Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale

Abstract

Identification and characterization of small molecule–protein interactions is critical to understanding the mechanism of action of bioactive small molecules. Photo‐affinity labeling (PAL) enables the capture of noncovalent interactions for enrichment and unbiased analysis by mass spectrometry (MS). Quantitative proteomics of the enriched proteome reveals potential interactions, and MS characterization of binding sites provides validation and structural insight into the interactions. Here, we describe the identification of the protein targets and binding sites of a small molecule using small molecule interactome mapping by PAL (SIM‐PAL). Cells are exposed to a diazirine‐alkyne‐functionalized small molecule, and binding interactions are covalently captured upon UV irradiation. An isotopically coded, acid‐cleavable biotin azide handle is attached to the conjugated proteins using copper‐catalyzed azide‐alkyne cycloaddition. Biotin‐labeled proteins are enriched for on‐bead digestion and quantitative proteomics. Acid cleavage of the handle releases the bead‐bound conjugated peptides for MS analysis and isotope‐directed assignment of the binding site. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a small molecule–conjugated protein sample following treatment of live cells Alternate Protocol: Generation of a small molecule–conjugated protein sample following treatment of cell lysate Basic Protocol 2: Copper‐catalyzed azide‐alkyne cycloaddition functionalization and enrichment of labeled peptides Support Protocol 1: Synthesis of acid‐cleavable, isotopically coded biotin picolyl azide handle Support Protocol 2: Monitoring enrichment by immunoblotting Basic Protocol 3: Mass spectrometry analysis to identify interacting proteins and conjugation sites

Chemoenzymatic Probes Reveal Peptidoglycan Recognition and Uptake Mechanisms in Candida albicans

“…With the 940 muropeptide and LdtA enzyme in hand, we next focused on the preparation of functional d -amino acids to be installed. Initially, we conceived making the bifunctional-940 probe containing both a minimalist diazirine photo-crosslinker and a bio-orthogonal alkyne, as such moiety has been widely used for target identification of small-molecule ligands . For instance, MDP-based photoaffinity probes such as x-alk-MDP- L,D were synthesized and used in chemo-proteomic studies in mammalian cells to successfully reveal the cognate NOD2 target .…”

“…Initially, we conceived making the bifunctional-940 probe containing both a minimalist diazirine photocrosslinker and a bio-orthogonal alkyne, as such moiety has been widely used for target identification of small-molecule ligands. 32 For instance, MDP-based photoaffinity probes such as x-alk-MDP-L,D were synthesized and used in chemoproteomic studies in mammalian cells to successfully reveal the cognate NOD2 target. 33 However, no such probes have been made on natural Gram-negative peptidoglycan fragments such as 940 muropeptide.…”

Protein Chemistry Combined with Mass Spectrometry for Protein Structure Determination

“…We have also observed a higher yield of the modification reaction and a more narrow distribution of the modification sites in the target protein sequences for reactions in frozen samples compared to when the reactions were conducted at ambient temperature. The method can be applied on a proteome-wide scale for the identification of the drug target protein candidates, especially if additional affinity, cleavable, and mass spectrometry identifiable reporter groups have been incorporated into the structure of the photoreactive probe conjugates. , The modified sites determined by photoaffinity labeling can be used to generate ligand–protein distance constraints in flexible-docking ligand–protein structure modeling …”

“…The method can be applied on a proteome-wide scale for the identification of the drug target protein candidates, especially if additional affinity, cleavable, and mass spectrometry identifiable reporter groups have been incorporated into the structure of the photoreactive probe conjugates. 41,42 The modified sites determined by photoaffinity labeling can be used to generate ligand−protein distance constraints in flexible-docking ligand− protein structure modeling. 43…”

Proteomics in the pharmaceutical and biotechnology industry: a look to the next decade

“…This is due to the low insertion efficiency of available photo-reactive moieties as well as the fact that the carbene radical-based, random insertion process tends to give rise to a mixture of molecular modification products even for a single binding pocket and a given peptide sequence. Apart from improved data analysis strategies, experimental workflows have been introduced to aid with this process, e.g., the SIM-PAL workflow which uses introduction of unique isotopic patterns to identify probe-labeled peptides in digested enriched samples [87].…”