Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Deglycosylation of glycoproteins with trifluoromethanesulfonic acid (TFMS): unravelling the chemistry allows extension of the protocol to the analysis of released glycan chains using standard proteomics methods (#74)

Paul D Veith 1 , Richard AJ O'Hair 2 , Michael G Leeming 3 , Shuai Nie 3 , Gavin E Reid 2 4 , Eric C Reynolds 1
  1. Melbourne Dental School, Bio21 Institute, University of Melbourne, Parkville, Victoria, Australia
  2. School of Chemistry, BIO21 Institute, University of Melbourne, Parkville, Victoria, Australia
  3. Mass Spectrometry and Proteomics Facility, Bio21 Institute, University of Melbourne, Parkville, Victoria, Australia
  4. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

During the deglycosylation of glycoproteins with TFMS under anhydrous conditions, the glycan chains are cleaved but the protein remains intact. It is therefore used widely to analyse the deglycosylated protein and the site of glycosylation. There have been some reports of detecting N-acetylated sugars in the released glycans, but in general, analysis of the released glycans has not been reported, presumably due to a lack of understanding of the chemistry associated with the deglycosylation process. In our extensive MS analyses of glycoprotein virulence factors of the bacterial pathogen, Porphyromonas gingivalis we discovered that the toluene or other arene which is included in the TFMS reaction reacts with reducing sugars to form a 1,1-diaryl-1-deoxyalditol. The reaction goes to near completion allowing the expected products to be readily detected by MS at abundances much higher than intermediates and side-products. The di-aryl addition allows the modified glycans to bind to C18 columns and hence the reaction products can be washed and concentrated using C18 solid phase extraction kits and analysed by LC-MS. Furthermore, glycan chains that have the di-aryl addition produce a reliable diagnostic ion at m/z 209.13 (C16H17) which aids in their identification. While the chemistry was thought to be specific to the C-1 of reducing sugars, the arenes appear to also react with non-reducing sugars such as fructose and analyses are continuing to determine if the reaction products are reproducibly different. Sugars that are N-acetylated at C-2 are also cleaved and react with arenes producing additional diagnostic ions. The unravelling of this chemistry significantly extends the utility of the TFMS deglycosylation procedure by providing additional information on the released glycans.