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
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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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
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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