Light‐activated protein‐imprinted hydrogels capable of translating protein surface accessibility into missing‐peptide footprints

Mass spectrometry (MS)-based structural proteomics can reveal higher-order structural features in complex biological samples, but many covalent footprinting and crosslinking strategies require specialized workflows to identify modified or crosslinked species. Here, we introduce a hydrogel-based strategy that converts protein surface accessibility into a simple signal-off readout in standard label-free LC-MS/MS. We synthesize protein-permeable polyethylene glycol molecularly imprinted hydrogels (MIHs) and install perfluorophenyl azide (PFPA) photoreactive groups after polymerization. When proteins are adsorbed to the imprinted cavities, UV irradiation crosslinks nearby protein surface segments to the gel. After extensive washing and in-gel digestion, peptides from crosslinked surface regions remain trapped in the gel matrix and are therefore depleted from the recovered digest, yielding a “missing-peptide” footprint without explicit identification of crosslinked species. Lysozyme-imprinted MIHs retain template recognition and report ligand-dependent protection of a carbohydrate-binding loop. A proteome-imprinted MIH generates > 3000 protein structural fingerprints from ~ 500 ng of HEK293T lysate and detects rapamycin-dependent changes in FK506-binding proteins (FKBPs), including an FKBP4 peptide near the rapamycin-binding interface. PFPA-functionalized MIHs thus provide an aqueous solid-phase transducer that makes structural proteomics compatible with routine bottom-up workflows.