Mass spectrometric characterization of protein structures and protein complexes in condensed and gas phase

Abstract

Proteins are essential for almost all physiological processes of life. They serve a myriad of functions which are as varied as their unique amino acid sequences and their corresponding three-dimensional structures. To fulfill their tasks, most proteins depend on stable physical associations, in the form of protein complexes that evolved between themselves and other proteins. In solution (condensed phase), proteins and/or protein complexes are in constant energy exchange with the surrounding solvent. Albeit methods to describe in-solution thermodynamic properties of proteins and of protein complexes are well established and broadly applied, they do not provide a broad enough access to life-science experimentalists to study all their proteins' properties at leisure. This leaves great desire to add novel methods to the analytical biochemist's toolbox. The development of electrospray ionization created the opportunity to characterize protein higher order structures and protein complexes rather elegantly by simultaneously lessening the need of sophisticated sample preparation steps. Electrospray mass spectrometry enabled us to translate proteins and protein complexes very efficiently into the gas phase under mild conditions, retaining both, intact protein complexes, and gross protein structures upon phase transition. Moreover, in the environment of the mass spectrometer (gas phase, in vacuo), analyte molecules are free of interactions with surrounding solvent molecules and, therefore, the energy of inter- and intramolecular forces can be studied independently from interference of the solvating environment. Provided that gas phase methods can give information which is relevant for understanding in-solution processes, gas phase protein structure studies and/or investigations on the characterization of protein complexes has rapidly gained more and more attention from the bioanalytical scientific community. Recent reports have shown that electrospray mass spectrometry provides direct access to six prime protein complex properties: stabilities, compositions, binding surfaces (epitopes), disassembly processes, stoichiometries, and thermodynamic parameters.

Mass Spectrometric and Bio-Computational Binding Strength Analysis of Multiply Charged RNAse S Gas-Phase Complexes Obtained by Electrospray Ionization from Varying In-Solution Equilibrium Conditions

“…Matching with this experimental observation, our in-silico simulations indicated that remaining forces that held the S-peptide complexed to the S-protein in the gas phase, i.e., after evaporation of solvent were consistent with previous estimations where subtle structural changes of protein surface amino acid residues (of protein complexes) in the gas phase were described to take place while simultaneously the protein ions maintained their over-all solution-like structures [24,32,33]. One limitation when choosing the force field for gas-phase simulations of protein structures is parametrization, as published parameter sets are available only for the neutral forms of amino acids.…”

“…Due to the rich data background on RNAse S, this commercially available non-covalent protein–peptide complex with clearly defined molecular composition is an ideal substance for developing methods by which supramolecular properties as well as influences of the solution environment can be elucidated in both the condensed and the gas phase. The desire to apply mass spectrometry for determining binding strengths of non-covalent forces, i.e., affinities between biomolecules was expressed already from early on [ 6 ] taking solution chemistry and biophysical concepts as models [ 24 ]. Our quantitative data on RNAse S dissociation in the gas phase substantiate the interpretation that interfering with polar interactions between S-peptide and S-protein by in-solution pH changes is resembled by a shift in mean charge states of the protein complex in the gas phase.…”

“…The direct transition of analytes from the condensed phase into the gas phase, i.e., desorption, has been recognized as the key process for successful mass spectrometric analysis of bio-macromolecules with desorption occurring shortly after or simultaneous to ionization [ 13 , 14 ]. Consequently, ESI processes that influenced the transition of intact non-covalent complexes into the gas phase as multiply charged supramolecular species have to be precisely controlled [ 23 , 24 ]. By keeping desorption and ionization under control, gas-phase complex monitoring methods might be regarded as equal to in-solution techniques for determining kinetic and thermodynamic properties of protein–protein complexes [ 22 , 25 ].…”

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

“…By contrast, the relatively low amounts of samples required (19) and the rapidity (6) by which mass spectrometric epitope mapping is executed is of great advantage in this respect (20). Chemical cross-linking mass spectrometry (21,22), hydrogen/deuterium exchange (HDX) 1 mass spectrometry (23) and mass spectrometric methods that employ chemical modification on proteins, such as Fast Photochemical Oxidation of Proteins (FPOP) (24,25) or chemical modification of surface exposed residues (26,27) have been applied in epitope mapping experiments (28) and in determinations of protein -protein interaction sites in general (29), but their application may be limited when rather demanding chemistries are involved, or when performing such experiments becomes laborious, and/or requires sophisticated laboratory equipment (20,30). Significant advances in epitope mapping protocols/methods have been reached with the two most commonly used mass spectrometric methods: epitope extraction and epitope excision (20,31,32).…”

Apparent activation energies of protein–protein complex dissociation in the gas–phase determined by electrospray mass spectrometry

“…Interestingly the trends of the gas phase values pretty much resembled those from the in= As shown here, dissociation energies of protein=protein complexes in the gas phase that have been corrected for "external energy" contributions seem to represent in=solution properties of protein=protein complexes well. As was pointed out in a recent review [30], surface induced dissociation (SID) seems to be an alternative to CID breakage of non= covalent bonds in the gas phase [31,32]. However, in SID experiments charges are distributed proportionally to the masses of dissociated constituents [33].…”