Chemical Proteomics

Overview of Services

The Chemical Proteomics Core Facility (ChemProt) is the sole national facility for supporting drug discovery and development by proteome-wide discovery of targets and identification of action mechanisms. ChemProt is located at the Department of Medical Biochemistry and Biophysics (MBB) in the Biomedicum building. Please feel free to contact us 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.

We will be glad to discuss, assist projects related to identification of protein targets with proteomics, adoptation of thermal solubility and stability chages, or protein structural analysis by Hydrogen Exchange Mass spectrometry (HDX-MS). We can assist with experimental design, complete pipelines starting from cells and compounds, deliver high throughput and high quality results, and assist with data analysis up to publication quality figures.

ChemProt has recently developed new high throughput (HT) methods which outperform the previous ones for target identification of at least tenfold and decrease by the same factor the time of analysis of the sample amount needed for each biological replicate. The novel methods can use label-free and latest quantitative 16-plex TMT-labelling technology for several biological replicates. 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. ChemProt has recently miniaturized workflows and can analyze extremely low amounts of cells with deep analyses for primary cells, and is walking not the implementation into iPSC-derived 2D and 3D cultures for drug development. ChemProt also provides HDX-MS for binding site identification and epitope mapping in purified protein systems.

Currently Offered Technologies and Services

A) Chemical Proteomics for Target Discovery and Determination of Mechanism of Action (MoA) and Off-Target landscape

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

A) Chemical Proteomics for Target Discovery, Determination of Mechanism of Action (MoA) and Off-Target landscape

General proteomics workflow for chemical proteomics

• Consultation, project description generation and experiment design
• Cell culture and drug / compound treatments
• Cells / tissue sample processing (method-specific)
• Sample preparation for mass spectrometry-based proteomics (method-specific)
• Tandem Mass Tag (TMT) reporter ion labelling for multiplexed quantitative proteomics (label-free and stable isotope labelling in cell culture also available). Up to 16 biological replicates in one multiplexed experiment (TMTpro 16-plex) and coordination of multiplexed experiments are available
• Sample fractionation for deep analysis
• nLC-MS analysis of samples / sample fractions
• MS-based protein identification and quantification (method-specific)
• Functional data analysis and interpretation
• Consultation and follow-up support for further experimental design and publications

A1) Orthogonal methods of deep MS-based proteome analysis for:

• protein solubility/stability alteration 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 (the first three are developed in-house *):

• Proteome Integral Solubility Alteration (PISA) Assay *, high throughput method based on protein solubility/stability upon thermal treatment (J Proteome Res 2019, DOI: 1021/acs.jproteome.9b00500) with no need of chemical engineering of compounds (now up to 16-plex with TMTpro 16-plex, i.e. 16 biological samples in one multiplex analysis);

• Functional Identification of Target by Expression Proteomics (FITExP)*, based on compound-specific proteome responses (Sci Rep 2015, DOI: 1038/srep11176) and with no need of chemical engineering of compounds (now up to 16-plex with TMTpro 16-plex)

• ProTargetMiner *, high throughput method to study anticancer compounds based on FITExP-derived database of proteome signatures using a library of anticancer molecules (Nat Commun 2019, DOI: 1038/s41467-019-13582-8);

• PISA-Express *: high throughput method combining PISA and Expression proteomics, even in all-in-one multiplexed experiment with up to 4 replicates per condition. PISA data can be normalized on global protein amount variation, extending the use of PISA for longer treatments or conditioning of cell cultures. Today available also for low number of cells and applicable to primary cells and 3D organoids. 

• Identification of interactions and protein complexes after affinity-based approaches using chemical engineered probes (now up to 16-plex with TMTpro 16-plex) 

• Thermal Proteome Profiling (TPP), based on protein melting curve fitting and Tm extrapolation (Savitski et al. Science 2014, DOI: 1126/science.1255784) 

• RedOx Proteomics, for specific proteome changes in the reduction-oxidation balance

A2) Application of chemical proteomics methods for the identification of substrate interaction, protein-protein interactions and with other molecules.

• “System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis” (SIESTA), a new method developed in-house(Nat Commun 2021, DOI: 1038/s41467-021-21540-6) for identification of enzyme protein substrates. It relyes on the enzymatic post-translational modifications of substrate proteins changing their solubility and it assesses solubility changes of proteins in cell lysate when a recombinant protein and a co-factor are simultaneously added to the lysate in excess.

A3) Additional proteomics services where needed in coordination with the above (e.g. PTMs, etc.)

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

HDX-MS (hydrogen-deuterium exchange mass spectrometry) is a technology for protein structure analysis. It measures the dynamics of H/D exchange of purified proteins, compared alone or mixed with a ligand, to identify the region of binding or conformational changes. The technology makes use of automated liquid handling for deuteration of proteins with and without ligand, automated digestion into peptides on a column and in-line LC-MS for peptide analysis. LC-MS will measure and monitor the incorporation of deuterium into the amide group of a polypeptide chain.  Deuterium incorporation into the backbone amide groups of a polypeptide chain depends on solvent accessibility, intramolecular hydrogen bonding and the physicochemical characteristics of the buffer (temperature and pH). The binding of a ligand to a protein can be detected by comparing the deuterium kinetics of the target protein in the presence and absence of the ligand. 

HDX-MS workflows are optimized at ChemProt for:

B1) Binding site mapping and characterization for: protein-small molecule interactions; protein-peptide interactions; protein-protein interactions, including epitope mapping;

B2) Conformational changes monitoring to study effects of mutations, to analyze misfolding and for biosimilar characterization.

Personnel

Roman Zubarev, Ph.D.   (Facility Director)
Massimiliano Gaetani, Ph.D.   (Head of Facility)
Susanna Lundström, Ph.D.   (Senior Staff Proteomics Specialist)
Olga Lytovchenko, Ph.D.   (Staff Research Engineer)
Zhaowei Meng   (Staff LC-MS Engineer)
Qinyu Jia   (Associated HDX-MS Specialist)
Pan Fang, Ph.D.   (Staff HDX-MS Research Engineer)
Giorgia Palano, Ph.D   (Staff Research Engineer- Stem Cells and 3D cultures) 

Location and hours of operation

Links and Resources

1. ChemProt KI official site
2. Chemical Proteomics (National Facility) - SciLifeLab webpage
3. Chemical Protoemics -Swedish National Infrastructure for Biological Mass Spectrometry (BioMS)

Contacts