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Chemical Proteomics

National facility

The Chemical Proteomics facility (ChemProt) is the national expert facility for supporting drug discovery and development by proteome-wide deconvolution of targets and action mechanisms of small molecules.

ChemProt has recently invented and offers high throughput (at least 10 times higher than previous art) chemical proteomics methods which could enable projects otherwise impracticable or difficult tu sustain with other approaches. The cost of each tested biological sample is also reduced by at least ten times per biological replicate respect to previous approaches, such as thermal proteome profiling. These novel methods developed by ChemProt use high number of biological replicates respect to previous approaches, thus providing more reproducible results. These new approaches also need much lower sample amount and allow a high level of multiplexing. The last can be also used for combined analysis of different compounds or biological systems. The LC-MS insturment park has recently been renewing and increased for better supporting the new opportunities opened by of our new high throughput methodologies.

 

LINKS

- National BioMS infrastructure and Chemical Proteomics technology

- ChemProt website at Karolinska Institutet 

- See what proteomics people say about our PISA assay:https://proteomicsnews.blogspot.com/2019/12/pisa-multiplex-thermal-proteome.html

 

SERVICES

ChemProt makes use of proteome-wide orthogonal approaches to find out how small molecules work when incubated in cell cultures and in lysates. Most approaches can be provided with no molecular modification for chemical probe engineering. ChemProt can provide full pipelines reproducing the biological treatments of the phenotype of interest or operating the treatment planned with the users.  ChemProt also offers hydrogen-deuterium exchange mass spectrometry (HDX-MS) for mapping and characterizing the target binding site, and monitoring other structural changes. Our outcomes include full data analysis, high confidence identification of most reproducible and treatment-specific MoA signatures and target candidates. Follow up support for consequent experimental and publication purposes are provided; consultation is always provided on request and free of charge, also to guide further planning.

 

Summary of Current Technologies and Services:

A) Deconvolution of compound targets, determination of mechanism of Action (MoA) and off-target landscape through orthogonal methods of mass spectrometry-based deep proteomics:

  • protein solubility/stability alteration signatures in treated cell cultures and lysates;
  • treatment-specific expression/degradation proteome signatures;
  • changes in proteome redox balance in treated cell cultures.

Matches over two orthogonal approaches dramatically increase the chances to find the correct target. These methods are here listed:

  • Proteome Integral Solubility Alteration (PISA) Assay, high throughput method based on protein solubility/stability upon thermal treatment, with no need of chemical engineering of compounds (now up to 16-plex, i.e. 16 biological samples in one multiplex analysis) (Gaetani M et al., J Proteome Res, 2019, DOI: 1021/acs.jproteome.9b00500);
  • FITExP, based on compound-specific proteome responses and with no need of chemical engineering of compounds (now up to 16-plex) (Chernobrovkin A et al., Sci Rep, 2015, DOI:1038/srep11176 );
  • ProTargetMiner, high throughput method to study anticancer compounds based on FITExP-derived database of proteome signatures using a library of anticancer molecules (Saei AA et al., Nat Commun, 2019; DOI:1038/s41467-019-13582-8);
  • Identification of interactions and protein complexes after affinity-based approaches using chemical engineered probes (now up to 16-plex)
  • Thermal Proteome Profiling (TPP), based on protein melting curve fitting and Tm extrapolation (Savitski MM et al., Science, 2014, DOI: 1126/science.1255784)
  • RedOx Proteomics, for specific proteome changes in the reduction-oxidation balance

B) Protein Structural Analysis using Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Binding site mapping and characterization for: protein-small molecule interactions; protein-peptide interactions; protein-protein interactions, including epitope mapping;
  • Conformational changes monitoring to study effects of mutations, to analyze misfolding and for biosimilar characterization

C) Identification of enzyme protein substrates using a thermal profiling (TPP or PISA) based approach in cell lysate and recombinant enzymes and their co-factors (Saei AA et al, submitted; BioRxiv: DOI: 1101/423418 )

D) Additional proteomics services related to / coordinated with the above (e.g. PTMs, deep quantitative proteomics, etc.)

 

Our Division at Medical Biochemistry and Biophysics (MBB) Department of Karolinska Institutet can also provide other types of proteomics services through the regional core facility Proteomics Biomedicum.

 

EQUIPMENT

ChemProt  laboratories are at located at the Department of Medical Biochemistry and Biophysics (MBB) of Karolinska Institutet, Biomedicum. ChemProt represents an innovative facility model offering complete pipelines and including different laboratories for cell culture, sample preparation, LC-MS and bioinformatic analysis.

 

Cell culture lab Instruments: Two laminar flow cabinets, Two CO2 incubators, Two Microscopes, Eppendorf centrifuge, BioRad cell counter, etc.

Centrifuges: Beckmann XPN-80 ultracentrifuge, Speedvac centrifuges , Eppendorf centrifuges for large and small tubes

Mass Spectrometers:

  • Two Orbitrap Fusion Lumos, Thermo Scientific
  • One Orbitrap Fusion, Thermo Scientific
  • Two Orbitrap Q Exactives HF, Thermo Scientific
  • One Orbitrap Q Exactive, Thermo Scientific
  • One LTQ Velos Pro Orbitrap Elite, Thermo Scientific

UPLC:

  • Two Ultimate 3000 RSLCnano for double column operation
  • Five UltiMate 3000 RSLCnano / nEasy-LC 1000 for nLC-MS
  • Ultimate 3000 for off-line high pH fractionation;
  • Three UltiMate 3000 Nano LC or nEasy-LC 1000 for nLC-MS

HDX-MS Robot: CTC PAL Liquid Handling System connected to a Peltier-cooled box

Data Analysis Harware and Software: Four PC stations, Proteome Discoverer 2.4, BioPharma Finder, Peaks, MaxQuant, Mascot, SIMCA, GraphPad PRISM, Excel, R, etc.

 

 

RECENT PROJECTS

Proteome Integral Solubility Alteration: A High-Throughput Proteomics Assay for Target Deconvolution (Gaetani M et al.  J Proteome Res. 2019, doi: 1021/acs.jproteome.9b00500)

ProTargetMiner as a proteome signature library of anticancer molecules for functional discovery (Saei AA et al. Nat. Commun. 2019doi: 10.1038/s41467-019-13582-8)

Thermal proteome profiling identifies oxidative-dependent inhibition of the transcription of major oncogenes as a new therapeutic mechanism for select anticancer compounds (Peuget S et al. Cancer Res. 2020doi: 10.1158/0008-5472.CAN-19-2069)