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.
Highlights
Aberrant protein glycosylations are known to be associated with tumorigenesis and cancer progression steps including oncogenic transformation, tumor invasion, and metastasis [1]
A Proof of Concept Study of Sialylated Glycopeptide Isolation Using a Modified solid phase extraction of glycopeptides (SPEG) Method—To identify proteins preferentially sialylated in breast cancer tissues, the sialylated glycopeptides were isolated
We used a low concentration of sodium periodate to selectively oxidize the terminal sialic acids of sialoglycopeptides to aldehydes followed by immobilization of oxidized glycopeptides to solid support [30] and released the formerly N-linked sialoglycopeptides by PNGase F
Summary
The following materials were used: hydrazide resin (Bio-Rad); sodium periodate (Bio-Rad); PNGase F (New England Biolabs, Ipswich, MA); sequencing grade trypsin (Promega, Madison, WI); Sep-Pak Vac C18 cartridge (Waters, Milford, MA); BCA assay kit (Pierce); ␣-Cyano4-hydroxycinnamic acid (CHCA) (Agilent, Palo Alto, CA); neuraminidase (sialidase) (Roche Applied Science); agarose-bound Sambucus nigra lectin (SNA) (Vector Laboratories, Burlingame, CA); rabbit polyclonal anti-human versican (Abcam, Cambridge, MA); monoclonal antibody 2B1 against versican (Seikagaku, Tokyo, Japan); EnVisionϩ system horseradish peroxidase (diaminobenzidine, DAB) (DakoCytomation, Kjelsas, Oslo, Norway); negative control mouse IgG (DakoCytomation, Kjelsas, Oslo); SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Rochford, IL); and human serum and other chemicals (Sigma-Aldrich). ABI 4800 MALDI-TOF/ TOF analyzer was from Applied Biosystems (Foster City, CA). The LTQ ion trap mass spectrometer was from Thermo Finnigan (San Jose, CA)
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