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

0518861 - MBÚ 2020 RIV US eng J - Journal Article
Moller, I.R. - Slivacka, M. - Hausner, Jiří - Nielsen, A.K. - Pospíšilová, Eliška - Merkle, P.S. - Lišková, Růžena - Polák, Marek - Loland, C.J. - Kádek, Alan - Man, Petr - Rand, K.D.
Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.
Analytical Chemistry. Roč. 91, č. 17 (2019), s. 10970-10978. ISSN 0003-2700. E-ISSN 1520-6882
R&D Projects: GA ČR(CZ) GA16-20860S; GA MŠMT(CZ) LQ1604; GA MŠMT(CZ) ED1.1.00/02.0109; GA MŠMT(CZ) LD15089
Institutional support: RVO:61388971
Keywords : aspartic protease nepenthesin-1 * x-ray-structure * conformational-analysis
OECD category: Biochemistry and molecular biology
Impact factor: 6.785, year: 2019
Method of publishing: Limited access
https://pubs.acs.org/doi/10.1021/acs.analchem.9b00973

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.
Permanent Link: http://hdl.handle.net/11104/0303882