Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Development of a fully automated magnetic workflow for phosphoproteome profiling (#862)

Stoyan Stoychev 1 2 , Previn Naicker 2 , Ireshyn Govender 2 , Sindisiwe Buthelezi 2 , Sipho Mamputha 2 , Isak Gerber 2 , Jusitn Jordaan 1
  1. Proteomics, Resyn Biosciences, Pretoria, Gauteng, South Africa
  2. CSIR, Pretoria, GAUTENG, South Africa

The research aims to implement a seamless fully automated workflow for phosphoproteome profiling, covering all steps from extracted protein to the mass spectrometry analysis. Automation of this workflow increases throughput and reproducibility. Each component of the pipeline was evaluated individually, and subsequently integrated to form the seamless workflow. We started with protein isolation from a complex lysate using different strategies, employing three bead chemistries for solid phase extraction, strong anion exchange (SAX), hydrophilic affinity (HILIC), and amine for on-bead protein aggregation capture (PAC/SP3). Based on 200ug starting material the three approaches exhibited similar recoveries. However, their performance varies based on sample source, amount of protein, concentration, extraction conditions, and additives. We further plan to assess the ability to perform peptide fractionation using SAX, potentially allowing for deeper phosphoproteome mining. For phosphopeptide enrichment we evaluate the complementarity of Ti-IMAC, Zr-IMAC and TiO2. We provide alternate buffering conditions that can result in increased phosphopeptide recovery. The potential complementary of the phosphopeptide enrichment chemistries is explored, where various bead combinations are tested in an attempt to provide more comprehensive phoshopeptide coverage in a single enrichment. All of the above-mentioned methods including protein capture, clean-up and on-bead digestion as well as phosphopeptide enrichment have been adapted for use in a magnetic handling stations (KingFisher Duo) allowing for fully automated sample processing. The methods are readily transferable to a range of liquid handling robots fitted with magnetic handling platforms.