A new instrument configuration for native ion mobility-mass spectrometry (IM-MS) is described. Macromolecule ions are generated by using a static ESI source coupled to an RF ion funnel, and these ions are then mobility and mass analyzed using a periodic focusing drift tube IM analyzer and an Orbitrap mass spectrometer. The instrument design retains the capabilities for first-principles determination of rotationally averaged ion-neutral collision cross sections and high-resolution measurements in both mobility and mass analysis modes for intact protein complexes. Operation in the IM mode utilizes FT-IMS modes (originally described by Knorr ( Knorr , F. J. Anal. Chem . 1985 , 57 ( 2 ), 402 - 406 )), which provides a means to overcome the inherent duty cycle mismatch for drift tube (DT)-IM and Orbitrap mass analysis. The performance of the native ESI-FT-DT-IM-Orbitrap MS instrument was evaluated using the protein complexes Gln K (MW 44 kDa) and streptavidin (MW 53 kDa) bound to small molecules (ADP and biotin, respectively) and transthyretin (MW 56 kDa) bound to thyroxine and zinc.
Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated.Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold-and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.
Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the β-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag+ ions are more weakly bound than are the other four. Higher order Ag i -MT complexes (i = 7–17) are formed in solutions that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addition of NEM to this solution to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Ag i -MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag+ increases.
Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ∼5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ∼60 is obtained for pyruvate kinase (MW ∼ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ∼ 68 kDa) is less than ∼20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.
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