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Characterization of hydrophobic peptides in the presence of detergent by photoionization mass spectrometry.

Abstract : The characterization of membrane proteins is still challenging. The major issue is the high hydrophobicity of membrane proteins that necessitates the use of detergents for their extraction and solubilization. The very poor compatibility of mass spectrometry with detergents remains a tremendous obstacle in studies of membrane proteins. Here, we investigated the potential of atmospheric pressure photoionization (APPI) for mass spectrometry study of membrane proteins. This work was focused on the tetraspanin CD9 and the multidrug transporter BmrA. A set of peptides from CD9, exhibiting a broad range of hydropathicity, was investigated using APPI as compared to electrospray ionization (ESI). Mass spectrometry experiments revealed that the most hydrophobic peptides were hardly ionized by ESI whereas all peptides, including the highly hydrophobic one that corresponds to the full sequence of the first transmembrane domain of CD9, were easily ionized by APPI. The native protein BmrA purified in the presence of the non-ionic detergent beta-D-dodecyl maltoside (DDM) was digested in-solution using trypsin. The resulting peptides were investigated by flow injection analysis of the mixture followed by mass spectrometry. Upon ESI, only detergent ions were detected and the ionic signals from the peptides were totally suppressed. In contrast, APPI allowed many peptides distributed along the sequence of the protein to be detected. Furthermore, the parent ion corresponding to the first transmembrane domain of the protein BmrA was detected under APPI conditions. Careful examination of the APPI mass spectrum revealed a-, b-, c- and y- fragment ions generated by in-source fragmentation. Those fragment ions allowed unambiguous structural characterization of the transmembrane domain. In conclusion, APPI-MS appears as a versatile method allowing the ionization and fragmentation of hydrophobic peptides in the presence of detergent.
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Aïcha Bagag, Jean-Michel Jault, Nazha Sidahmed-Adrar, Matthieu Réfrégiers, Alexandre Giuliani, et al.. Characterization of hydrophobic peptides in the presence of detergent by photoionization mass spectrometry.. PLoS ONE, Public Library of Science, 2013, 8 (11), pp.e79033. ⟨10.1371/journal.pone.0079033⟩. ⟨hal-01326069⟩

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