Using hydrogen-deuterium exchange mass spectrometry to characterize Mtr4 interactions with RNA

Ball, Darby; Nguyen, Trang Vu Thien; Zhang, Naifu; D’Arcy, Sheena

doi:10.1016/bs.mie.2022.04.002

Cited by 6 publications

(5 citation statements)

References 86 publications

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“…We should also note that the H/D exchange efficiency has been reported to be pH-dependent [ 53 ]. For example, amides demonstrate a minimum exchange rate around a pH of 2.5 [ 54 ], while carbohydrates, primarily composed of hydroxyls, exhibit a minimum exchange rate around a pH of 6.5, with the exchange rate increasing as the solution becomes more acidic or basic [ 55 ]. The pH of the mobile phases used in the HILIC method was approximately 3, aligning with the observed pH-dependent trends in amides and carbohydrates.…”

Section: Resultsmentioning

confidence: 99%

“…The increased ratio of [M(D x-1 )+D] + /[M(D x )+D] + ions in the partial HILIC-HDX-MS method can be attributed to the presence of non-deuterated mobile-phase modifiers providing hydrogen atoms, thereby reducing the yield of fully deuterated ions.We should also note that the H/D exchange efficiency has been reported to be pHdependent[53]. For example, amides demonstrate a minimum exchange rate around a pH of 2.5[54], while carbohydrates, primarily composed of hydroxyls, exhibit a minimum exchange rate around a pH of 6.5, with the exchange rate increasing as the solution becomes more acidic or basic[55]. The pH of the mobile phases used in the HILIC method was approximately 3, aligning with the observed pH-dependent trends in amides and carbohydrates.…”

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confidence: 86%

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Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Cajka,

Hricko,

Rakusanova

et al. 2024

IJMS

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Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time–m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

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Section: Resultsmentioning

confidence: 99%

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confidence: 86%

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Cajka,

Hricko,

Rakusanova

et al. 2024

IJMS

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“…First attempts at collecting this dataset saw that the Mg 2+ /DMSO induced extensive peptide carryover. Efforts to reduce carryover following previously described guidelines 41,42 were made and we obtained high-quality data from a pulsed HDX experiment using a 251s labelling reaction at pH 6.0 (83 peptides, 92.9% protein coverage). This analysis showed that similar key regions across multiple domains of AcrA S exhibited reduced structural dynamics by NSC 60339 in the presence of Mg 2+ , for example 75 IILKRNFKEGSD 86 in site IV and 308 VPQQGVTRTPRGDATVL 32 in the MP domain away from site IV showed the characteristic strong HDX protection ( Figure S11 ).…”

Section: Resultsmentioning

confidence: 99%

Mg²⁺-dependent mechanism of environmental versatility in a multidrug efflux pump

Russell Lewis,

Uddin,

Kuo

et al. 2024

Preprint

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Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump fromEscherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

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“…In higher-order conformations, more structured and solvent inaccessible regions are protected from exchange compared to disordered and solvent accessible regions of the molecule [5]. The m/z shifts arising from isotopic exchange within the latter regions are currently exploited by HDX-MS to study structures of challenging systems [18][19][20][21][22][23][24][25][26] folding dynamics [27][28][29][30][31], protein-protein and protein-oligonucleotide interactions [32][33][34][35][36][37][38][39][40], as well as protein-ligand interactions and any associated conformational changes [41][42][43][44][45][46][47][48][49].…”

Section: Introductionmentioning

confidence: 99%

Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry

Attanayake,

Mahmud,

Banerjee

et al. 2024

Preprint

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Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.

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Using hydrogen-deuterium exchange mass spectrometry to characterize Mtr4 interactions with RNA

Cited by 6 publications

References 86 publications

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Mg²⁺-dependent mechanism of environmental versatility in a multidrug efflux pump

Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry

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Using hydrogen-deuterium exchange mass spectrometry to characterize Mtr4 interactions with RNA

Cited by 6 publications

References 86 publications

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Hydrophilic Interaction Liquid Chromatography–Hydrogen/Deuterium Exchange–Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry

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Mg²⁺-dependent mechanism of environmental versatility in a multidrug efflux pump