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Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis

Abstract

The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts ‘to mark’ the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast photochemical oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a ‘snapshot’ of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography–tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.

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Fig. 1: Major applications of FPOP.
Fig. 2: Schematic illustration of the laser optics setup.
Fig. 3: Red arrow flag for checking the laser alignment.
Fig. 4: Schematic illustration of a two-valve LC system.
Fig. 5: Schematic illustration of the FPOP workflow.
Fig. 6: Anticipated FPOP results.

Data availability

A calmodulin data set that contains a global-level measurement by Bruker maXis and a peptide- and residue-level measurement by Thermo Fisher Scientific Q Exactive is freely available online in the Mendeley data (https://data.mendeley.com/) with DOI: 10.17632/xfd5sh76pm.1.

Software availability

The Protein Metrics Suite is commercially available at https://www.proteinmetrics.com.

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Acknowledgements

This work was supported by National Institutes of Health National Institute of General Medical Sciences grants 5P41GM103422 and R24GM135766. Instrumentation was supplied by 1S10OD016298-01A1 (to M.L.G.). We are grateful to H. Rohrs for help in identifying parts for the HPLC and to Protein Metrics for software support.

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Authors

Contributions

X.R.L., D.L.R. and M.L.G. developed the protocol. X.R.L. and M.L.G. wrote and edited the manuscript.

Corresponding authors

Correspondence to Xiaoran Roger Liu or Michael L. Gross.

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Competing interests

The authors declare an ongoing collaboration with Protein Metrics in establishing an HDX data processing platform, a topic that is not related to this protocol. The recommendation of using Protein Metrics as preferred data processing software for FPOP data predates the ongoing collaboration. M.L.G. is an unpaid member of the scientific advisory board of GenNext Technologies, which provides products and services for hydroxyl radical footprinting.

Additional information

Peer review information Nature Protocols thanks Richard W. Vachet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Hambly, D. M. et. al. J. Am. Soc. Mass Spectrom. 16, 2057–2063 (2005): https://doi.org/10.1016/j.jasms.2005.09.008

Espino, J. A. et. al. Anal. Chem. 87, 7971–7978 (2015): https://doi.org/10.1021/acs.analchem.5b01888

Li, K. S. et. al. J. Am Chem. Soc. 138, 12090–12098 (2016): https://doi.org/10.1021/jacs.6b07543

Niu, B. et. al. J. Am. Soc. Mass Spectrom. 28, 389–392 (2017): https://doi.org/10.1007/s13361-016-1552-4

Liu, X. R. et. al. Anal. Chem. 91, 12560–12567 (2019): https://doi.org/10.1021/acs.analchem.9b03491

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Liu, X.R., Rempel, D.L. & Gross, M.L. Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis. Nat Protoc 15, 3942–3970 (2020). https://doi.org/10.1038/s41596-020-0396-3

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