Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry

Jianliang Zhou, Weiming Yang, Yingwei Hu, Naseruddin Höti, Yang Liu, Punit Shah, Shisheng Sun, David Clark, Stefani Thomas, Hui Zhang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)7623-7630
Number of pages8
JournalAnalytical Chemistry
Volume89
Issue number14
DOIs
StatePublished - Jul 18 2017

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Glycopeptides
Mass spectrometry
Lectins
Glycoproteins
Cells
Proteins
Chromatography
Labeling

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry. / Zhou, Jianliang; Yang, Weiming; Hu, Yingwei; Höti, Naseruddin; Liu, Yang; Shah, Punit; Sun, Shisheng; Clark, David; Thomas, Stefani; Zhang, Hui.

In: Analytical Chemistry, Vol. 89, No. 14, 18.07.2017, p. 7623-7630.

Research output: Contribution to journalArticle

Zhou, Jianliang ; Yang, Weiming ; Hu, Yingwei ; Höti, Naseruddin ; Liu, Yang ; Shah, Punit ; Sun, Shisheng ; Clark, David ; Thomas, Stefani ; Zhang, Hui. / Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry. In: Analytical Chemistry. 2017 ; Vol. 89, No. 14. pp. 7623-7630.
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abstract = "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.",
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AU - Yang, Weiming

AU - Hu, Yingwei

AU - Höti, Naseruddin

AU - Liu, Yang

AU - Shah, Punit

AU - Sun, Shisheng

AU - Clark, David

AU - Thomas, Stefani

AU - Zhang, Hui

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