Structural characterization of proteins and protein complexes by hydrogen-deuterium exchange and hydroxyl radical footprinting
Supervision
Petr Novák
BIOCEV, 1st Supervisor
Eduard Sabido
CRG, 2nd Supervisor
Objectives
Ideal instrument method for HDX MS and hydroxyl radical footprinting MS data acquisitions employing ion mobility trace. Optimized MS data export and further downstream interpretation for HDX and hydroxyl radical footprinting experiments. Visualization of HDX and hydroxyl radical footprinting results on PDB structures.
Methodology
Method optimization of two arms robot for reliable sample preparation automated pipetting. Tunning UPLC methods for high reproducible LC separation columns and gradient selection. Adjustment of timsTOF parameters to reach high confidence quantitative mass spectrometric data hardware set up. Design of the visual basic script to convert the raw data in to .txt file. Manual validation of acquired MS data as a benchmark for AI processing.
Required skills
The candidate should have a background in analytical or experimental sciences, with experience in automation and method optimization. Familiarity with instrument setup and data acquisition, as well as basic programming or data processing skills, is desirable. Strong attention to detail and ability to validate and interpret experimental data are essential.
Expected Results
The development of reliable software for processing mass spectrometric data will streamline interpretation, benefiting academia by enhancing understanding of biological system dynamics and aiding the pharma industry in boosting R&D and manufacturing efficiency.
Planned Secondments
Host: CRG (E. Sabido), Duration: 2 Months; When: Year 1, Goal: Targeted mass spectrometric acquisition methods for structural proteomics.
Host: FHOOE (V. Dorfer), Duration: 1 Month, When: Year2, Goal: Redesign of current search engine for the needs of FPOP analysis.
Host: BRUKER (D. Trede), Duration: 1 Month; When: Year 3, Goal: Tool implementation into commercial software.
Enrolment in doctoral programs
UNIVERZITA KARLOVA
References
1 Yassaghi G, Kukačka Z, Fiala J, Kavan D, Halada P, Volný M, Novák P. Top-Down Detection of Oxidative Protein Footprinting by
Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation. Anal Chem. 2022 Jul
19;94(28):9993-10002. doi: 10.1021/acs.analchem.1c05476.
2 Polák M, Yassaghi G, Kavan D, Filandr F, Fiala J, Kukačka Z, Halada P, Loginov DS, Novák P. Utilization of Fast Photochemical
Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription FactordsDNA Complex. Anal Chem. 2022 Feb 22;94(7):3203-3210. doi: 10.1021/acs.analchem.1c04746.
3 Fojtík L, Fiala J, Pompach P, Chmelík J, Matoušek V, Beier P, Kukačka Z, Novák P. Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. J Am Chem Soc. 2021 Dec 15;143(49):20670-20679. doi:
10.1021/jacs.1c07771.
4 Kukačka Z, Rosůlek M, Jelínek J, Slavata L, Kavan D, Novák P. LinX: A Software Tool for Uncommon Cross-Linking Chemistry. J
Proteome Res. 2021 Apr 2;20(4):2021-2027. doi: 10.1021/acs.jproteome.0c00858.
5 Loginov DS, Fiala J, Chmelik J, Brechlin P, Kruppa G, Novak P. Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis. ACS Omega. 2021
Apr 8;6(15):10352-10361. doi: 10.1021/acsomega.1c00732.