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

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.