PROBE-Select: A Web Server for Selecting Optimal Protein-Specific Labeling Reagents to Maximize Structural Information Obtained from Covalent Labeling Mass Spectrometry

Covalent labeling mass spectrometry (CL-MS) reports the solvent exposure of individual residues, but how much structural information an experiment returns depends on which labeling reagent is used. Currently, there are no tools available to guide the reagent choice. Here, we present PROBE-Select (PRotein-Oriented Best-reagent Evaluator for Selective labeling), a computational workflow that selects labeling reagents for a given protein sequence and is available as a web application on the Rosetta Online Server that Includes Everyone (ROSIE). Starting from a sequence, the workflow generates a ColabFold ensemble of candidate structures under diversity-promoting settings, removes models dominated by nonregular secondary structure, clusters the remaining models, and selects three representative conformations. The workflow then retains as reporters all residues whose predicted solvent exposure differs between at least two of the three representatives by more than a threshold T. Reporters are mapped to compatible labeling reagents, and each reagent is scored by the number of reporters it can label for each conformation pair. On a benchmark of 11 proteins with experimentally characterized alternative conformations, the workflow identified 182 reporter residues at T = 5. The number of reporters increased with the structural separation between representatives, and the number of reporters available to each reagent differed between proteins and between pairs of representatives. Diethylpyrocarbonate was the top-ranked residue-selective reagent in 8 of the 10 benchmark proteins that had reporters. PROBE-Select returns the reagent ranking, the residue-level exposure data behind it, and the three representative structures. PROBE-Select is freely available for academic and non-profit use, and can be accessed with a GitHub account via this link ( r2.graylab.jhu.edu/apps/submit/probe-select ).