Altered Expression of Sialylated Glycoproteins in Breast Cancer Using Hydrazide Chemistry and Mass Spectrometry

Abstract

Sialylation is one of the altered protein glycosylations associated with cancer development. The sialoglycoproteins in cancer cells, however, largely remain unidentified because of the lack of a method for quantitative analysis of sialoglycoproteins. This manuscript presents a high throughput method for quantitative analysis of N-linked sialoglycoproteins using conditional hydrazide chemistry, liquid chromatography, and tandem mass spectrometry. We further applied the sialoglycoproteomic method to the profiling of breast cancer tissues and compared findings with the results from the total glycoproteomic analysis using the original hydrazide chemistry method. We identified altered expression of sialoglycoproteins, as well as the total glycoprotein changes associated with breast cancer. Using lectin and Western blot analysis, we characterized one of the sialoglycoproteins, versican, and confirmed that versican was most sialylated and elevated in breast cancer. Furthermore, we showed that versican was detected in both cancer epithelial cells and peritumoral stromal cells using immunohistochemistry. Tissue microarray analysis revealed that epithelial expression of versican had significant relations to lymph node metastasis and pathological stages. This is the first quantitative sialoglycoproteomic and glycoproteomic analysis of breast cancer and noncancerous tissues. These findings present a significant addition of the method to the identification of altered expression of sialylated glycoproteins associated with breast cancer development. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.011403, 1-10, 2012.Aberrant protein glycosylations are known to be associated with tumorigenesis and cancer progression steps including oncogenic transformation, tumor invasion, and metastasis (1). In breast cancer, elevated concentrations of highly glycosylated proteins, such as mucins, are associated with increased tumor burden and poor prognosis (2). Several glycosylation changes including sialylation, fucosylation, increased branching of N-glycans, and incomplete biosynthesis resulting in truncated glycans are commonly found in cancer (3). By existing as terminal sugars on glycans attached to proteins and lipid moieties, sialic acids play critical roles in intermolecular interactions and the formation of cellular characteristics (4). Altered expression of sialylated glycoproteins has been discovered in many carcinomas such as colon, acute myeloid, leukemia, cervix, and brain tumors (5-14). Furthermore, partial removal of sialic acids on cell surface has also been shown to increase both cell adhesion and aggregation in the pancreatic cancer cells (15), which confirmed that sialylation indeed contributes to cell metastasis.Recent development in MS technology has fueled high throughput analyses of glycoproteins (16,17). Current strategies in studying glycoproteins generally start with enrichment of glycoproteins or glycopeptides from complex mixtures using different physical-chemical methods followed by identification and quanti...

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine

“…Apart from the variations occurred in the protein cores of versican due to alternative splicing, versican exhibits significant structural alterations on its glycosylation in various tumors [37–39]. In breast cancer, versican is differentially glycosylated, containing more sialic acid [40]. In most cases stromal cells are the main source of versican in tumor stroma although some cancer cells can synthesize versican themselves.…”

“…Disruption of the HA–CD44 interaction with HA oligomers may be used for targeting tumor progression making HA oligomers promising inhibitors of cancer dissemination [370]. Furthermore, a novel versican isoform V4 is highly expressed in breast cancer [36], whereas versican is also differentially glycosylated in breast cancer because it contains more sialic acid [40]. This alternative splice variant of versican or the presence of unusual glycosylation may comprise possible targets for therapeutic intervention in breast cancer with antibody-related agents.…”

Metabolic Flux Increases Glycoprotein Sialylation: Implications for Cell Adhesion and Cancer Metastasis

“…More specifically, to identify individual proteins whose sialylation status was impacted by analog-driven flux, the formerly Nlinked total glycopeptides and sialoglycopeptides were isolated from cells that had been pretreated or not with 1,3,4-OBu 3 ManNAc, labeled with iTRAQ reagents, and analyzed by mass spectrometry (13)(14)(15)22). Two proteins, CD44 and integrin ␣6, were used as examples to illustrate this process, as shown in Fig.…”

Post-translational Modifications and Mass Spectrometry Detection

“…To overcome this limitation, a strategy based on mild periodate oxidation was developed for the characterization of sialic acid-containing glycopeptides (150). With this procedure it is possible to maintain the glycan structure stable with the exception of sialic acid moieties (151). For the characterization of Oglycoproteins, lectins have been largely exploited (152).…”