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

Abstract

We investigated the influence of a solvent’s composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex’s gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes’ gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis

“…The integration of experimental data with computational approaches enables one to draw conclusions with atomic level resolution . With the combination of mass spectrometry and molecular dynamics simulations, structural rearrangements of noncovalent protein complexes have been studied with respect to their transition from the condensed phase to the gas phase with single amino acid resolution . Moreover, fine mapping of antibody–epitope interactions using molecular dynamics in conjunction with mass spectrometry, ITEM-TWO analysis, in particular, lead to the differentiation of orthodox binding and unorthodox binding, depending on small alterations of a recognition motif that were caused by single amino acid exchanges …”

“…56 With the combination of mass spectrometry and molecular dynamics simulations, structural rearrangements of noncovalent protein complexes have been studied with respect to their transition from the condensed phase to the gas phase with single amino acid resolution. 39 Moreover, fine mapping of antibody−epitope interactions using molecular dynamics in conjunction with mass spectrometry, ITEM-TWO analysis, in particular, lead to the differentiation of orthodox binding and unorthodox binding, depending on small alterations of a recognition motif that were caused by single amino acid exchanges. 37 ■ CONCLUSION AND OUTLOOK ITEM-TWO studies outline a strategy by which one experimentally can test whether a predicted binding motif, that is, a respective surface atom assembly, actually will be recognized by an antibody.…”

“…Then the centrifugal filters were inverted, placed into new vials, and centrifuged for 5 min at 4500 rpm and 23 °C to collect the retentate volumes of approximately 20 μL (antibody stock solutions). The protein concentrations of the antibody stock solutions were determined using the Qubit 2.0 Fluorometer assay. , The anti-HpTGEKP antibody concentration was between 3.44 and 4.17 μM, depending on replicate variations. The anti-Histag antibody concentration was 4.56 μM.…”

“…Mixtures were incubated at room temperature for at least 1 h. For all measurements ∼3 μL of antibody and/or immune-complex-containing mixture was loaded into nanoESI capillaries using microloader pipet tips (Eppendorf, Hamburg, Germany) and (phospho-) hexapeptide binding behaviors toward the anti-HpTGEKP antibody as well as binding of the Histag peptide binding to the anti-Histag antibody (control) were investigated by ITEM-TWO analyses. 33,39 Offline NanoESI-MS Instrument Settings, Data Acquisition, and Data Analysis. As previously described, 33,37 ITEM-TWO measurements were performed using a Synapt G2S mass spectrometer (Waters MS-Technologies, Manchester, UK) with the following instrumental settings: source temperature, 50 °C; capillary voltage, 1.8 kV; source offset voltage, 130 V; sample cone voltage, 130 V; nanoflow gas, 0.05 bar; trap gas flow (argon), 8 mL/min; transfer collision cell voltage difference, 2 V; trap collision cell voltage difference (V): 0, 2, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 145, 165, 180, 200, and 220.…”

“…The protein concentrations of the antibody stock solutions were determined using the Qubit 2.0 Fluorometer assay. 38,39 The anti-HpTGEKP antibody concentration was between 3.44 and 4.17 μM, depending on replicate variations. The anti-Histag antibody concentration was 4.56 μM.…”