Atsuhiko ‘Ash’ Toyama, Shimadzu Corporation, Singapore/Japan
Chris Bowen, Shimadzu Scientific Instruments, Australia
Two presentations will be given covering the following topics:
“Breaking the sensitivity barrier to unravelling the novel roles of lipid mediators in pathophysiology” by Ash Toyama, Ph.D.
1) A brief overview of Shimadzu’s targeted omics solution portfolio, each addressing the difficulty of the targeted class of compounds by unique approaches.
2) The recent progress using the LC/MS/MS Lipid Mediators Method Package in a collaborative work with the University of Tokyo, illustrating that method reproducibility is the most critical factor for delivering scientifically meaningful outcome.
3) Tools to assist data processing and interpretation
“The Q-TOF technologies to reproduce the benefits of targeted workflows in high-resolution analyses” by Chris Bowen
1) Ultra-fast data acquisition and ion accumulation
2) UFgrating and iRefTOF – simple ideas made possible by meticulous engineering
3) Flight tube thermostability for exceptionally stable mass measurement accuracy
4) DIA capabilities
5) Nexera Mikros, the microflow solution with great ease
Dissecting the roles of glycosylation on bacterial proteins within the Burkholderia genus
Dr Nichollas Scott
Department of Microbiology and Immunology, The University of Melbourne
Bio: Dr Scott completed his PhD at the University of Sydney (2007 to 2012) on the development of MS approaches for the study of bacterial protein modifications. The key focus of his doctorate was the establishment of a protocol for enriching bacterial glycopeptides irrespective of glycan composition. In 2012 with the aid of a NHMRC Early Career Fellowship he moved to the University of British Columbia to undertake a postdoc with Prof Leonard Foster. Within the Foster lab Dr Scott began applying quantitative proteomics approaches to study protein interactions and bacterial glycosylation systems at a systems level. From 2012 to 2015 Dr Scott spearheaded projects to characterise bacterial glycosylation systems across Gram-negative pathogens including, Acinetobacter baumannii, Burkholderia cenocepacia and Ralstonia solanacearum. In 2016 Dr Scott returned to Australia where he completed a second postdoc in the laboratory of Prof Elizabeth Hartland, developing novel enrichment approaches to track Arginine-glycosylation. In 2017, on the back of two national awards from the Australasian Proteomics Society (the 2016 International Early Career Award & the 2017 Ken Mitchelhill Award), NHMRC and ARC funding Dr Scott established his independent research group in the Department of Microbiology and Immunology at the University of Melbourne. Since 2017 Dr Scott’s lab has sought to identify and track microbial glycosylation events to understand the role of glycosylation in microbial pathogenesis. In 2019 in recognition of his contribution to the field of glycoconjugate research Dr Scott was awarded the 2019-IGO Young Glycoscientist Award from the International Glycoconjugate Organization.
Abstract: Protein glycosylation, the chemical addition of sugars to proteins, is an important but poorly understood aspect of bacterial physiology. Recently we identified that multiple human pathogens of the Burkholderia genus possess a conserved O-linked glycosylation system. High-throughput screening studies have shown the inhibition of glycosylation profoundly impacts virulence in Burkholderia, yet little is known about the underlying mechanism. Using the Cystic fibrosis pathogen B. cenocepacia as a model we have sought to improve our understanding of the role of glycosylation within Burkholderia species. Using state of the art proteomics approaches we have now identified >70 proteins are glycosylated at over 110 sites across strains of B. cenocepacia. Mutagenesis studies have shown that the loss of glycosylation leads to profound effects on the proteome with quantitative proteomics/degradomics revealing that a subset of glycoproteins require glycosylation for stability. Using thermal proteomics, we have also begun to track the effect of glycosylation on protein stability within the proteome. Our quantitative proteomics, degradomics and thermal proteomics results support that glycosylation plays multiple roles in the biology of B. cenocepacia. By understanding how glycosylation contributes to B. cenocepacia protein function this work will improve our ability to harness bacterial glycosylation to create bespoke glycoconjugates.
Direct measurement of protein translation and degradation rates In vivo at proteomic scales in the clinic and beyond; new approaches to stable isotope metabolic labelling and analysis
Dr Owen Duncan
School of Molecular Sciences, The University of Western Australia
Bio: Dr Duncan is a Post-doctoral Research Fellow working in Harvey Millar's laboratory at the University of Western Australia. His main area of work is the development of mass spec based data acquisition and processing focusing on metabolic incorporation of stable isotopes for measuring flux at the protein level (protein synthesis and degradation rates). His PhD studies were supervised by Prof. Ryan Lister and Jim Whelan where he used early QTOF instruments combined with an intricate free flow electrophoresis based subcellular fractionation workflow to establish the mitochondrial outer membrane proteome in higher plants. He has since worked with nanoflow proteomics daily and have recently been involved in the establishment and commissioning of the Western Australian Proteomics core facility established with NCRIS funding in December 2019 which is focused on the delivery of flux proteomics to researchers nationally.
Abstract: Protein abundances are established by the combination of synthesis and degradation; the direct measurement of which has been complex and time consuming for individual proteins and simply not possible at scale. Advances at the University of Western Australia using stable isotope labelling combined with mass spectrometry has allowed the parallel measurement of synthesis and degradation rates of hundreds to thousands of protein species in a single sample. In collaboration with researchers at the Fiona Wood Foundation we've refined the use of heavy hydrogen in the form of deuterium oxide (²H₂O) coupled to skin biopsies and mass spectrometry to measure the synthesis and degradation rates of various isoforms of collagen in human burns patients over time in scar vs non scar skin with the aim of both determining when the most effective times are to apply drug based interventions in scar formation and to measure their efficacy. The second part of this talk will cover experimental design and other examples of protein flux measurements.