Poster Presentation 25th Annual Lorne Proteomics Symposium 2020

High-confidence identification of protein methylation in Giardia duodenalis, a neglected gastrointestinal parasite (#101)

Samantha J Emery-Corbin 1 , Joshua J Hamey 2 , Marc R Wilkins 2 , Aaron R Jex 1 3
  1. Walter and Eliza Hall Institute, Parkville, VICTORIA, Australia
  2. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
  3. Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, Victoria, Australia

Protein methylation coordinates epigenetic gene expression and drives cell development. For many human parasites, limited drug classes and resistance to frontline drugs mean new avenues for inhibiting parasite life-cycles are urgently required. Methyltransferase enzymes represent prime drug targets in Giardia— the most common human gastrointestinal parasite causing diarrheal disease in 200 million people worldwide. Giardia is a unique model for protein methylation; this deep-branching protist has a diverged lysine methylome (K-Me) and is the first eukaryote with no arginine methylome.

We acknowledge large-scale methyl-site identifications using LC-MS/MS are subject to high false discovery rates (FDR) in methylpeptide spectrum matches (methyl-PSMs)1. Ideally, ‘true’ methyl-sites are identified by orthogonal methyl-peptide identification through isotopic labelling of methylation sites via heavy-methyl SILAC. These are not feasible for non-model organisms and parasites which have limited in vitro cultures and/or undefined media. Therefore, we implemented new methyl-site confidence-filtering pipelines in Giardia. Filtering is implemented post-database searching with Maxquant2, complimented by avoiding methanol- and SDS-based sample preparation (which introduce methyl-artefacts1), and high-mass accuracy peptide fragmentation with higher-energy collisional dissociation (HCD). Filtering removes common sources of false positive methyl-PSMs, and retains sites with robust MS/MS evidence. We validated methyl-site filters with mouse controls, demonstrating filtering enriches known (‘true’) mouse methyl-sites.

These confidence filters identified over 200 K-Me sites in 160 proteins in Giardia. These Giardia K-Me proteins are mostly species- and lineage-specific with only 58/160 and 40/160 shared orthologs in H. sapiens and S. cerevisiae, respectively. Many K-Me proteins in Giardia specific gene-families were associated with cytoskeletal regulation. Significant enrichment for coiled-coil features demonstrated many K-Me sites specifically occurred within coiled-coil heptads in solvent-facing positions, with intriguing implication for regulation of this versatile protein folding motif.


  1. Hart-Smith G, Yagoub D, Tay AP, Pickford R, Wilkins MR. Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates. Mol Cell Proteomics 15, 989-1006 (2016).
  2. Tyanova S, Temu T, Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11, 2301-2319 (2016).