Structural characterization of the protein-material interfacial interactions by using lysine reactivity profiling-mass spectrometry

The exploration of interfacial molecular interactions of protein-material integrations and how material modulate the protein structure and activity are essential to the safety evaluation of biomedical micro/nanomaterials, toxicity estimation and design of nano-drugs, and catalytic activity improvement of bio-inorganic functional hybrids. However, characterizing the interfacial molecular details of protein-micro/nanomaterial hybrids remains a great challenge. Herein, we introduce the protocol of lysine reactivity profiling-mass spectrometry (LRP-MS) strategy for probing the interfacial molecular structures between proteins and micro/nanomaterials. LRP-MS utilizes lysine residues as the endogenous probes to characterize the protein localization orientation, interaction sequence regions, binding sites, and modulated protein structures in the protein-material hybrids, which cannot be achieved by traditional spectroscopy methods. We describe the optimized heavy and light two-step isotope dimethyl labeling strategy for protein-material hybrids under their native and denaturing conditions in sequence. The comparative quantification results of lysine reactivity (referred as NLE) are only dependent on the native microenvironments of lysine local structures. We also highlight other critical steps including protein digestion, elution from materials, data processing, and interfacial structure analysis. The two-step isotope labeling steps need about 5 h, and the whole protocol including digestion, liquid chromatography-tandem mass spectrometry, data processing, and structure analysis needs about 3-5 days.