This study utilized multiple mass spectrometry techniques to characterise necroptotic cell death which is mediated by the pseudokinase, mixed lineage kinase domain-like (MLKL). The mass spec data enabled more precise models using Small-Angle X-Ray Scattering analysis.

AbstractActivated MLKL oligomer stoichiometry was directly observed using native mass spectrometry on the maXis II ETD UHR-QTOF.Additionally, chemical cross-linking was then used investigate the orientation of the oligomeric MLKL complex on the membrane with LC-MS (XL-MS) analysis on the Impact II UHR-QTOF [1]. Finally, Small Angle X-ray Scattering analysis coupled to inline sizeexclusion chromatography (SEC) using constraints from the native mass spectrometry and XL-MS data enabled modeling of this complex.

Conclusions• By combining multiple complementary mass spectrometry techniques, we were able to comprehensively characterize the transition of MLKL from an inactive basal monomer to an activated tetramer capable of eliciting necroptotic cell death.• The analysis of MLKL under native conditions using the high-resolution maXis II ETD UHR-QTOF allowed us to observe ATP binding to monomeric MLKL and also demonstrated that the activated MLKL oligomer was a tetramer. Infusion of samples using an off-line nano electrospray ionisation source maximisedthe sensitivity of detection.• Modelling the orientation of the MLKL tetramer was enabled by analysis of inter-subunit chemical cross-links using the Impact II UHR-QTOF. The constraint information provided by the XL-MS analysis was supported by the native mass spectrometry results and Small-Angle X-Ray Scattering analysis.• These results demonstrate that the Bruker Ultra-High Resolution Quadrupole Time-of-Flight instruments are an exceptional platform for the analysis and determination of the structure and composition of protein complexes.