Chapter 10
Hands on Native Mass Spectrometry Analysis
of Multi-protein Complexes
Ste ´ phane Erb, Sarah Cianfe ´ rani, and Julien Marcoux
Abstract
By maintaining intact multi-protein complexes in the gas-phase, native mass spectrometry provides their
molecular weight with very good accuracy compared to other methods (typically native PAGE or
SEC-MALS) (Marcoux and Robinson, Structure 21:1541–1550, 2013). Besides, heterogeneous samples,
in terms of both oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly
describe the analysis of several oligomeric protein complexes ranging from a 16 ¼ kDa dimer to a 801-kDa
tetradecameric complex on different instrumental setups.
Key words Structural mass spectrometry, Noncovalent mass spectrometry, Oligomeric states, Stoichiometry, Subcomplexes
1 Introduction
Mass spectrometry (MS) plays a pivotal role for the characterization
of multi-protein complexes. Routinely coupled with reversed-phase
liquid chromatography (LC-MS), it allows purity and homogeneity
assessment of biological complexes. Classical LC-MS workflow
requires organic solvents (usually H 2 O/acetonitrile) and acidic
conditions (e.g., acetic or formic acid) to achieve best
chromatographic resolutions along with optimal MS detection.
However, those experimental conditions induce dissociation of
multi-protein noncovalent complexes. In the early 1990s, MS
approaches performed in ammonium acetate buffer maintaining
quaternary structures of multi-protein complexes have been first
described [1–3], opening the way for a new field of application
called noncovalent or “native” MS [4, 5]. By transferring intact
noncovalent assemblies into the gas phase of the mass spectrometer, native MS enables multi-protein complex stoichiometry assessment thanks to accurate mass measurements. Mostly performed on
TOF and Q-TOF [6] instruments in its early years, it has recently
benefited from technological improvements to reach highArnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
173
Hands on Native Mass Spectrometry Analysis
of Multi-protein Complexes
Ste ´ phane Erb, Sarah Cianfe ´ rani, and Julien Marcoux
Abstract
By maintaining intact multi-protein complexes in the gas-phase, native mass spectrometry provides their
molecular weight with very good accuracy compared to other methods (typically native PAGE or
SEC-MALS) (Marcoux and Robinson, Structure 21:1541–1550, 2013). Besides, heterogeneous samples,
in terms of both oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly
describe the analysis of several oligomeric protein complexes ranging from a 16 ¼ kDa dimer to a 801-kDa
tetradecameric complex on different instrumental setups.
Key words Structural mass spectrometry, Noncovalent mass spectrometry, Oligomeric states, Stoichiometry, Subcomplexes
1 Introduction
Mass spectrometry (MS) plays a pivotal role for the characterization
of multi-protein complexes. Routinely coupled with reversed-phase
liquid chromatography (LC-MS), it allows purity and homogeneity
assessment of biological complexes. Classical LC-MS workflow
requires organic solvents (usually H 2 O/acetonitrile) and acidic
conditions (e.g., acetic or formic acid) to achieve best
chromatographic resolutions along with optimal MS detection.
However, those experimental conditions induce dissociation of
multi-protein noncovalent complexes. In the early 1990s, MS
approaches performed in ammonium acetate buffer maintaining
quaternary structures of multi-protein complexes have been first
described [1–3], opening the way for a new field of application
called noncovalent or “native” MS [4, 5]. By transferring intact
noncovalent assemblies into the gas phase of the mass spectrometer, native MS enables multi-protein complex stoichiometry assessment thanks to accurate mass measurements. Mostly performed on
TOF and Q-TOF [6] instruments in its early years, it has recently
benefited from technological improvements to reach highArnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
173