Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.

Individual ion mass spectrometry (IMS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, IMS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. IMS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (IMS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent IMS acquisition.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra.

Improved Signal Processing for Mass Shifting Ions in Charge Detection Mass Spectrometry.

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.

Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (IMS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions.

Determining Collisional Cross Sections from Ion Decay with Individual Ion Mass Spectrometry.

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 (IMS) provides charge detection and measures ion lifetimes across the length of an FT-MS detection event.

Automated imaging and identification of proteoforms directly from ovarian cancer tissue.

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) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform.

Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry.

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry.

Combining Surface-Induced Dissociation and Charge Detection Mass Spectrometry to Reveal the Native Topology of Heterogeneous Protein Complexes.

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 (/). 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.

Electronic Structure of Heteronuclear Cerium-Platinum Clusters.

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, CeOPt, CePt, and CePt, are presented and interpreted with supporting density functional theory calculations.

Automated Control of Injection Times for Unattended Acquisition of Multiplexed Individual Ion Mass Spectra.

Charge detection mass spectrometry (CDMS) provides mass domain spectra of large and highly heterogeneous analytes. Over the past few years, we have multiplexed CDMS on Orbitrap instruments, an approach termed Individual Ion Mass Spectrometry (IMS). Until now, IMS required manual adjustment of injection times to collect spectra in the individual ion regime.

Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS.

SARS-CoV-2 Omicron (B.1.1.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells.

Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells.

Decoding the protein composition of whole nucleosomes with Nuc-MS.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.

Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry.

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.

Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.

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 (IMS) 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.

Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry.

Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (IMS) and spectral output directly into the mass domain.

Native vs Denatured: An in Depth Investigation of Charge State and Isotope Distributions.

New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of so-called "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher / with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions.

STORI Plots Enable Accurate Tracking of Individual Ion Signals.

Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration.

Standard Proteoforms and Their Complexes for Native Mass Spectrometry.

Native mass spectrometry (nMS) is a technique growing at the interface of analytical chemistry, structural biology, and proteomics that enables the detection and partial characterization of non-covalent protein assemblies. Currently, the standardization and dissemination of nMS is hampered by technical challenges associated with instrument operation, benchmarking, and optimization over time. Here, we provide a standard operating procedure for acquiring high-quality native mass spectra of 30-300 kDa proteins using an Orbitrap mass spectrometer.

Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins.

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.