scholarly journals Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry

2017 ◽  
Vol 474 (11) ◽  
pp. 1867-1877 ◽  
Author(s):  
Glenn R. Masson ◽  
Sarah L. Maslen ◽  
Roger L. Williams
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Phosphoinositide 3 Kinase ◽  
Hdx Ms

Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.

scholarly journals Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 286
Author(s):  
Oliver Ozohanics ◽  
Attila Ambrus
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Ligand Interaction ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

scholarly journals Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

2018 ◽  
Author(s):  
Kelly R. Karch ◽  
Mariel Coradin ◽  
Levani Zandarashvili ◽  
Zhong-Yuan Kan ◽  
Morgan Gerace ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Nucleosome Assembly ◽  
Histone Tail ◽  
Deuterium Label ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms

AbstractUntil recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

scholarly journals Construction of a Dual Protease Column, Subzero (-30 oC) Chromatography System and Multi-channel Precision Temperature Controller for Hydrogen-Deuterium Exchange Mass Spectrometry

2020 ◽  
Vol 125 ◽  
Author(s):  
Jeffrey W. Hudgens
Keyword(s):  
Mass Spectrometry ◽  
Three Dimensional ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Precision Temperature ◽  
Hydrogen Deuterium ◽  
3D Printed ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers that regulate to ±0.05 oC are also included in this tutorial.

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