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Native MS: investigating macromolecular complexes
Elisabetta Boeri Erba
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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
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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
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I. RIP2 kinase: stoichiometry
10 Pellegrini E, Signor L, Singh S, Boeri Erba E, Cusack S. PLoS One. 2017
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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
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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
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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
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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
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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
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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.
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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
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21 Longchamp et al. PNAS 2017
MS separation soft-landing of proteins onto surfaces imaging of individual proteins
Imaging of soft-landed proteins
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Solution VS gas phase structure
22 Boeri Erba et al. Anal Chem. 2011.
Influence on oligomeric state: •Solution pH •Solution concentration
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Solution VS gas phase abundance
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• Changing solution conditions: different peak intensities
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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
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Information by native MS
Boeri Erba E, Petosa C. Protein Sci. 2015, 24(8):1176-92 26