Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

An ACF multicentre testing initiative: exposing the limitations of shotgun proteomics (#183)

Erwin Tanuwidjaya 1 , Giuseppe Infusini 2 , Mark R Condina 2 , Belinda Schiller 3 , Tara Pukala 4 , Matt P Padula 5 , Ben Crossett 6 , Ralf B Schittenhelm 1
  1. Monash Proteomics & Metabolomics Facility, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
  2. Mass Dynamics, Melbourne, Victoria, Australia
  3. Australian Proteome Analysis, Macquarie University, Macquarie Park, NSW, Australia
  4. School of Physical Sciences, Faculty of Sciences, Engineering and Technology, University of Adelaide, Adelaide, South Australia, Australia
  5. School of Life Sciences, Faculty of Science, University of Technology Sydney, Broadway, NSW, Australia
  6. Sydney Mass Spectrometry, The University of Sydney, The Hub, NSW, Australia

The Australasian Core Facilities (ACF) group has been established in 2019 to discuss challenges, opportunities and best practices in core facility management and mass spectrometric applications. In line with these goals, the ACF group has conducted during 2022 their first Multicentre Testing Initiative, which was aimed to (i) examine reproducibility across Australasian mass spectrometric core facilities and (ii) to identify optimal strategies and workflows – but also potential pitfalls and issues – in data-dependent acquisition (DDA) mass spectrometry, which is commonly referred to as shotgun proteomics.

To that end, two tryptic peptide samples of defined composition and differences were distributed to 20 participating mass spectrometric core facilities to be acquired in technical triplicates on any LC-MS/MS system(s) operated in DDA mode. Any acquisition method or workflow was admissible provided that (i) the linear separation gradient did not exceed 120 min, (ii) the samples were not further fractionated and (iii) less than 2 ug were injected per replicate. Following these guidelines, the ACF group has received a total 66 complete datasets acquired on a multitude of mass spectrometers and LC systems using a variety of parameters and settings. Upon receipt, all data were de-identified by ACF’s data custodian, and forwarded to MassDynamics to be comparatively analysed and visualised using their standardised, automated pipelines.

In this poster we will comprehensively describe the findings of this first Multicentre Testing Initiative. We will furthermore use these benchmarking datasets to expose significant limitations of shotgun proteomic approaches, but also provide advice on seemingly optimal DDA strategies and workflows.