Department of Pathology, Johns Hopkins University , Baltimore 21287, Maryland United States.
Department of Traditional Chinese Medicines, Zhejiang Institute for Food and Drug Control , Hangzhou 310052, China.
Anal Chem. 2017 Jul 18;89(14):7623-7630. doi: 10.1021/acs.analchem.7b01493. Epub 2017 Jul 3.
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.
糖基化（Fuc）在调节蛋白质功能中起着重要作用，并且与几种癌症类型的发展有关，包括前列腺癌（Pca）。因此，近年来，糖基化蛋白的研究在分析领域受到了越来越多的关注。在此，建立了一种基于凝集素亲和富集完整糖肽，然后进行质谱分析的策略，用于评估各种 Fuc 结合凝集素对非侵袭性（NAG）和侵袭性（AG）Pca 细胞系糖基化特异性 Fuc 分析的特异性。单独或串联使用凝集素（LCA、PSA、AAL、LTL、UEA I 和 AOL）和 MAX 提取试剂盒评估 Fuc 糖肽的富集特异性。我们的结果表明，与 MAX 富集相比，使用凝集素富集显著增加了糖基化糖肽与总糖肽的比率。此外，与单独使用凝集素富集相比，串联使用凝集素后再使用 MAX 增加了 Fuc 糖肽的鉴定数量。LCA、PSA 和 AOL 比 AAL、LTL 和 UEA I 具有更强的结合能力。此外，LCA 和 PSA 特异性结合核心 Fuc，而 AOL、AAL 和 UEA I 显示与核心 Fuc 和支链 Fuc 均有结合。然后，将优化的富集方法与 LCA 串联富集，然后再用 MAX（LCA-MAX）应用于两种 NAG 和两种 AG Pca 细胞系的 Fuc 糖蛋白组学检测。通过使用串联质量标记（TMT）标记和纳升液相色谱-质谱（nanoLC-MS/MS）分析，从 252 个 Fuc 蛋白中鉴定和定量了 973 个完整的 Fuc 糖肽。进一步的数据分析表明，与 NAG 细胞相比，51 个 Fuc 糖肽在 AG 细胞系中的表达量增加了 2 倍以上。蛋白核心岩藻糖基化的分析对于帮助我们理解 AG Pca 的侵袭活性具有很大的潜力，并可能导致开发用于 AG Pca 的诊断方法。
