Abstract
Understanding how proteins and materials interact is useful for evaluating the safety 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. This protocol describes the lysine reactivity profiling-mass spectrometry strategy for determining which parts of a protein are interacting with the micro/nanomaterials. Lysine residues occur frequently on hydrophilic protein surfaces, and their reactivity is dependent on the accessibility of their amine groups. The accessibility of a lysine residue is lower when it is in contact with another object; allosteric effects resulting from this interaction might reduce or increase the reactivity of remote lysine residues. Lysine reactivity is therefore a useful indicator of protein localization orientation, interaction sequence regions, binding sites and modulated protein structures in the protein-material hybrids. We describe the optimized 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 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 ~5 h, and the whole protocol including digestion, liquid chromatography-tandem mass spectrometry, data processing and structure analysis needs ~3–5 d.
Key points
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Lysine residues occur frequently on hydrophilic protein surfaces. The reactivity of lysine residues to dimethylation depends on their accessibility. Reaction conditions can be optimized such that the heterogeneity of lysine reactivity is reflected by labeling efficiency.
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In combination with mass spectrometry, this approach—lysine residue profiling-mass spectrometry—can be used to determine interfacial molecular details for protein-protein and protein-material complexes.
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Data availability
The key data used in this protocol as examples or anticipated results are available within the paper and at https://doi.org/10.5281/zenodo.7697479. All relevant figures are available within the paper and at https://doi.org/10.6084/m9.figshare.22199440. Any additional data that supports the findings can be provided from the corresponding author upon request.
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Acknowledgements
We acknowledge financial support from the National Key R&D Program of China (2022YFC3400502), the National Natural Science Foundation of China (32088101, 22288201, 92253304 and 22204165) and grants from DICP (DICPI202242). The authors acknowledge the technological support of the biological mass spectrometry station of the Dalian Coherent Light Source.
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Z.L., S.Y., L.Z. and F.W. developed the protocol and wrote the manuscript. M.H. and Y.B. optimized the protocol. S.Z. contributed to the manuscript modification.
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Nature Protocols thanks Junfeng Ma and the other, anonymous, reviewer(s) for their contribution.
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Key references using this protocol
He, M. et al. J. Energy Chem. 70, 437–443 (2022): https://doi.org/10.1016/j.jechem.2022.03.002
Shang, X. et al. Nat. Catal. 4, 607–614 (2021): https://doi.org/10.1038/s41929-021-00654-6
Zhang, G. et al. Nat. Nanotechnol. 17, 993–1003 (2022): https://doi.org/10.1038/s41565-022-01177-2
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Liu, Z., Yang, S., Zhou, L. et al. Structural characterization of protein-material interfacial interactions using lysine reactivity profiling-mass spectrometry. Nat Protoc 18, 2600–2623 (2023). https://doi.org/10.1038/s41596-023-00849-0
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DOI: https://doi.org/10.1038/s41596-023-00849-0
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