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