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Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

Intrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered NTAIL domain and the phosphoprotein X domain (PXD) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire NTAIL domain bound to PXD at atomic resolution. [Figure not available: see fulltext.]

A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.