Nanoelectrospray—More than just a minimized-flow electrospray ionization source

Abstract. The question whether electrosprayed protein ions retain solution-like conformations continues to be a matter of debate. One way to address this issue involves comparisons of collision cross sections (Ω) measured by ion mobility spectrometry (IMS) with Ω values calculated for candidate structures. Many investigations in this area employ traveling wave IMS (TWIMS). It is often implied that nanoESI is more conducive for the retention of solution structure than regular ESI. Focusing on ubiquitin, cytochrome c, myoglobin, and hemoglobin, we demonstrate that Ω values and collisional unfolding profiles are virtually indistinguishable under both conditions. These findings suggest that gas-phase structures and ion internal energies are independent of the type of electrospray source. We also note that TWIMS calibration can be challenging because differences in the extent of collisional activation relative to drift tube reference data may lead to ambiguous peak assignments. It is demonstrated that this problem can be circumvented by employing collisionally heated calibrant ions. Overall, our data are consistent with the view that exposure of native proteins to electrospray conditions can generate kinetically trapped ions that retain solution-like structures on the millisecond time scale of TWIMS experiments.

Section: Twims Of Native Proteins Using Regular Esi and Nanoesimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

Sun

Vahidi

Sowole

et al. 2015

“…It has been stated that nanospray, a lowflow subclass of ESI, is even softer [6,7] and has a greater tolerance for non-volatile salts [8] than conventional electrospray, thereby making it even more suitable for the study of non-covalent interactions. There are now a number of reports that detail the observation of non-covalent interactions using mass spectrometry, and this topic has been recently reviewed by several groups [9 -13].…”

mentioning

confidence: 99%

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Smith

Siu

Rafferty

2004

The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H 4 B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H 4 B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 3 Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions. (J Am Soc Mass Spectrom 2004, 15, 629 -638)

“…However, extensive dissociation during ESI is also observed for some electrostatically stabilized complexes, even under the most gentle ionization conditions [34]. ESI-MS can also produce false-positive results, i.e., some analytes tend to form noncovalent gas-phase assemblies during ESI, despite the nonexistence of the corresponding complexes in solution [35,36]. Hu et al have demonstrated that the nonspecific binding of metal ions to proteins can be a severe problem when the ionization conditions are not chosen properly [37].…”

mentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)