Fucosylation (Fuc) of glycoproteins plays an important role in regulating protein function and has been associated with the development of several cancer types including prostate cancer (Pca). Therefore, the research of Fuc glycoproteins has attracted increasing attention recently in the analytical field. Herein, a strategy based on lectin affinity enrichments of intact glycopeptides followed by mass spectrometry has been established to evaluate the specificities of various Fuc-binding lectins for glycosite-specific Fuc analysis of nonaggressive (NAG) and aggressive (AG) Pca cell lines. The enrichment specificities of Fuc glycopeptides using lectins (LCA, PSA, AAL, LTL, UEA I, and AOL) and MAX extraction cartridges alone, or in tandem, were evaluated. Our results showed that the use of lectin enrichment significantly increased the ratio of fucosylated glycopeptides to total glycopeptides compared to MAX enrichment. Furthermore, tandem use of lectin followed by MAX increased the number of identifications of Fuc glycopeptides compared to using lectin enrichment alone. LCA, PSA, and AOL showed stronger binding capacity than AAL, LTL, and UEA I. Also, LCA and PSA bound specifically to core Fuc, whereas AOL, AAL, and UEA I showed binding to both core Fuc and branch Fuc. The optimized enrichment method with tandem enrichment of LCA followed by MAX (LCA-MAX) was then applied to examine the Fuc glycoproteomes in two NAG and two AG Pca cell lines. In total, 973 intact Fuc glycopeptides were identified and quantified from 252 Fuc proteins by using the tandem-mass-tags (TMT) labeling and nanoliquid chromatography-mass spectrometry (nanoLC-MS/MS) analysis. Further data analysis revealed that 51 Fuc glycopeptides were overexpressed more than 2-fold in AG cell lines compared to NAG cells. The analysis of protein core fucosylation has great potential for aiding our understanding of invasive activity of AG Pca and may lead to the development of diagnostic approaches for AG Pca.
Bibliographical noteFunding Information:
We thank assistance from Dr. Guizhong Xin from China Pharmaceutical University for the OPLS-DA analysis. This work was supported by the National Institutes of Health, National Cancer Institute, the Early Detection Research Network (EDRN, Grant U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC, Grant U24CA160036), National Heart Lung and Blood Institute, Programs of Excellence in Glycosciences (PEG, Grant P01HL107153), and the National Institute of Allergy and Infectious Diseases (Grant R21AI122382) and by amfAR, The Foundation for AIDS Research on Bringing Bioengineers to Cure HIV (Grant amfAR 109551-61-RGRL), by Maryland Innovation Initiative (MII), and by The Patrick C. Walsh Prostate Cancer Research Fund.
© 2017 American Chemical Society.