane transport, and cell adhesion proteins, such as CD44, CD166, integrins, anexins, calnexin, etc. In contrast, a small portion of proteins identified using our targeted approach were classified as having endoplasmic reticulum/Golgi location (∼2%). A large portion of proteins were classified as intracellular (>50%), either cytoplasm or nucleus, whereas 18 proteins for N2 and 13 proteins for ML2 remain unclassified. Recently, Sardana et al. conducted a cellular “secretome” analysis in which the conditioned medium of three prostate cancer cell lines (including PC3) was examined. They identified ∼2000 proteins of which 12% were classified as known extracellular proteins (35). Our surface glycoprotein approach observed 32 proteins that were in common with this “secretome” study and that 85% of these were classified as membrane proteins. These findings underscore the value of our targeted strategy to capture and isolate cell-surface glycoproteins.

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