Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes

Abstract

Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαβ, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (10.1007/s13361-018-2015-x) contains supplementary material, which is available to authorized users.

Top or Middle? Up or Down? Toward a Standard Lexicon for Protein Top-Down and Allied Mass Spectrometry Approaches

“…Like complex-up and complex -down, this method relies on native (electrospray) ionization of monomeric proteins and noncovalent assemblies (although reports of ionization of intact peptide oligomers and protein complexes using MALDI have recently been published [38, 39]). In contrast to the aforementioned methods, the higher order structure is largely assumed to be retained during backbone cleavage in native top-down (nTD) experiments.…”

Mass Spectrometry Analysis of Intact Proteins from Crude Samples

“… 47 , 48 Thus, while intact proteins can be ionized in the presence of detergents and high salt concentrations, 48 the preservation of protein–protein or protein–ligand interactions is challenging. 25 , 49 …”

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

“…Native MS under optimised conditions preserves protein quaternary structure, the result of delicate non-covalent interactions, allowing the analysis of folded proteins and protein-ligand complexes [40][41][42]. Native MS is almost universally performed by nanoESI, although other ion sources have been investigated [43][44][45], and can be combined with LESA by use of an appropriate solvent system. Native LESA MS was demonstrated for the analysis of purified protein complexes exceeding 800 kDa from a glass substrate [46].…”