Altered expression of sialylated glycoproteins in breast cancer using hydrazide chemistry and mass spectrometry.
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.
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Yuan
Y
Tian
Francisco J
FJ
Esteva
Jin
J
Song
Hui
H
Zhang
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002846:Chromatography, Affinity; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D008207:Lymphatic Metastasis; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009961:Orosomucoid / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D046888:Tissue Array Analysis; D053675:Versicans / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D006834:Hydrazines; D009961:Orosomucoid; D010446:Peptide Fragments; C508322:VCAN protein, human; D053675:Versicans; D019158:N-Acetylneuraminic Acid