Oral Presentation 29th Annual Lorne Proteomics Symposium 2024

Circulating plasma sub-proteome analysis uncovers novel insights into the mechanisms underlying high-dose sodium ascorbate in sepsis (#25)

Samantha Emery-Corbin 1 2 , Jumana Yousef 1 2 , Yugeesh R Lankadeva 3 4 , Rinaldo Bellomo 4 5 6 7 8 , Fumitaka Yanase 5 6 , Mark P Plummer 9 , Clive N May 4 10 , Laura F Dagley 1 2
  1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
  3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia
  4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia
  5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia
  6. Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Victoria, Australia
  7. Department of Intensive Care, Royal Melbourne Hospital, Parkville, Victoria, Australia
  8. Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Victoria, Australia
  9. Department of Intensive Care, Royal Adelaide Hospital, Adelaide, South Australia, Australia
  10. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia

Sepsis remains the leading cause of in-hospital deaths (30-50%), causing ~11 million deaths annually. Current care is supportive/palliative, meaning new, effective, and specific treatments are key to reducing global mortality. In pre-clinical ovine trials, megadose sodium ascorbate (NaAscorbate) was shown to reduce vasopressor (noradrenaline) support, restore renal function and reverse acute kidney injury (AKI) (1). Despite hypotheses surrounding NaAscorbate benefits, its molecular mechanism is unknown.

To elucidate mechanisms of NaAscorbate, we have performed unbiased plasma proteomics from human plasma collected during a pilot, single-dose, double-blind, randomised controlled trial (2) of a single intravenous megadose NaAsorbate (60g) compared to placebo in patients with septic shock (n=18). Patient plasma was collected pre-treatment (baseline, 0hr), and 1, 4, 6 and 24 hrs (n = 90). In addition, we have analysed plasma-derived, circulating extracellular vesicles (cEVs) using a size-exclusion and antibody-based (ExoNet, INOVIQ) and SAX-based (MagNet, ReSynBio, (3)) mag-bead approach, noting precedent of EVs in sepsis in systemic inflammation and immune cell responses (4, 5). For neat plasma, technical replicates and pooled controls were included, whereas for cEV plasma was pooled across patients and timepoints to create cohort pools based on sepsis source, treatment and placebo. Neat plasma and cEV fractions were processed via on-bead digestion (6) and analysed on a timsTOFpro MS (Bruker) diaPASEF method utilising a 30-minute analytical gradient (48 minute cycle time) with a 15cm C18 fused silica column (Aurora, IonOpticks), with data searched library-free in DIA-NN (7).

After pre-processing and filtration, a non-redundant 2231 total proteins were identified across methods (599 Plasma, 762 MagNet, 1921 ExoNet), of which 320 (14%) were common across all methods and 1254 (56%) were uniquely identified in ExoNet. We observed variation in precursor and protein IDs in ExoNet and MagNet, where ExoNet clearly captured inter-patient and intra-patient (timepoint) variation, whereas patient-pooled cEV samples shared high Pearson correlations > 0.9. After normalisation, principal component analysis (PC1) in all three approaches could separate placebo and treatment on the PC1, but source of sepsis infection contributed a significant variation to data. We have performed pairwise (limma) and continuous (maSigPro, moanin) statistical analyses of treatment and time course to evaluate molecular progression of sepsis over time (placebo) as well as between placebo and megadose NaAscorbate. Together, we believe these data will provide a molecular reference for cEV methods and human sepsis pathology, and will critically inform the best approach for upcoming human NaAscorbate clinical trials.

  1. Y. R. Lankadeva et al., Reversal of the Pathophysiological Responses to Gram-Negative Sepsis by Megadose Vitamin C. Crit Care Med 49, e179-e190 (2021).
  2. F. Yanase et al., Mega-dose sodium ascorbate: a pilot, single-dose, physiological effect, double-blind, randomized, controlled trial. Crit Care 27, 371 (2023).
  3. C. W. Christine et al., Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome. bioRxiv, 2023.2006.2010.544439 (2023).
  4. P. Raeven, J. Zipperle, S. Drechsler, Extracellular Vesicles as Markers and Mediators in Sepsis. Theranostics 8, 3348-3365 (2018).
  5. M. Burgelman, C. Vandendriessche, R. E. Vandenbroucke, Extracellular Vesicles: A Double-Edged Sword in Sepsis. Pharmaceuticals (Basel) 14, (2021).
  6. L. F. Dagley, G. Infusini, R. H. Larsen, J. J. Sandow, A. I. Webb, Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics. J Proteome Res 18, 2915-2924 (2019).
  7. V. Demichev et al., dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nature Communications 13, 3944 (2022).