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

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.

Site‐ and Structure‐Specific Quantitative N‐Glycoproteomics Using RPLC‐pentaHILIC Separation and the Intact N‐Glycopeptide Search Engine GPSeeker

“…After labeling, the intact N-glycopeptides are enriched by zicHILIC according to reported procedures (Liu et al, 2017;Xue et al, 2018) with minor modifications.…”

“…Selective fragmentation of both the peptide backbone and the N-glycan moiety (Banazadeh, Veillon, Wooding, Zabetmoghaddam, & Mechref, 2017;Nilsson, 2016) can be achieved with different methods (such as CID and ETD; Scott et al, 2011) or with different energies of the same method (such as HCD with stepped NCEs; Cao et al, 2014;Liu et al, 2017).…”

“…The past few years have brought rapid developments in site-specific N-glycoproteomics analysis of intact N-glycopeptides providing information about the amino acid sequence of the peptide backbone as well as the N-glycosite and monosaccharide composition of the N-glycan moiety. Large-scale characterization of differential N-glycosylation in various diseases (Kontro, Joenvaara, Haglund, & Renkonen, 2014;Shah et al, 2015;Zhou et al, 2017;Zhu et al, 2019) has been successfully carried out using various bioinformatics tools, such as GPFinder 3.0 (Strum et al, 2013), Glycopeptide Search (Chandler, Pompach, Goldman, & Edwards, 2013), MAGIC (Lih et al, 2016), I-GPA (Park et al, 2016), Byonic (Zhou et al, 2017), pGlyco 2.0 (Liu et al, 2017), SugarQb (Stadlmann et al, 2017), GPQuest 2.0 (Hu, Shah, Clark, Ao, & Zhang, 2018), MoFi (Skala, Wohlschlager, Senn, Huber, & Huber, 2018), and Mascot (Bollineni, Koehler, Gislefoss, Anonsen, & Thiede, 2018). However, fragmentation of N-glycan moieties was not or only partially interpreted, and putative N-glycan structures together with their monosaccharide compositions were only sometimes reported.…”

Comparison of three glycoproteomic methods for the analysis of CHO cells treated with 1,3,4-O-Bu3ManNAc

“…Because each glycoprotein can exhibit multiple glycosylation sites (glycosites) and each glycosite can exhibit a number of glycan structures, glycosylation is also the most complex modification, with the potential for a single protein isoform to exhibit thousands of glycosylation combinations (2). Further, specific glycan structures at specific glycosites have been associated with altered function and disease (3), and thus comprehensive systematic analysis is both challenging and crucial.…”

Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides

“…However, information related to both glycosites and the attached glycans at specific glycosylation sites need be determined. More recently, analyses of intact glycopeptides have been used to study glycosylation heterogeneity from a specific glycoprotein or complex samples [18][19][20][21][22][23][24] Recently, several software tools 25 have been developed to assist the assignment of glycopeptides, such as Byonic 26 , Glycopeptide Search (GPS) 27 , Glycopep Grader 28 , SpectraST 29 , GPQuest 30 , and pGlyco 31,32 , allowing for high throughput identification of intact glycopeptides.…”