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Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

ABSTRACT: The use of submicrometer capillaries for nanoelectrospray ionization of native proteins and protein complexes effectively reduces the number of nonspecific salt adducts to biological molecules, therefore increasing the apparent resolution of a mass spectrometer without any further instrument modifications or increased ion activation. However, the increased interaction between proteins and the surface of the capillary has been shown to promote protein expansion and therefore loss of native structure. Here, we compare the effect of micrometer and submicrometer sized capillaries on the native structures of the protein complexes streptavidin, concanavalin A, and C-reactive protein under charge reducing conditions. We observe that the use of submicrometer capillaries did not result in a significantly higher charge state distribution, indicative of expansion, when compared to micrometer sized capillaries for complexes in 100 mM ammonium acetate and 100 mM triethylammonium acetate and for streptavidin in 200 mM ammonium acetate with no charge reduction. Additionally, no significant differences in collision cross sections were observed using ion mobility mass spectrometry. Finally, the dissociation behaviors of protein complexes ionized using micrometer and submicrometer capillaries were compared to determine if any structural perturbation occurred during ionization. Protein complexes from both capillary sizes displayed similar surface-induced dissociation patterns at similar activation energies. The results suggest that submicrometer capillaries do not result in significant changes to protein complex structure under charge reducing conditions and may be used for native mass spectrometry experiments. Submicrometer capillaries can be used to resolve small mass differences of biological systems on a QTOF platform; however, a laser tip puller is required for pulling reproducible submicrometer capillaries, and disruption in spray due to clogging was observed for larger protein complexes.

SUBMITTER: Panczyk EM

PROVIDER: S-EPMC7203676 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

Panczyk Erin M EM Gilbert Joshua D JD Jagdale Gargi S GS Stiving Alyssa Q AQ Baker Lane A LA Wysocki Vicki H VH

Analytical chemistry 20200121 3

The use of submicrometer capillaries for nanoelectrospray ionization of native proteins and protein complexes effectively reduces the number of nonspecific salt adducts to biological molecules, therefore increasing the apparent resolution of a mass spectrometer without any further instrument modifications or increased ion activation. However, the increased interaction between proteins and the surface of the capillary has been shown to promote protein expansion and therefore loss of native struct ...[more]

PMID: 31909984

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Project description:We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.

Dataset Information

Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

Publications

Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

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