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

Li, Huilin; Nguyen, Hong Hanh; Loo, Rachel R. Ogorzalek; Campuzano, Iain D. G.; Loo, Joseph A.

doi:10.1038/nchem.2908

Cited by 185 publications

(271 citation statements)

References 63 publications

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“…Native top-down MS can also reveal the specific site(s) of noncovalent ligand binding and the location of surface residues [31, 37–41]. ECD fragmentation dissociates covalent backbone bonds but preserves non-covalently bound ligands.…”

Section: Introductionmentioning

confidence: 99%

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

Nshanian

Lantz

Wongkongkathep

et al. 2018

J. Am. Soc. Mass Spectrom.

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Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer's disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. Graphical Abstract ᅟ.

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Section: Introductionmentioning

confidence: 99%

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

Nshanian

Lantz

Wongkongkathep

et al. 2018

J. Am. Soc. Mass Spectrom.

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“…Conversely, in “native” ExD, focus will shift toward larger, more challenging systems than the small monomeric proteins that have heretofore received most attention. This evolution is already apparent, with several recent reports of ECD and ETD of large noncovalent complexes and highly dynamic proteins . In this context, the specifics of the mechanism will surely continue to be investigated, and more sophisticated experimental methods such as interrogation of radical intermediates via action spectroscopy might provide more insight .…”

Section: Future Perspectivesmentioning

confidence: 97%

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

Lermyte

Valkenborg

Loo

et al. 2018

Mass Spectrometry Reviews

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130

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In recent years, electron capture (ECD) and electron transfer dissociation (ETD) have emerged as two of the most useful methods in mass spectrometry-based protein analysis, evidenced by a considerable and growing body of literature. In large part, the interest in these methods is due to their ability to induce backbone fragmentation with very little disruption of noncovalent interactions which allows inference of information regarding higher order structure from the observed fragmentation behavior. Here, we review the evolution of electron-based dissociation methods, and pay particular attention to their application in "native" mass spectrometry, their mechanism, determinants of fragmentation behavior, and recent developments in available instrumentation. Although we focus on the two most widely used methods-ECD and ETD-we also discuss the use of other ion/electron, ion/ion, and ion/neutral fragmentation methods, useful for interrogation of a range of classes of biomolecules in positive- and negative-ion mode, and speculate about how this exciting field might evolve in the coming years.

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“…The sign # denotes consecutive H2O or NH3 losses from bn or bn* fragments. 30 The bn and yn fragments that do not contain the PSS chain are attributed to fragmentation of the doubly protonated PLL-PSS complex by charge separation [92,93] which generates two charged fragments, one bound to PSS non-covalently and the other without the PSS.…”

Section: Instrumental Conditionsmentioning

confidence: 99%

“…Tandem mass spectrometry (MS 2 ) is employed extensively to investigate the noncovalent interactions in protein-protein and protein-ligand complexes [25][26][27][28][29][30]. Although the most widely mode of activation used in these studies has been collisionally activated dissociation (CAD) [29], newer MS 2 techniques such as electron capture dissociation (ECD) [12] and electron transfer dissociation (ETD) [14] have proven to be promising alternatives for the elucidation of non-covalent interactions [25,31,32].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of supramolecular peptide-polymer bioconjugates using multistage tandem mass spectrometry

Wei

Gerişlioğlu

Atakay

et al. 2019

International Journal of Mass Spectrometry

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Mass spectrometry (MS) is an essential analytical tool for the characterization of the structure of biological macromolecules, including protein-protein and protein-ligand complexes. One-dimensional MS separates gas-phase analyte ions based on their mass to charge ratio (m/z); however, to obtain more detailed structural information, tandem MS (MS/MS), which involves isolation and subsequent fragmentation of a precursor ion, is required.In this thesis, electrospray ionization multistage tandem mass spectrometry (ESI-MS n ) was employed to examine the non-covalent complexes between poly(styrene sulfonate) (PSS) and poly-L-lysine (PLL). During single-stage ion activation, the PLL peptide chain mainly underwent backbone cleavages without disruption of the noncovalent interaction which could only be broken via sequential application of electron transfer dissociation (ETD) and collisionally activated dissociation (CAD), indicating strong binding interactions between the two polyelectrolyte chains. Such binding properties make PSS a potential "non-covalent (supramolecular) label" for determining the surface accessibility of basic residues on a peptide or protein. To probe this premise, noncovalent complexes of substance P and PSS were characterized by ESI-MS n using different ion activation methods. Both MS 2 and MS 3 experiments on the substance P + PSS complex resulted in the formation of bn (on CAD) or cn (on ETD) fragments attached non-covalently to the intact PSS chain. All peptide fragments containing the intact PSS chain included Arg1, Lys3, and Gln5, pointing out that these residues, which are located near the Nterminus, are most likely involved in the noncovalent interaction with PSS. In contrast, iv Gln6 was excluded from this fragment series, attesting a much weaker interaction with PSS due to lesser accessibility. The strong tendency of PSS to bind peptides noncovalently at sites that can be elucidated by MS n demonstrates a proof-of-concept for the capacity of this approach to unveil higher order structure in proteins.

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

Cited by 185 publications

References 63 publications

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

Characterization of supramolecular peptide-polymer bioconjugates using multistage tandem mass spectrometry

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