A simple heated‐capillary modification improves the analysis of non‐covalent complexes by Z‐spray electrospray ionization

Abstract

Rationale The observation of intact non-covalent complexes by ESI-MS hinges on the ability to minimize in-source activation processes that take place during analyte desolvation. We explored the merits of replacing the sampling cone of a standard Z-spray source with a heated capillary that makes the desolvation process slower and more gradual. We employed well-characterized protein-RNA and DNA-DNA assemblies to compare the alternative configurations. Methods Mass analysis evaluated the complexes’ integrity, whereas traveling wave ion mobility experiments assessed the stability of biomolecular structure. Analyses were performed back-to-back on the same samples on a Synapt G2 HDMS equipped with either the standard sampling cone or the heated-capillary apparatus. In each configuration, the source/capillary temperature was varied in controlled fashion, while keeping all other desolvation parameters constant to monitor the in-source dissociation of selected DNA duplexes. Ion mobility data were obtained from the same precursor by using the alternative configurations under the same settings. Results Monitoring the percentage of associated complex demonstrated that the heated capillary provided softer desolvation that was more conducive to the detection of intact non-covalent interactions. This configuration failed to produce complete dissociation of 14bp and 24bp duplexes, even when the source/capillary temperature was increased well above their solution melting points. Analyzed by IMS-MS, a selected construct displayed just one conformation with the heated capillary, but two with the standard sampling cone. Conclusions The heated capillary minimizes in-source activation processes that can lead to unintended dissociation of complexes and perturbation of biomolecular structure, which rely on the integrity of non-covalent interactions. This effect can be attributed to the attenuation of the supersonic expansion typical of the Z-spray geometry and the greater ability to control the energy imparted to the system. This hardware modification will be expected to benefit the analysis of biomolecular structure performed on this particular instrumental platform.

Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance

“…Again, the Orbitrap and FT-ICR possess heated transfer capillaries which transport the ions from atmosphere to partial vacuum. A heated transfer capillary modification to a Q-ToF platform 31 may prove beneficial for native Nd analysis. Conversely, the operating pressures and therefore the mean-free-path at which the MSP1D1-Nd (and bacteriorhodopsin) activation and subsequent desolvation (and protein liberation, in the case of bacteriorhodopsin) occurs are also very different and must be considered.…”

The use of chromium(III) complexes to enhance peptide protonation by electrospray ionization mass spectrometry

“…Interestingly, experiments with the Z‐spray ionization source show no enhanced protonation regardless of the parameters employed. In addition, Fabris and coworkers have reported that the Z‐spray source can produce unwanted in‐source dissociation of noncovalent complexes (ie, some ions have sufficient energy to dissociate) . Therefore, the Z‐spray ionization process may destabilize the peptide‐Cr(III)‐water complex before an “extra” proton is transferred to peptide ions.…”

“…The Water ligands have an unusually long lifetime around Cr(III) due to a kinetic inertness to displacement. [11][12][13] The residence time of a water molecule in the first hydration shell 74 77 Therefore, the Z-spray ionization process may destabilize the peptide-Cr(III)-water complex before an "extra" proton is transferred to peptide ions. The absence of enhanced protonation in the Z-spray source further supports our hypothesis that this is a gas-phase or desolvation-phase effect, as opposed to a solution-phase effect occurring before the ESI event.…”

High‐resolution ion mobility spectrometry‐mass spectrometry of isomeric/isobaric ribonucleotide variants

“…A stainless‐steel wire was inserted into the emitter back end to provide typical spray voltages in the 0.9 to 1.2‐kV range. Source temperature and desolvation voltages were finely adjusted by monitoring the incidence of ammonium adducts and water clusters . The ion mobility experiments were completed in the 25‐cm TW cell by using a 90 mL/min flow of N 2 and 180 mL/min of He to maintain a constant pressure of ~4.40 mbar (uncalibrated gauge reading).…”

“…Source temperature and desolvation voltages were finely adjusted by monitoring the incidence of ammonium adducts and water clusters. 42 The ion mobility experiments were completed in the…”