Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Strategic applications of data dependent and targeted multistage fragmentation in dual modes to expedite mass spectrometry-based sequencing of glycopeptides (#135)

Kay-Hooi Khoo 1
  1. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

While glycomics aims at mapping the entire range of glycan entities expressed at any one patho-physiological stage of a particular cell type, tissue or organism, glycoproteomics ventures a step further in locating their protein site-specific distribution. Although recent advances in mass spectrometry (MS) have enabled meaningful glycoproteomic undertakings, many technical limitations remain unsolved. Among these, ability to efficiently sequence the peptide backbone for de novo identification, delineating multiple N- and O-glycosylation sites on single glycopeptides and deriving more glycan structure information to discriminate isomeric glycoforms, are well acknowledged practical problems to be urgently tackled. We have been exploring all different kinds of experimental workflows for a most productive LC-MS/MS-based glycopeptide analysis, focusing particularly on the multiple glycosylated peptides and those carrying sulfated glycans. We have examined the complementarity of various fragmentation modes and most recently the judicious applications of negative ion mode sequencing to supplement a robust positive mode LC-MS2/MS3 workflow. We found that sulfation and sialylation drastically alter the fragmentation pattern in negative ion mode and the characteristic features identified can be utilized to program the most informative MS3. Moreover, facile elimination of the O-glycan moieties under MS2 affords an easy way to discover additional O-glycosylation on a glycopeptide that would otherwise not obvious. By high sensitivity detection of the characteristic glycan fragmentation ions produced by N- or O-glycans in combination with readily distinguishable elimination of the entire O-glycans in negative ion mode, we demonstrated in this work that unambiguous MS/MS sequencing of intact glycopeptides could be extended to those bearing both N- and O-glycans, or multiple O-glycans. Ability to advance from definitive identification of single to multiple site-specific glycosylation pattern on the same peptide backbones is anticipated to have a significant impact on the level of structural and biological insights one can gain in glycoproteomics applications.