Translational proteomics experiments are designed to perform qualitative and quantitative analysis on all proteins across samples. Data acquisition and processing strategies are required to confidently generate a refined list of statistically relevant proteins to drive appropriate study-relevant conclusions. Key workflow aspects to ensure reproducible data acquisition and generalizable conclusions include: optimized, reproducible sample preparation, efficient acquisition strategies, accurate analyte identification, and precise quantification. The ideal method to evaluate these aspects involves synthetic peptides to determine workflow precision and accuracy.<br/>For translational experiments involving glycoproteomics, we focused on the following glycosylation in biological samples: O-GalNAc, Gal-GalNAc, O-GlcNAc, S-ManNAc, and S-GlcNAc. We addressed the following limitations for development of qualitative and quantitative approaches. No glycopeptide synthetic standards exist for O-glycosylation identification and quantitation; we synthesized a library of 40 glycopeptides with variations of GalNAc or Gal-GalNAc on Serine or Threonine, O-S-linked GlcNAc/GalNAc and S-linked ManNAc peptides. We included an N-glycopeptide in the repertoire to address another limitation for translational workflows: sample preparation for O-glycopeptide enrichment from complex mixtures. Our method for O-glycopeptide enrichment employed strong anion exchange, anti-GlcNAc antibody pull-downs, and Retain AX-cartridges. This approach was effective for samples containing 200 micrograms total protein and offered advantages over lectin capture approaches requiring significantly higher protein concentrations. A range of issues with this antibody routinely used for pull-down experiments has been addressed in our work using microarray technology and LC-MSMS. Lack of fragment ions to distinguish between different forms of O-glycosylation is another limitation that we have addressed in our work. The glycan oxonium ion 204 (HexNAc) is common to these O-glycosylated peptides; the ratios of the oxonium ion fragments are used in our work to distinguish the different types of O-glycosylation.<br/>We will highlight the advantages of using both microarray technology and LC-MSMS for optimizing GlcNAc and GalNAc glycosylation in our work.

Systems biology approaches for O-linked glycosylation using LC-MS/MS and microarray technologies

256th National Meeting (2018)

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