Post on 25-Apr-2020
Native MS: investigating macromolecular complexes
Elisabetta Boeri Erba
Outline
• MS analyses of intact proteins
• Native MS:
- Assessment of stoichiometry
- Dynamics of macromolecular complexes
- Gas-phase vs solution results Boeri Erba E*. Proteomics, 2014. Boeri Erba E*, Petosa C. Protein Science, 2015. Boeri Erba E*, Signor L, Oliva M F, Hans F, Petosa C. Methods in Molecular Biology, 2018
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MS: different types • Proteomics: digestion • Denaturing MS: intact proteins
• Native MS: map of assemblies
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LC-ESI-TOF: <100 kDa
MALDI-TOF: >100 kDa
Boeri Erba E*, Signor L, Oliva M F, Hans F, Petosa C. Methods in Molecular Biology, 2018
MS analyses of intact proteins
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D-, 13C15N- labeling of TTR
5 Experiments done by Luca Signor Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem Int Ed Engl. 2016, 55(32):9292-6
LC-ESI-TOF
Pellegrini E, Signor L, Singh S, Boeri Erba E, Cusack S. PLoS One. 2017
Intact proteins: degree of phosphorylation LC-ESI-TOF
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Outline
• MS analyses of intact proteins • Native MS
- Assessment of stoichiometry: human RIP2 kinase - Dynamics of macromolecular complexes
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Pellegrini E, Signor L, Singh S, Boeri Erba E, Cusack S. PLoS One. 2017
LC-ESI-TOF
I. Inactive and active RIP2 kinase
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I. RIP2 kinase: phosphorylation sites
MALDI-TOF
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LC-ESI-TOF
Pellegrini E, Signor L, Singh S, Boeri Erba E, Cusack S. PLoS One. 2017
I. RIP2 kinase: stoichiometry
10 Pellegrini E, Signor L, Singh S, Boeri Erba E, Cusack S. PLoS One. 2017
Outline
• MS analyses of intact proteins • Native MS
- Assessment of stoichiometry - Dynamics of macromolecular complexes
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TTR (transthyretin)
• Fibrillation behaviour:
point mutations: T119M and S52P
Tafamidis, treatment of familial amyloid polyneuropathy
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• 4mer in blood plasma and cerebrospinal fluid
• Transport of the thyroxine and vitamin A
• Aggregation into amyloid fibrils
Trevor Forsyth, ILL, Grenoble Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem, 2016 Yee AW…, Boeri Erba E, Forsyth VT. Nat. Comm, 2019
D-, 13C15N-TTR labeling
13 Experiments done by Luca Signor Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem Int Ed Engl. 2016, 55(32):9292-6
LC-ESI-TOF
Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem Int Ed Engl. 2016, 55(32):9292-6
Structures of H- and D-TTR
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Backbone atom alignment
Exchange of TTR subunits
15 Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem, 2016 Yee AW…, Boeri Erba E, Forsyth VT. Nat. Comm, 2019
Nano-ESI-Q-TOF
Exchange of TTR subunits
16 Yee AW…, Boeri Erba E, Forsyth VT. Angew Chem, 2016 Yee AW…, Boeri Erba E, Forsyth VT. Nat. Comm, 2019
Nano-ESI-Q-TOF
Neutron structures of TTR mutants
17 Yee AW…, Boeri Erba E, Forsyth VT. Nat. Comm, 2019
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Is the gas-phase structure similar to the structure in solution?
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• Removal of water for milliseconds • No immediate unfolding of protein and
protein complexes
Is the gas-phase structure similar to the structure in solution?
Meyer et al. 2009 An atomistic view to the gas phase proteome. Structure 17, 88–95.
20 Ouyang et al.Science 301, 1351 (2003)
After MS separation, soft-landing of proteins onto surfaces
Soft-landed enzymes retained biological activity
Lysozyme and trypsin retain their activity after the ionization, mass selection, and soft-landing processes
21 Longchamp et al. PNAS 2017
MS separation soft-landing of proteins onto surfaces imaging of individual proteins
Imaging of soft-landed proteins
Solution VS gas phase structure
22 Boeri Erba et al. Anal Chem. 2011.
Influence on oligomeric state: •Solution pH •Solution concentration
Solution VS gas phase abundance
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• Changing solution conditions: different peak intensities
Conclusions
I. Effect of point mutations
II. Dynamics of complexes
• MS analyses of intact proteins
• Native MS
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Acknowledgments • Luca Signor • Carlo Petosa • Stephen Cusak • Erika Pellegrini
• Ai Woon Yee • Trevor Forsyth • Cécile Breyton • Nathan Epalle
Information by native MS
Boeri Erba E, Petosa C. Protein Sci. 2015, 24(8):1176-92 26