Florian Marty (Presenter)
Biognosys AG
Bio: Dr. Florian Marty studied Developmental Biology at the University of Zurich (UZH), Switzerland with a focus on shotgun proteomics. For his PhD he started a collaborative project with the laboratory of Prof. Dr. Ron M.A. Heeren at the AMOL/FOM-Institute Amsterdam and the Laboratory of Prof. Dr. Konrad Basler at the Institute of Molecular Life Sciences at the UZH, Switzerland. In his project he worked on imaging mass spectrometry to elucidate the distribution of small molecules on developing organs. After successfully finishing his PhD he joined Dualsystems AG as a Senior Scientist to optimize their ligand-receptor deorphanizing system (LRC-TriCEPS). Since June 2016, Dr. Florian Marty, works at Biognosys AG as a Product Support Manager.
Authorship: Florian Marty
Biognosys AG, Wagistrasse 25, 8952 Schlieren, Zurich, Switzerland
| Short Abstract Comprehensive data-independent acquisition (DIA) and targeted data analysis with retention-time-normalized spectral libraries have become the methods of choice for discovery and quantitative proteomics. We demonstrate here how thousands of proteins are reproducibly quantified from as little as 12`000 liver derived cells using Spectronaut 9.0. In this study, liver micro tissues were used to show that liver toxicity markers are up-regulated below the acetaminophen’s therapeutic dose. In contrast, proteins expected to contribute to the therapeutic effect are only affected at concentrations higher than the therapeutic dose. |
Long Abstract
Mass spectrometry based discovery proteomic workflows have long relied on data dependent acquisition (DDA) to identify and quantify proteins of interest. In a classical DDA experiment, thousands of peptides and proteins can be identified but their quantitative analysis is usually limited by the low analytical reproducibility of the workflow. Due to its semi stochastic nature, on average, only 30-60% of the peptides are reproducibly identified in technical replicates. Recently, data-independent acquisition (DIA or SWATH) has rapidly evolved as more powerful mass spectrometric approach for comprehensive, reproducible and precise proteome quantitation.
In contrast to DDA where single ions are isolated for further analysis, in DIA mode the mass spectrometer cycles through broad precursor windows fragmenting multiple peptide ions together. This results in a comprehensive digital map of all detectable peptides present in a sample and more importantly in a highly reproducible fashion. DIA data is complete but highly convoluted and the peptide signals can be extracted using retention-time-normalized spectral libraries. This SWATH-type DIA workflow is called hyper reaction monitoring (HRM) and employs Spectronaut software for analysis and quantification.
In this study, HRM workflow was used for analysis of acetaminophen toxicity in liver microtissues. Acetaminophen was applied in 5 different concentrations from sub-therapeutic to toxic doses. Nine LC MS runs were acquired in a DDA mode for spectral library generation and 15 quantitative analysis DIA runs representing five samples in triplicates were acquired on the Thermo Scientific Q Exactive HF. Spectral library generation and the DIA data analysis were performed using Spectronaut 9. More than 3’200 proteins were reproducibly quantified in each sample with technical replicates CVs as low as 7.5%. We could identify several proteins being upregulated already at a sub-therapeutic concentration of 14µM APAP. Fuzzy c-means clustering followed by pathway enrichment analysis of the regulated candidates confirmed that these proteins are mainly involved in the metabolism of Xenobiotics by Cytochrome P450. Furthermore, we could show that acute inflammatory response proteins are only downregulated at concentrations close to the therapeutic dose (123µM) or higher. The findings also demonstrated the power of Biogsnoys’ HRM workflow in drug metabolism and drug toxicity studies.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Biognosys AG |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |