Detecting Protein–Small Molecule Interactions Using Limited Proteolysis–Mass Spectrometry (LiP-MS)

Protein–small molecule interactions control many cellular processes, such as allosteric regulation of enzyme activity. One method to study protein–small molecule interactions in a complex sample is limited proteolysis–mass spectrometry (LiP-MS). In this approach, a small molecule is added to a complex sample such as a cell lysate, which is then briefly incubated with a nonspecific protease. Since binding of a small molecule can locally alter the protease accessibility of its target proteins, this yields condition-specific cleavage products. The size of the fragments is then reduced by secondary digestion with trypsin and peptide abundances are measured by MS, yielding structural fingerprints for all detected proteins. Comparing structural fingerprints plus and minus the small molecule identifies target proteins and enables pinpointing of binding sites. Use of a concentration gradient of the small molecule can further distinguish between proteins with different relative binding affinities. Peptide-level resolution: Alterations in structural fingerprints can be identified with a resolution of ~12 amino acids, suggesting small-molecule-binding sites. Enables the generation of mechanistic hypotheses: Detects and discriminates among different types of protein–small molecule interactions, including allosteric, enzyme–substrate, enzyme–product, and drug–target interactions. In situ analysis: Structural changes are probed in a native-like environment such as a cell lysate. Proteome-wide scale: Structural fingerprints are simultaneously measured for all MS-detectable proteins in a sample. Broad applicability: The method is not limited to detecting protein–small molecule interactions but can be applied to identify different types of protein structural changes upon a range of perturbations, such as temperature or growth medium. Requires high sequence coverage of the target protein: To identify a binding site, the peptide at the binding interface must be detected by MS. Thus, the method is biased towards medium/high-abundance proteins. Conformational changes hamper binding site identification: If the small molecule induces a structural change beyond the binding region, the interaction is detected but the binding site cannot be accurately pinpointed. Binding may not alter protease susceptibility: Target identification is based on the principle that small-molecule binding alters the protease accessibility of the target protein. If this does not occur, the target cannot be identified. P.P. is funded by a Personalized Health and Related Technologies (PHRT) grant (PHRT-506), a Sinergia grant from the Swiss National Science Foundation (SNSF grant CRSII5_177195), and an ERC consolidator grant (866004).