Are Charge-State Distributions a Reliable Tool Describing Molecular Ensembles of Intrinsically Disordered Proteins by Native MS?

Natalello, Antonino; Santambrogio, Carlo; Grandori, Rita

doi:10.1007/s13361-016-1490-1

Cited by 40 publications

(44 citation statements)

References 82 publications

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“…The intrinsically disordered nature of the protein appears to enable a high degree of charging that is more typical of globular proteins measured from denaturing solution conditions [46]. …”

Section: Resultsmentioning

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: Resultsmentioning

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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“…[47][48][49][50][51][52][53][54] The present review, instead, focuses specifically on CSD analysis for structural characterization of IDP/Rs. The reliability of this approach describing disordered conformational ensembles in solution has been object of debate, [55] but rapidly increasing evidence from the comparison with other methods provides support to structural interpretation of native-MS data collected on IDPs. Recent examples are discussed here and an MS-based index for IDP comparison and classification is described.…”

Section: Doi: 101002/pmic201800060mentioning

confidence: 99%

Conformational Characterization and Classification of Intrinsically Disordered Proteins by Native Mass Spectrometry and Charge‐State Distribution Analysis

et al. 2018

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Intrinsically disordered proteins (IDPs) are systematically under‐represented in structural proteomics studies. Their structural characterization implies description of the dynamic conformational ensembles populated by these polymers in solution, posing major challenges to biophysical methods. “Native” MS (native‐MS) has emerged as a central tool in this field, conjugating the unique MS analytical power with structurally meaningful descriptors, like solvent‐accessible surface area (SASA) and collisional cross section (CCS). This review summarizes recently published papers comparing native‐MS and solution methods, with a focus on charge‐state‐distribution (CSD) analysis for IDP conformational analysis. The results point to substantial agreement, supporting structural interpretation of native‐MS spectra of IDPs. The discussion is integrated with data from our group on “synthetic” IDPs, obtained by reduction and alkylation of natively folded proteins, whose fold is stabilized by disulfide bridges. Finally, an MS‐based compaction index (CI) is introduced, evaluating SASA with reference to globular and fully disorder proteins. Such a parameter can be calculated for single conformers or the whole conformational ensemble, offering a continuous index for IDP comparison and classification.

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“…More importantly, a lower charge state is preferable for maintaining the native folded conformation of the protein by lowering the Coulombic repulsion among surface charges. 37 The only experimental difficulty in pursuing the low charge state with a minimal acid concentration in our setup is clogging of the ESI capillary. Innovations in the ESI source for producing low charge more stable ions and for increasing ion fluxes are thus highly desirable.…”

Section: Discussionmentioning

confidence: 99%

Doping of Green Fluorescent Protein into Superfluid Helium Droplets: Size and Velocity of Doped Droplets

et al. 2017

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We report doping of green fluorescent protein from an electrospray ionization (ESI) source into superfluid helium droplets. From analyses of the time profiles of the doped droplets, we identify two distinct groups of droplets. The faster group has a smaller average size, on the order of 106 helium atoms/droplet, and the slower group is much larger, by at least an order of magnitude. The relative populations of these two groups depend on the temperature of the droplet source: from 11 K to 5 K, the signal intensity of the slower droplet group gradually increases, from near the detection limit to comparable to that of the faster group. We postulate that the smaller droplets are formed via condensation of gaseous helium upon expansion from the pulsed valve, while the larger droplets develop from fragmentation of ejected liquid helium. Our results on the size and velocity of the condensation peak at higher source temperatures (> 7 K) agree with previous reports, but those at lower temperatures (< 7 K) seem to be off. We attribute this discrepancy to the masking effect of the exceedingly large droplets from the fragmentation peak in previous measurements of droplet sizes. Within the temperature range of our investigation, although the expansion condition changes from subcritical to supercritical, there is no abrupt change in either the velocity distribution or the size distribution of the condensation peak, and the most salient effect is in the increasing intensity of the fragmentation peak. The absolute doping efficiency, as expressed by the ratio of ion doped droplets over the total number of ions from the ESI source, is on the order of 10−4, while only hundreds of doped ions have been detected. Further improvements in the ESI source are key to extending the technology for future experiments. On the other hand, the separation of the two groups of droplets in velocity is beneficial for size selection of only the smaller droplets for future experiments of electron diffraction.

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Are Charge-State Distributions a Reliable Tool Describing Molecular Ensembles of Intrinsically Disordered Proteins by Native MS?

Cited by 40 publications

References 82 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

Conformational Characterization and Classification of Intrinsically Disordered Proteins by Native Mass Spectrometry and Charge‐State Distribution Analysis

Doping of Green Fluorescent Protein into Superfluid Helium Droplets: Size and Velocity of Doped Droplets

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