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

Kód výsledku v 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>

Nalezeny alternativní kódy

RIV/00216208:11310/19:10402879

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

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

Rok uplatnění

2019

Kód důvěrnosti údajů

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

Název periodika

Analytical Chemistry

ISSN

0003-2700

e-ISSN

—

Svazek periodika

91

Číslo periodika v rámci svazku

17

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

10970-10978

Kód UT WoS článku

000484644800010

EID výsledku v databázi Scopus

2-s2.0-85071718948