An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I
MS) method for ...charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.
It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing ...the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.
Electronic Structure of Heteronuclear Cerium-Platinum Clusters Mason, Jarrett L.; Huizenga, Caleb D.; Ray, Manisha ...
The journal of physical chemistry. A, Molecules, spectroscopy, kinetics, environment, & general theory,
08/2023, Letnik:
127, Številka:
32
Journal Article
Recenzirano
Beyond the now well-known strong catalyst-support interactions reported for ceria-supported platinum catalysts, intermetallic Ce-Pt compounds exhibit fascinating properties such as heavy fermion ...behavior and magnetic instability. Small heterometallic Ce-Pt clusters, which can provide insights into the local features that govern bulk phenomena, have been less explored. Herein, the anion photoelectron spectra of three small mixed Ce-Pt clusters, Ce2OPt–, Ce2Pt–, and Ce3Pt–, are presented and interpreted with supporting density functional theory calculations. The calculations, which are readily reconciled with the experimental spectra, suggest the presence of numerous close-lying spin states, including states in which the Ce 4f electrons are ferromagnetically coupled or antiferromagnetically coupled. The Pt center is consistently in a nominal −2 charge state in all cluster neutrals and anions, giving the Ce-Pt bond ionic character. Ce-Pt bonds are stronger than Ce-Ce bonds, and the O atom in Ce2OPt– coordinates only with the Ce centers. The energy of the singly occupied Ce-local 4f orbitals relative to the Pt-local orbitals changes with cluster composition. Discussion of the results includes potential implications for Ce-rich intermetallic materials.
Abstract
The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based ...multiplexed workflow for top-down tandem MS (MS
2
) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS
2
identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.
Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in ...distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high m/z by >2.5-fold and >1.6-fold, respectively.
Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with ...the mass-to-charge ratio (m/z). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.
The quality of data in charge detection mass spectrometry depends on accurate determination of ion charge. While the method of selective temporal overview of resonant ions (STORI) has proven to be ...highly enabling for determining the charge of ions that survive for variable amounts of time, it assumes that the ion frequency exactly matches the frequency being used in the calculation. Any mismatches result in low charge estimates. To address this, the misSTORI method was developed to correct these discrepancies. This can significantly reduce the charge measurement errors for samples with unstable masses. As an example, the misSTORI approach can eliminate a 5.7% charge determination error for a VP3-only AAV capsid that shifts 25 ppm in mass.
Collision cross section (CCS) measurements determined by ion mobility spectrometry (IMS) provide useful information about gas-phase protein structure that is complementary to mass analysis. Methods ...for determining CCS without a dedicated IMS system have been developed for Fourier transform mass spectrometry (FT-MS) platforms by measuring the signal decay during detection. Individual ion mass spectrometry (I
MS) provides charge detection and measures ion lifetimes across the length of an FT-MS detection event. By tracking lifetimes for entire ion populations, we demonstrate simultaneous determination of charge, mass, and CCS for proteins and complexes ranging from ∼8 to ∼232 kDa.
PDF
