Macromolecular Structure/Chemoproteomics Joint Seed Grant

Applications accepted for Track 1 (budget of $50,000) or Track 2 (budget of up to $25,000) split between Macromolecular Structure and Chemoproteomics

Identifying the molecular targets and binding sites of bioactive compounds represents a major challenge in therapeutic development. Mass spectrometry-based proteomics provides powerful approaches to assess the biological effects of candidate drugs, it allows the identification of target proteins, as well as the elucidation of interaction sites on a proteome-wide scale. Complementary structural biology techniques, such as biophysical analysis of the ligand-protein interaction and estimation of the binding affinity of the complex, as well as changes in the target protein's ternary and quaternary architecture in complex with the ligand, are integrated with X-ray crystallography to further characterize biomolecular interactions. Together, these methodologies provide a deeper understanding of drug-target interactions across the proteome and they offer detailed molecular insights on a molecular level that can guide the characterization and design of effective therapies.

Successful proposals should seek to identify and validate interactors of biomolecular targets of interest in therapeutics, engineering, or technological development.

The Macromolecular Structure part of the proposal should include the following to characterize a protein in complex with interactors.

  • Interactors: the molecules to be tested as ligands will be provided by the applicant. Although preliminary data demonstrating the binding capacity of the ligand to a target biomolecule is not required, hypotheses on how the ligand would interact with the target should be put forward.
  • Cell Cultures: bacterial, mammalian, or baculovirus expressing the target biomolecule recombinantly. Cell strains should be provided by the applicant or would be cultured in the facility (provided a BSL2 or lower).
  • Protein Expression: protein samples will be made available as an inducible plasmid that encodes the 6x His-tagged target protein. This plasmid can be used to overexpress the target protein in a suitable host cell strain (preferably, but not restricted to, E. coli).
  • Protein Purification: the target protein will be isolated to high purity using chromatographic methods. Protein quality control will be performed with electrophoresis, dynamic light scattering (DLS), and mass photometry (MP).
  • Binding Affinity Assay: the target protein will be assayed in native state for binding affinity towards the interactor using microscale thermophoresis (MST) or MP. Alternatively, the inhibitory activity of the interactor will be tested using absorbance-based assays or fluorescently based methods like differential scanning fluorimetry (DSF).
  • Structural Biology: the target protein and the interactor binding in complex will be analyzed at the molecular level using X-ray crystallography.

Macromolecular Structure in-kind support will include one-on-one training, consumables, and access to instrumentation at each stage of the protein production, purification, crystallization, and characterization.

 

The Chemoproteomics part of the proposal should focus on protein characterization with LC-MS/MS (select one or two of the methods below):

  • Protein abundance profiling: cellular responses to compound/protein/stress condition of interest (increase or decrease of protein abundance) can be analyzed with DDA or DIA LC-MS/MS
  • Activity-based protein profiling: interaction sites of probes analyzed with DDA or DIA LC-MS/MS
  • PTM profiling: post-translational modifications and how they change in response to a compound/protein/stress condition analyzed with DDA (or DIA) LC-MS/MS
  • Interactome profiling: perform a pull-down (affinity purification or immunoprecipitation) experiment and characterize interactors with DDA or DIA-LC-MS/MS
  • Identify conformational changes/binding sites: perform a Limited proteolysis-coupled MS experiment to characterize proteome-wide conformational changes as a response to a compound/protein/stress condition. This method can also be used to narrow down interactors and identify binding interfaces
  • Thermal Stability Proteomics: changes in protein stability upon interaction can be studied with thermal proteome profiling MS
  • Absolute protein quantification: Absolute quantification of proteins of interest (heavy labeled standard peptides will have to be purchased separately)
  • Or other LC-MS/MS-based protein characterizations: Maybe the method you were thinking of is not covered in this list? Describe what you would like to do in your proposal.

Chemoproteomics in-kind support will include one-on-one training, sample preparation, LC-MS/MS measurement time, data analysis (access to licensed software, help with coding for data analysis), and project-associated consumables.

The proposal must identify at least one graduate student or postdoctoral scholar who will devote at least 20% of time to the project. The student or postdoctoral scholar should expect to spend that time in the Nucleus lab space and further analyzing data remotely.

Questions about proposal development should be directed to Dina Schuster (Chemoproteomics, dschust@stanford.edu) and Daniel Fernandez (Macromolecular Structure, danilo@stanford.edu).