Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.
Anal Chem
; 91(17): 10970-10978, 2019 09 03.
Article
en En
| MEDLINE
| ID: mdl-31408320
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-MS-compatible 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
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Antiportadores
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Proteínas de Escherichia coli
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Proteínas de Drosophila
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática
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Proteínas de Transporte de Serotonina en la Membrana Plasmática
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio
Límite:
Animals
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Humans
Idioma:
En
Revista:
Anal Chem
Año:
2019
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Estados Unidos