Genomics:GTL

Protein production, identification

Post-translational modifications

Sequence of polymorphisms, isoforms

Cloning artifacts

Required cofactors, ligands, binding partners
(combinatorial approaches)

Stability (cofactors, ligands, binding partners)

Folding (cofactors, ligands, binding partners)

Storage and handling conditions

Mass spectrometry, affinity tag reaction (e.g., arrays, microfluidics, gels), light scattering, spectral matching (IR, UV), 1D/2D gels, liquid chromatography (e.g., affinity, ion exchange)

Centrifugation, light scattering, spectroscopy methods (UV, CD)

Screening level (UV-CD, dye binding, partial proteolysis/MS, isotope exchange/MS, FT-IR, SAXS/SANS, WAXS, EM)

Robotic HT combinatorial methods (e.g., pH, temperature, salts, buffers, solvents), test with stability diagnostics

Prepurification

(See items below under postpurification)

Postpurification

Binding partners; identification of reconstitution conditions, intermolecular interactions (dissociation constants)

Identification of monomeric or multimeric state

Probe of folding landscape, identification of motifs, folding stability, thermodynamics, ordered and disordered regions

Discovering substrates (orphan enzymes)

Identification of cofactors (e.g., metals, NADH, ATP, ligands)

Biological effect of post-translational modifications

Identification of DNA and RNA binding, sequence motifs

Assignment of function to proteins

HT screening: Dye binding, internal fluorescent labels, metabolite and molecular cocktails/MS (i.e., agonists and antagonists), affinity arrays, MS, biochemical and binding assays, ATP binding, kinase activity, affinity reagent effect on protein activity (neutral or inhibitory)

HT, high fidelity: Dye binding, internal fluorescent labels, metabolite and molecular cocktails/MS (i.e., agonists and antagonists), affinity arrays, MS, biochemical and binding assays, ATP binding, kinase activity

HT, high fidelity: UV-CD, dye binding, partial proteolysis/MS, isotope exchange/MS, FT-IR, fluorescence emission/lifetime (FlE/L), FRET, SAXS/SANS, WAXS, EM, calorimetry, size-exclusion chromatography coupled with laser light scattering (SEC-LLS)

Affinity reagent on protein activity (neutral or inhibitory)

Protein primary, secondary, tertiary, and quaternary structures

Structural-activity relations

Assignment of functions

Computational modeling and simulation

Analyses from GTL facilities

HT structural measurements: X-ray crystallography, NMR, cryoEM, scanning probe microscopy, FRET, single-molecule spectroscopies

Design of affinity reagents

Protein and molecular machine redesign or refinement

Pathway redesign

Engineering into nanomaterials and devices

Computational modeling and simulation

Analyses from GTL facilities

Functionalization of nanomaterials, synthetic biology, directed evolution

Microbial and cell-free systems design and engineering