All

What are you looking for?

All
Projects
Results
Organizations

Quick search

  • Projects supported by TA ČR
  • Excellent projects
  • Projects with the highest public support
  • Current projects

Smart search

  • That is how I find a specific +word
  • That is how I leave the -word out of the results
  • “That is how I can find the whole phrase”

Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments

The result's identifiers

  • Result code in IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F19%3A00518861" target="_blank" >RIV/61388971:_____/19:00518861 - isvavai.cz</a>

  • Alternative codes found

    RIV/00216208:11310/19:10402879

  • Result on the web

    <a href="https://pubs.acs.org/doi/10.1021/acs.analchem.9b00973" target="_blank" >https://pubs.acs.org/doi/10.1021/acs.analchem.9b00973</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.1021/acs.analchem.9b00973" target="_blank" >10.1021/acs.analchem.9b00973</a>

Alternative languages

  • Result language

    angličtina

  • Original language name

    Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments

  • Original language description

    Insight into the structure function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MScompatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence, however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.

  • Czech name

  • Czech description

Classification

  • Type

    J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database

  • CEP classification

  • OECD FORD branch

    10608 - Biochemistry and molecular biology

Result continuities

  • Project

    Result was created during the realization of more than one project. More information in the Projects tab.

  • Continuities

    P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Others

  • Publication year

    2019

  • Confidentiality

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Data specific for result type

  • Name of the periodical

    Analytical Chemistry

  • ISSN

    0003-2700

  • e-ISSN

  • Volume of the periodical

    91

  • Issue of the periodical within the volume

    17

  • Country of publishing house

    US - UNITED STATES

  • Number of pages

    9

  • Pages from-to

    10970-10978

  • UT code for WoS article

    000484644800010

  • EID of the result in the Scopus database

    2-s2.0-85071718948