Replication in Bioanalytical Studies with Hdx MS: Aim as High as Possible

Moroco, Jamie A.; Engen, John R.

doi:10.4155/bio.15.46

Cited by 18 publications

(22 citation statements)

References 16 publications

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“…Deuterium incorporation levels were determined using DynamX 3.0 software (Waters) along with the manual inspection of every spectrum to ensure accurate assignments of isotope clusters. The low variability in deuterium incorporation between the two independent manipulation replicates (0.12 Da LCAT; 0.18 Da ApoA-I) supports the reproducibility of our studies, and highlights the effects of LCAT binding to HDL 55 . For LCAT, we identified 115 peptides with an overall 75.2% coverage and 5.0 redundancy score (average number of overlapped peptides per amino acid residue; Supplementary Fig.…”

Section: Methodssupporting

confidence: 76%

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

et al. 2020

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Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogendeuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

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Section: Methodssupporting

confidence: 76%

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

et al. 2020

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“…The measured GMPPNP and GDP content for the samples analyzed here is shown in Supplemental FIGURE S4a. To characterize the spread of the HDX MS data as a result of different levels of nucleotide loading, we performed multiple biological replicates [49, 50] wherein each replicate was composed of an independent expression of protein followed by purification, nucleotide switching and characterization by HPLC, and finally HDX MS analysis. The variability of the measured deuterium levels in these replicates (Supplemental FIGURE S4b) was relatively low for the GDP-bound form, but was high in some regions for the GMPPNP-bound forms.…”

Section: Resultsmentioning

confidence: 99%

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching

Harrison

Carrasco

et al. 2016

Journal of Molecular Biology

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Structural dynamics of Ras proteins contribute to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras targeting strategies, experimental methods to measure Ras dynamics are required. Here we demonstrate the utility of hydrogen-deuterium exchange mass spectrometry to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main chain movement. Hydrogen-deuterium exchange provides a means of evaluating Ras protein dynamics which may be useful for understanding mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.

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“…Producing a deuterium-labeled sample is simple and requires no specialized equipment or chemicals, other than D 2 O. However, the labeling reaction is highly sensitive to experimental conditions, such as pH and temperature fluctuations, among other factors 34 . It requires a number of controls and a great amount of care to ensure that the reaction is conducted in a manner that does not bias the results or lead to imprecise measurements.…”

Section: Mainmentioning

confidence: 99%

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments

et al. 2019

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Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

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Replication in Bioanalytical Studies with Hdx MS: Aim as High as Possible

Abstract: "While more complicated and time consuming, the higher order the replication, the more biologically relevant the information."

Cited by 18 publications

References 16 publications

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments

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