Membrane proteins (MP) play a crucial role in cell functions and represent a significant portion of the drug target landscape yet remain challenging therapeutic targets. Native Mass Spectrometry (nMS) analysis requires the release of MPs from their solubilizing environment without affecting the gas phase conformation. Mass Photometry (MPhoto) provides insight into MP oligomeric states without requiring prior buffer exchange and release of the MP from its solubilizing environment.

 

Here we focus on the combination of a rapid online buffer exchange (OBE) by size exclusion chromatography (SEC) workflow, coupled to nMS of two MP complexes: the pentameric receptor channel Glic and the nicotinic AcetylCholine receptor (nAChR) from Torpedo Marmorata.

 

OBE SEC-nMS analyses were carried out using NativePAC OBE-1 (50mm x 2.1µm) on an Acquity UPLC H-class system (Waters, Wilmslow, UK), coupled to an Orbitrap Exactive Plus EMR (ThermoFisher Scientific, Bremen, Germany). Fractions after OBE were collected manually for Mass Photometry analysis, conducted on a TwoMP instrument (Refeyn Ltd).

 

On one side MPhoto highlited the loss of pentameric structure of Glic through the OBE workflow, as nMS provided the characterization of Glic up to the monomeric subunit at 36,547 ± 2 Da.



On the other side, for nAChR, MPhoto showed the preservation of the pentamer structure in different detergents.  Monomeric characterization of 2 of the hetero-pentamer nAChR subunits could be achieve at 51903.2 ±0.2 Da and 55,528.6 ± 0.5 Da. After optimization of the MS method, a mass of 276707 ± 169 Da could be measured for the nAChR pentamer solubilized in DDM. The signal obtained remained highly heterogeneous due to high amounts of complex glycosylation characterized at the subunits level in denaturing-MS.



In conclusion, MPhoto provided key insights into the preservation of the oligomeric state of the samples through the optimization of the OBE workflow, before nMS characterization of MP complexes.