title: Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift.
abstract: We reported an improved combinatorial approach for identifying site-specific O-glycosylation using both glycan cleaved and non-cleaved methods. In this approach, a non-reducing β-elimination kit coupled with non-specific enzymes performed efficient digestion, O-glycan cleavage, and partial dephosphorylation without significant side reactions, thus enabling an automatic database search for the cleaved O-glycosylation or serine/threonine (S/T) phosphorylation sites. From the same sample concurrently prepared without β-elimination, the corresponding intact O-glycopeptides were mapped by accurate precursor ion mass using an in-house glycan database majorly composed of GalNAc (mucin-type) core and the retention-time shift (ΔRt). Each glycopeptide assignment was verified by the detection of glycan-specific fragments using collision-induced dissociation (CID) to estimate False Discovery Rate (FDR). Using fetuin as a model, all identified S/T elimination sites were matched to multiple intact glycopeptides with a 31% FDR. This considerably reduced to 0% FDR by ΔRt filtering. This approach was then applied to a protein mixture composed of therapeutic Factor IX and Enbrel(®) mixed with fetuin and kappa-casein. A total of 26 glycosylation sites each of which corresponds to 1-4 glycans were positively mapped and confirmed. The FDR decreased from 33% to 3.3% by ΔRt filtering and exclusion of repeated peptide tags that covered the same glycosylation sites. Moreover, the phosphorylation and O-glycosylation on the same site such as T159 of Factor IX and T170 of kappa-casein were able to be unambiguously differentiated. Thus, our approach is useful for in-depth characterization of site-specific O-glycosylation of a simple mixture such as protein-based therapeutics.
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Huang
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Jen-Hui
authors.initials: JH
authors.lastname: Lin
authors.affiliation: Department of Communication Engineering, National Central University, Jhongli, Taiwan.
authors.forename: Jung-Heng
authors.initials: JH
authors.lastname: Tsai
authors.affiliation: Department of Communication Engineering, National Central University, Jhongli, Taiwan.
authors.forename: Yen-Yin
authors.initials: YY
authors.lastname: Chu
authors.affiliation: Department of Communication Engineering, National Central University, Jhongli, Taiwan.
authors.forename: Yen-Wen
authors.initials: YW
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Shun-Li
authors.initials: SL
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan. Electronic address: shchen@mail.ncku.edu.tw.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011134:Polysaccharides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002364:Caseins; D006020:Glycopeptides; D011134:Polysaccharides
keywords: Enbrel(®); LC–MS; O-glycosylation; O-glycosylation site; β-Elimination