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An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes


Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

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Figure 1: Macromolecular protein complex with molecular weights up to 1.86 MDa can be transmitted and detected by FTICR mass spectrometry under native ESI condition.
Figure 2: Structure of glutamate dehydrogenase.
Figure 3: Native top-down MS analysis of GP.
Figure 4: Structure of bovine glutamate dehydrogenase (PDB 1HWZ).
Figure 5: Native top-down MS analysis of GDH.
Figure 6: Native top-down MS analysis of β-galactosidase.
Figure 7: Native top-down ECD reveals the structural features of protein complexes.

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The authors thank C. Wan and R. Malmirchegini for discussions and M. Penichet for the hTfR sample. The authors acknowledge support from the US National Institutes of Health (R01 GM103479 and S10 RR028893 to J.A.L.), the US Department of Energy (UCLA/DOE Institute for Genomics and Proteomics; DE-FC03-02ER63421) and the American Society for Mass Spectrometry Postdoctoral Research Award (to H.L.).

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H.L. and J.A.L. conceived and designed the experiments. H.L. performed experiments and analysed data. H.H.N. contributed to analysis tools. I.D.G.C. prepared and contributed the GroEL sample. H.L., R.R.O.L. and J.A.L co-wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Huilin Li or Joseph A. Loo.

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The authors declare no competing financial interests.

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Li, H., Nguyen, H., Ogorzalek Loo, R. et al. An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes. Nature Chem 10, 139–148 (2018).

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