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Chemical Proteomics Core Facility

The Chemical Proteomics facility (ChemProt) is a recent national facility expert on supporting drug discovery and development by proteome-wide deconvolution of targets and mechanism of action (MoA) of small molecules or any active compounds.

ChemProt is the reference facility for chemical proteomics within SciLifeLab and BioMS national infrastructures.

The main goal of ChemProt is to decipher proteome signatures induced by an active compound for MoA elucidation and target identification. For these aims, ChemProt has adopted the latest mass spectrometry(MS)-based proteomic technologies and developed new high-throughput (HT) methods.

ChemProt also provides the HDX-MS (hydrogen-deuterium exchange mass spectrometry) technology for protein structure analysis on purified proteins with the goal of mapping the site of interaction with active compound of conformational changes in response to the applied system perturbation.

Using multidisciplinary competences and resources, ChemProt represents an innovative facility model offering complete pipelines from perturbations in cell cultures, to MS-based proteomics and data analysis, including data bioinformatics processing, publication figures and data representation.

ChemProt has recently developed high throughput (HT) chemical proteomics methods which outperforms the previous methods for target deconvolution of at least tenfold and decreases by the same factor the time of analysis of the sample amount needed for each biological replicate. The novel methods can use a high level on multiplexing for several biological replicates, up to 16 per experiment according to the latest state of the art for quantitative proteomics. Different compounds or more than one biological system can be tested simultaneously.

The LC-MS instrument park has been renewed  for better supporting the new high throughput chemical proteomics.

Instruments of the Chemical Proteomics Core Facility.

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) Assayhigh 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);

  • ProTargetMinerhigh 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 can also provide other types of proteomics services through the regional core facility Proteomics Biomedicum.

Equipment

The ChemProt  laboratories are at located at Karolinska Institutet's research building 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, Sabatier P, Saei AA, Beusch CM, Yang Z, Lundström SL, et al
J. Proteome Res. 2019 Nov;18(11):4027-4037

ProTargetMiner as a proteome signature library of anticancer molecules for functional discovery.
Saei AA, Beusch CM, Chernobrovkin A, Sabatier P, Zhang B, Tokat ÜG, et al
Nat Commun 2019 12;10(1):5715

Thermal proteome profiling identifies oxidative-dependent inhibition of the transcription of major oncogenes as a new therapeutic mechanism for select anticancer compounds.
Peuget S, Zhu J, Sanz G, Singh M, Gaetani M, Chen X, et al
Cancer Res. 2020 Feb;():

Using the Facility

Please feel free to contact us (massimiliano.gaetani@ki.se) and come visit us at KI Biomedicum. You will receive consultation free of charge, finding out about feasibility, details and better solutions for your project or your potential one.

ChemProt is also the national facility expert of Chemical Proteomics within the Swedish infrastructure for biological mass spectrometry (BioMS). ChemProt is responsible for the entire Chemical Proteomics technology and also provider of protein structural analysis through HDX-MS. Through BioMS and SciLifeLab we can support your project and cover relevant costs of it by national funding.

To book services of our facility please create a new application at the BioMS portal and when requested in the application form please select "Chemical Proteomics” as technology and then add “M.Gaetani” as BioMS contact.

Visiting Address

Karolinska Institutet
Department of Medical Biochemistry and Biophysics (MBB)
Biomedicum, quarter A9, floor 9
Solnavägen 9
Stockholm, Sweden

Contact

Massimiliano Gaetani

Head of Facility

Personnel

Roman Zubarev, Ph.D. (Facility Director)
Massimiliano Gaetani, Ph.D. (Head of Facility)
Susanna Lundström, Ph.D. (Senior Staff Proteomics Specialist)
Juan Astorga-Wells, Ph.D. (Senior Associated HDX-MS Specialist)
Olga Lytovchenko, Ph.D. (Staff Research Engineer)
Zhaowei Meng (Staff LC-MS Engineer)