Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Application of Innovative, Automated Workflows to Cardiac Ischemia Biomarker Discovery and Multiomics Data Visualization. (#169)

Scott Kuzdzal 1 , Junko Iida 2
  1. Shimadzu Scientific Instruments, Columbia, MD, United States
  2. LCMS Business Unit, Shimadzu Corporation, Kyoto, Japan

Protein sample preparation workflows for mass spectrometric analysis that involve proteolysis are labor-intensive, time-consuming and user-dependent. These workflows often involve digestion, solid phase extraction, drying, and re-suspension prior to reversed phase separation into the mass spectrometer. The introduction of variability at many of these steps hinders discovery initiatives as well as the ability to convert these discoveries into viable assays. An automated protein digestion platform was developed to accelerate the digestion process through use of immobilized enzyme reaction columns. Online digestion is integrated with desalting and reversed phase chromatography to create a seamless workflow to the mass spectrometer. This approach combines the selectivity of immunoassay approaches with the resolution of high performance liquid chromatography and is easily coupled to a mass spectrometer.

 

This automated platform [1] enables ultra-fast digestions with exceptional reproducibility. Complete, reproducible protein digestions in as little as 30 seconds have been demonstrated, allowing exceptional reproducibility (CV less than 10%). Comparison between the manual digestion and automated workstation workflows for the phosphorylated peptide ETYGEMADCCAK present in HSA used for a cardiac ischemia biomarker cohort study from Johns Hopkins School of Medicine shows increased reproducibility in the automated approach.

 

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Configuration and valve diagram for automated affinity capture, buffer exchange, trypsin immobilized enzyme reaction columns, desalting and reversed phase and for automated, on-line affinity enrichment and proteolysis.

 

The second part of this presentation highlights recent developments in the automated visualization of multiomics (metabolomics, proteomics, fluxomics and transcriptomics) data. We hereiin demonstrate automated visualization of multimoics data for three conditions of the Synechocystis sp. PCC 6803 strain (autotorphic, mixotrophic and photoheterotrophic conditions) in order to assess metabolic flux [2]. This automated multiomics visualization platform [3] greatly simplifies omics analyses.

 

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Automated visualization of multiomics data using an integrated, automated approach.

  1. [1] Using the Perfinity Workstation for High Throughput Biomedical Applications, Presentation by Dr. Christine Jelinek in Kyoto, Japan. https://www.youtube.com/watch?v=pnIXbaeeIQo
  2. [2] Nakajima. et al., Metabolic flux analysis of Synechocystis sp. PCC 6803 DeltanrtABCD mutant reveals a mechanism for metabolic adaptation to nitrogen-limited conditions..Plant Cell Physiol 58, 537-545 (2017).
  3. [3] Garuda is an open, community-driven, common platform that provides a framework to connect, discover and navigate through different applications, databases and services in biology and medicine. http://www.garuda-alliance.org.