scholarly journals Investigating the conformational response of the Sortilin receptor upon binding endogenous peptide- and protein ligands by HDX-MS

2018 ◽  
Author(s):  
Esben Trabjerg ◽  
Nadia Abu-Asad ◽  
Ziqian Wan ◽  
Fredrik Kartberg ◽  
Søren Christensen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Proximal Region ◽  
Binding Region ◽  
Exchange Mass Spectrometry ◽  
Protein Ligands ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
Membrane Proximal Region

AbstractSortilin is a multifunctional transmembrane neuronal receptor involved in sorting of neurotrophic factors and apoptosis signalling. So far, structural characterization of Sortilin and its endogenous ligands has been limited to crystallographic studies of Sortilin in complex with the neuropeptide Neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of Sortilin to binding biological ligands including the peptides Neurotensin and the Sortilin propeptide and the proteins Progranulin and pro-Nerve growth factor-β. The results show that the ligands employ two binding sites inside the cavity of the β-propeller of Sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of Sortilin. Our findings improves our molecular understanding of Sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.

scholarly journals Characterization of Ceramides with Phytosphingosine Backbone by Hydrogen-deuterium Exchange Mass Spectrometry

2019 ◽  
Vol 92 (3) ◽  
pp. 411-417
Author(s):  
Irena Dapic ◽  
Ivone Jakasa ◽  
Renata Kobetic ◽  
Lidija Brkljacic
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acyl ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Ionization Mode ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Ceramides are a lipid subclass of the sphingolipids that show large structural diversity. Structural characterization of the ceramides (CERs) can lead to better understanding of their role and function in the biological system. Here we investigated representatives of NP (CER III, CER IIIB) and AP ceramide classes (CER VI) that contain phytosphingosine (P) backbone. Ceramides were characterized in positive ionization mode by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Fragmentation in positive ionization mode of the CER III and CER VI resulted in abundant ions assigned to phytosphingosine moiety at m/z 282, 300 and 318. HDX-MS of fragments showed increase in m/z of corresponding ions confirming the exchange of deuterium. In negative ionisation spectra multiple fragment ions were assigned to fatty acyl (RCOO–) moiety. Presence of RCOO– allowed unambiguous identification of CER III and CER IIIB which were distinguished by the presence of double bond on fatty acyl chain.

scholarly journals Epitope Screening Using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): An Optimized Workflow for Accelerated Evaluation of Lead Monoclonal Antibodies

2021 ◽  
Author(s):  
Shaolong Zhu ◽  
Peter Liuni ◽  
Tricia Chen ◽  
Camille Houy ◽  
Derek Wilson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Epitope Mapping ◽  
Vaccine Development ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Background: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production has enabled candidate mAbs to be identified at a rapidly increasing rate resulting in a significant bottleneck in the characterization of ‘structural’ epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. Main methods and major results: The method is demonstrated on set of 6 candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes was unambiguously determined using two HDX mixing timepoints in 24 hours total run time, corresponding to substantial decrease in the instrument time required to map a single epitope using conventional HDX workflows. Conclusion: An accelerated HDX-MS epitope screening workflow was developed. The two-timepoint ‘screening’ workflow mapped all six mAbs and generated high confidence epitopes for five of the six mAbs assayed. The substantial improvement in the rate of data collection can advance HDX-MS for higher throughput investigations supporting the ability to evaluate a broader number of mAb candidates at an earlier stage of vaccine development.

scholarly journals Cataract-Associated New Mutants S175G/H181Q of βΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes

2020 ◽  
Vol 21 (18) ◽  
pp. 6504
Author(s):  
In-Kang Song ◽  
Seungjin Na ◽  
Eunok Paek ◽  
Kong-Joo Lee
Keyword(s):  
Structural Changes ◽  
Hydrogen Deuterium Exchange ◽  
Structural Protein ◽  
Exchange Mass Spectrometry ◽  
Human Lenses ◽  
Hydrogen Deuterium ◽  
Proteomic Dataset ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
New Mutations

β/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

scholarly journals Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 121
Author(s):  
Valentin Gogonea ◽  
Judith Peters ◽  
Gary S. Gerstenecker ◽  
Celalettin Topbas ◽  
Liming Hou ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Deuterium Exchange ◽  
Average Particle ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Incoherent Neutron Scattering ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
Incoherent Neutron

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

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.

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