Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Multiplexed single cell proteomics for investigating cellular heterogeneity during hypoxia (#102)

Craig P Barry 1 , Gert H Talbo 1 2 , Esteban Marcellin 1 2
  1. Australian Institute for Bioengineering and Nanotechnology, Fairfield, QLD, Australia
  2. Metabolomics Australia Queensland Node, The University of Queensland, MA, Brisbane, QLD, Australia

Cellular diversity is a ubiquitous property of biological systems and is exemplified in tumour cell heterogeneity. Investigating cellular heterogeneity requires single cell resolution in order to identify population subtypes or temporal phenomena, such as cellular differentiation. Single cell resolution is afforded by well-established sc-RNASeq methods, where transcript abundance is frequently taken as a proxy for protein abundance. Realistically, protein abundance is the integral of mRNA translation rate which limits the application of mRNA as a proxy of protein abundance to constitutively expressed genes. Methods for high throughput single cell proteomics using LC-MS have gained recent traction with developments in peptide multiplexing reagents and high resolution Orbitrap instruments.

Here, we present an implementation of a multiplexed single cell proteomics (scMS) workflow to semi-quantitatively investigate the diversity of oxygen-deprived HEK293 cells which are cultured in bioreactors. From this data, we were able identify hypoxia-driven heterogeneity in single cell proteomes. Meaningful protein profiles can be drawn from this data which corroborate literature findings from bulk-sampling methods. Here, we outline our implementation of an scMS workflow and discuss conclusions drawn from its utility in investigating hypoxia-driven stress in industrially relevant HEK293 cells.