Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

High-throughput sample preparation for proteomic analysis using 3d-printing (#67)

Dylan J Harney 1 , Amy T Hutchinson 2 , Luke Hatchwell 1 , Sean J Humphrey 1 , David E James 1 , Samantha Hocking 3 , Leonie K Helibronn 2 , Mark Larance 1
  1. School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia
  2. Discipline of Medicine, University of Adelaide, Adelaide, South Australia, Australia
  3. Central Clinical School, Faculty of Medicine and Health, University of Sydney, Camperdown, NSW, Australia

Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics, due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS. Spin96 is a 3D-printed device, designed to be compatible with any centrifuge with a deep-well adaptor. The design of the Spin96 minimizes sample loss and eliminates cross-contamination while enabling use of all commonly used chromatography resins such as C18, mixed-mode SDB-RPS and titanium dioxide. To demonstrate the efficacy of Spin96, we analysed human plasma from a clinical trial of Intermittent Fasting (IF). IF increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans is less clear. In this longitudinal study employing 8-weeks IF, we identified significant abundance differences induced by the IF intervention, including increased apolipoprotein A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3). These changes correlated with a significant decrease in plasma triglycerides after the IF intervention. Given that these proteins have a role in regulating apolipoprotein particle metabolism, we propose that IF had a positive effect on lipid metabolism through modulation of HDL particle size and function.  In addition, we applied a novel human protein variant database to detect common protein variants across the participants. We show that consistent detection of clinically-relevant peptides derived from both alleles of many proteins is possible, including some that are associated with human metabolic phenotypes. Together, these findings illustrate the power of our Spin96 device for high throughput proteomics to yield biological insight of clinical significance.