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 AC4ManNAz 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.
Highlights
These methods are effective at the enrichment and identification of broad classes of glycoproteins/glycopeptides, they still lack the specificity and selectivity required for analysis of specific cell surface glycoproteins that could serve as potential cancer biomarkers
Metastatic spread of cancer involves tumor cell extravasation and subsequent invasion of surrounding tissue, all processes that are tightly regulated by cell surface mechanisms [37, 38]
The identification of surface-accessible glycoproteins, which are altered in the metastatic process, will help to unfold the underlying biological events that support cancer metastasis
Summary
Materials—CompleteTM protease inhibitors were purchased from Roche Applied Sciences (Indianapolis, IN), sequencing grade trypsin was from Promega (Madison, WI), and Immobilon-FL PDVF membrane was from Millipore (Billerica, MA). Flow Cytometric Analysis of Cell-surface Sialic Acid Labeling— After metabolic labeling, N2 and ML2 cells were harvested, washed with 0.1% FBS/phosphate-buffered saline (PBS), and resuspended (106 cells) in 100 l click reaction solution with the indicated amount of each component. Membranes were blocked in LiCor blocking buffer (Rockland Immunochemicals, Gilbertsville, PA) diluted with PBS (1:1), incubated with anti-CDCP1 polyclonal (#4115, 1:1000, Cell Signaling Technology, Danvers, MA), or anti-integrin 1 mouse monoclonal (#610467, 0.1 g/ml, BD PharMingen, San Diego, CA) primary antibodies overnight at 4 °C. To generate the lists of cell-surface glycoproteins uniquely expressed in N2 or ML2 cells, identified proteins were required to meet the following 3 criteria: (a) identified from one cell line; (b) unique peptide Ն 1, and NXS/T motif Ն 1 or UniProt indicated N-linked or O-linked glycosylation; and (c) transmembrane region Ն 1, or UniProt membrane subcellular location. The list of identified proteins that were unique to each experimental group was subjected to the commercially available curator database software Ingenuity Pathways Analysis (IPA) to determine their molecular function and interacting networks
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