Oral Presentation 26th Annual Lorne Proteomics Symposium 2021

Investigating the role of histidine methylation on ribosome assembly using quantitative large-scale cross-linking mass spectrometry (#43)

Tara K Bartolec 1 , Joshua J Hamey 1 , Andrew Keller 2 , Juan D Chavez 2 , James E Bruce 2 , Marc R Wilkins 1
  1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Randwick, NSW, Australia
  2. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

Cross-linking mass spectrometry (XL-MS) involves the use of chemicals which covalently link amino acids within spatially constrained reactions. Recent technological advances have enabled XL-MS of complex samples and our group generated the first large-scale XL-MS dataset for yeast 1. This revealed novel insights into the protein interactome and unveiled the conformations of hundreds of unresolved protein structures. Studies of protein translation have revealed mounting evidence that ribosomes are both highly regulated and heterogeneous in structure, composition, interactions and post-translational modifications, making them an ideal system to explore using XL-MS. To investigate the function of one form of heterogeneity, protein methylation, we applied quantitative large-scale XL-MS to study methyl-histidine-243 of ribosomal protein uL3, which is deposited by the Hpm1 methyltransferase. 

Wild-type and Hpm1 knockout yeast were cultured using forward/reverse SILAC. Intact spheroplasts were crosslinked with the biotin-containing and MS-cleavable crosslinker PIR. Peptides were fractionated by SCX, enriched with avidin and analysed with stepped-HCD. Crosslinks were identified using the Mango/Comet/XlinkProphet pipeline, and crosslink abundance ratios extracted using MethylQuant/MassChroQ. This resulted in 8481 crosslink spectral matches at a 1% FDR, which represent 1268 unique lysine-lysine pairs. Over half of these were successfully quantified, with a significant proportion quantified in both biological replicates. Many of the detected changes were within or between ribosomal proteins/members of co-translational processes, indicating an altered ribosome structure/interactome. Interesting, we also detected large-scale yet reproducible changes in seemingly unrelated domains including the mitochondria and histones which may reveal unexplored aspects of Hpm1 biology.

This study is one of the few to date to perform differential interactomics analysis using in vivo quantitative large-scale XL-MS, and the first in yeast. It illustrates how this technique can be used to profile the diverse impacts of cellular perturbation, even as small as modulating a single methylation site.

  1. Cross-linking Mass Spectrometry Analysis of the Yeast Nucleus Reveals Extensive Protein–Protein Interactions Not Detected by Systematic Two-Hybrid or Affinity Purification-Mass Spectrometry. Tara K. Bartolec, Daniela-Lee Smith, Chi Nam Ignatius Pang, You Dan Xu, Joshua J. Hamey, and Marc R. Wilkins. Analytical Chemistry 2020 92 (2), 1874-1882. DOI: 10.1021/acs.analchem.9b03975