Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift

Li Juan Huang, Jen Hui Lin, Jung Heng Tsai, Yen Yin Chu, Yen Wen Chen, Shun Li Chen, Shu Hui Chen

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)136-145
Number of pages10
JournalJournal of Chromatography A
Volume1371
DOIs
Publication statusPublished - 2014 Dec 5

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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