Quantitative, ultra-sensitive protein footprinting in living cells

Researchers at Stanford have developed a technique to quantitatively measure protein structure and interactions in living cells by protein "footprinting"—monitoring the solvent accessibility of each residue under different environmental conditions. In contrast to existing methods (HDX and hydroxyl radical footprinting), this approach combines high-throughput cysteine mutagenesis with a cell-permeable, enrichable, mass-tagged, irreversible thiol-alkylating reagent that enables highly accurate mass spectrometric quantification. With these advances, solvent accessibility can be measured at high resolution for virtually all of a protein's residues within complex environments, including living cells.

This kind of footprinting can be applied to many areas of biological study where it is useful to understand protein conformation and how that changes under different environmental stimuli, including the addition of a protein or small molecule binding partner. It is particularly valuable for proteins involved in large complexes or membrane proteins that are difficult to purify or otherwise reconstitute in vitro.

Related Technologies:
Stanford docket S19-003 – describing hydroxamate-based affinity tags with size and cost advantages for purifying biological macromolecules.

Stanford docket S19-005 – describing improved tags for separating crosslinked peptides for mass spectrometry.

Stanford docket S19-007 – describing a first-of-its-kind family of protein crosslinkers that can be cleaved and easily detected by mass spectrometry.

Stage of Development
The researchers have demonstrated that their footprinting method in live cells can map a dynamic protein-protein interface.