Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry

Zöller, Jonathan; Hong, Sung Il; Eisinger, Martin Lorenz; Anderson, Malcolm; Radloff, Melanie; Desch, Kristina; Gennis, Robert B.; Langer, Julian D.

doi:10.1016/j.csbj.2022.09.036

Cited by 7 publications

(10 citation statements)

References 40 publications

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“…Here, all nine peptides from one-pass cIM exhibited intensity increases between 82 and 457% relative to their SYNAPT equivalents. This is consistent with previously published findings and is likely a significant factor contributing to the observed increase in peptide identification. The one-pass cIM data also showed a marked increase in precursor-product matching across all targets, thereby resulting in a greater proportion of peptides passing DynamX filtering (Figure S2d).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, via extended IM separations, cIM has potential to offer improved LC-MS peak capacity in bottom-up LC-MS applications such as HDX-MS. The design, operation, and multifunctional capabilities of cIM have been described in detail. , A handful of publications have now used the SELECT SERIES Cyclic IMS with cIM technology for the purpose of increasing LC-MS peak capacity during peptide mapping and other omics applications. − Despite this, to date, no systematic evaluation of cIM in HDX-MS has been performed nor has its multipass functionality been utilized online in any bottom-up LC-MS or omics application; only one-pass routines have previously been reported.…”

Section: Introductionmentioning

confidence: 99%

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Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications

Griffiths,

Anderson,

Richardson

et al. 2024

Anal. Chem.

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Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications

Griffiths,

Anderson,

Richardson

et al. 2024

Anal. Chem.

Self Cite

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“…Because of its outstanding efficiency, cyclic IMS was applied in various proteomics studies as well, both for peptide 56–62 and protein 27,63–69 analysis. This technique was shown to provide more identified peptides resulting in increased sequence coverage 56 . It was used to map ligand binding sites, 56 to differentiate isobaric synthetic peptides derived from biopharmaceuticals, 57 and to unambiguously assign the disulfide bonds of therapeutic mAb peptides 58 .…”

Section: Introductionmentioning

confidence: 99%

“…This technique was shown to provide more identified peptides resulting in increased sequence coverage 56 . It was used to map ligand binding sites, 56 to differentiate isobaric synthetic peptides derived from biopharmaceuticals, 57 and to unambiguously assign the disulfide bonds of therapeutic mAb peptides 58 . Further, cyclic IMS was found beneficial in the investigation of modified peptides to determine site localization (e.g., phosphorylation 59 or aspartic acid isomerization 60 ) and to unravel linkages in glycosylation 61,62 .…”

Section: Introductionmentioning

confidence: 99%

“…53 Separation of oligosaccharides resulted in a resolving power of approximately 120 using three passes, 54 while an estimated resolution of 920 could be achieved after 58 cycles. 55 Because of its outstanding efficiency, cyclic IMS was applied in various proteomics studies as well, both for peptide [56][57][58][59][60][61][62] and protein 27,[63][64][65][66][67][68][69] analysis. This technique was shown to provide more identified peptides resulting in increased sequence coverage.…”

Section: Introductionmentioning

confidence: 99%

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Optimum collision energies for proteomics: The impact of ion mobility separation

et al. 2023

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Ion mobility spectrometry (IMS) is a widespread separation technique used in various research fields. It can be coupled to liquid chromatography–mass spectrometry (LC–MS/MS) methods providing an additional separation dimension. During IMS, ions are subjected to multiple collisions with buffer gas, which may cause significant ion heating. The present project addresses this phenomenon from the bottom‐up proteomics point of view. We performed LC–MS/MS measurements on a cyclic ion mobility mass spectrometer with varied collision energy (CE) settings both with and without IMS. We investigated the CE dependence of identification score, using Byonic search engine, for more than 1000 tryptic peptides from HeLa digest standard. We determined the optimal CE values—giving the highest identification score—for both setups (i.e., with and without IMS). Results show that lower CE is advantageous when IMS separation is applied, by 6.3 V on average. This value belongs to the one‐cycle separation configuration, and multiple cycles may supposedly have even larger impact. The effect of IMS is also reflected in the trends of optimal CE values versus m/z functions. The parameters suggested by the manufacturer were found to be almost optimal for the setup without IMS; on the other hand, they are obviously too high with IMS. Practical consideration on setting up a mass spectrometric platform hyphenated to IMS is also presented. Furthermore, the two CID (collision induced dissociation) fragmentation cells of the instrument—located before and after the IMS cell—were also compared, and we found that CE adjustment is needed when the trap cell is used for activation instead of the transfer cell. Data have been deposited in the MassIVE repository (MSV000090944).

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Developments in rapid hydrogen–deuterium exchange methods

Chow

Wolf

Lento

et al. 2023

Essays in Biochemistry

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Biological macromolecules, such as proteins, nucleic acids, and carbohydrates, contain heteroatom-bonded hydrogens that undergo exchange with solvent hydrogens on timescales ranging from microseconds to hours. In hydrogen–deuterium exchange mass spectrometry (HDX-MS), this exchange process is used to extract information about biomolecular structure and dynamics. This minireview focuses on millisecond timescale HDX-MS measurements, which, while less common than ‘conventional’ timescale (seconds to hours) HDX-MS, provide a unique window into weakly structured species, weak (or fast cycling) binding interactions, and subtle shifts in conformational dynamics. This includes intrinsically disordered proteins and regions (IDPs/IDRs) that are associated with cancer and amyloidotic neurodegenerative disease. For nucleic acids and carbohydrates, structures such as isomers, stems, and loops, can be elucidated and overall structural rigidity can be assessed. We will provide a brief overview of technical developments in rapid HDX followed by highlights of various applications, emphasising the importance of broadening the HDX timescale to improve throughput and to capture a wider range of function-relevant dynamic and structural shifts.

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Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry

Cited by 7 publications

References 40 publications

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications

Optimum collision energies for proteomics: The impact of ion mobility separation

Developments in rapid hydrogen–deuterium exchange methods

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