Chemical Proteomics [CCDP™]

The binding interactions of small compounds to proteins within the cell is paramount to understanding a potential therapeutic compound's mechanism of action. It is therefore necessary to have tools and methods to survey the promiscuous behavior of compounds towards multiple proteins and posit such behavior as deterministic of either toxicity or efficacy (poly-pharmacology). We offer a new method for this purpose, called Compound- centric Displacement Proteomics (CCDP).

With CCDP, proteins are first non-selectively, and reversibly bound to a novel bead format. A subset of proteins can then be displaced upon introduction of soluble small compounds. Coupled to LC-MS, quantitative metrics of these affinity-eluted sub-proteomes help characterize and identify interacting proteins. These new methods gain efficiencies over prior covalent- based substitution methods and can serve applications in drug target deconvolution, on-target/off-target specificity, poly-pharmacology, and personalized medicine.

Client supplies:

typically 200 – 2000 µl of sample with a total soluble protein content between

Client supplies:

typically 200 – 2000 µl of sample with a total soluble protein content between 1 and 2 mg.

Our service includes:

Sample preparation – We verify that the protein amount is suitable for the analysis, and the sample is sufficiently clarified. We use the NuGel™ NRicher™ Mx product to capture the protein on bead.

Compound Displacement – The bead – bound proteome is thoroughly washed and at least three separations are performed in parallel.

A negative control, using only the final wash buffer.

A negative compound control, typically this is a non-specific chemical like Caffeine, at the same molarity as the challenge compound, in the same negative control buffer.

One (or more) challenge compounds at the same molarity, in the same negative control buffer.

LC/MS/MS (out-sourced Rutgers Center for Innovative Proteomics) – A nanoLC-MS/MS coupling a RSLC system (Dionex, Sunnyvale CA) interfaced with a LTQ Orbitrap Velos (ThermoFisher, San Jose, CA) with resolution of 60,000 FWHM. A single 2 hour gradient is used unless otherwise specified.

Data analysis: The data is searched against the species respective Ensembl databases using X!tandem with carbamidomethylation on cysteine as fixed modification and oxidation of methionine and deamidation on Asparagine as variable modifications using a 10 ppm precursor ion tolerance and a 0.4 Da fragment ion tolerance. Protein identifications that meet a suitable quality tolerance are compiled and reported with gene identifiers. The spectral counts of each identified protein is tabulated and relative ratios are calculated. This is the quantitative metric to compare protein amounts from the different displacement pools.

A Final Report – A full report including methods, data analysis and client responsive goals is prepared.