ABSTRACT
Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E.â coli GroEL molecules (801â kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40â µm diameter with a maximum flux of 25â molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL.
Subject(s)
Chaperonin 60 , Escherichia coli , Nanotechnology , Chaperonin 60/chemistry , Mass Spectrometry , Micro-Electrical-Mechanical Systems/instrumentation , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/analysisABSTRACT
Multidimensional multiple-stage tandem processing of ions is demonstrated successfully in a novel segmented linear ion trap. The enhanced performance is enabled by incorporating the entire range of ion activation methods into a single platform in a highly dynamic fashion. The ion activation network comprises external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals. Axial segmentation of the two-dimensional trapping field provides access to a unique functionality landscape through a system of purpose-designed regions for processing ions with maximum flexibility. Design aspects of the segmented linear ion trap, termed the Omnitrap platform, are highlighted, and motion of ions trapped by rectangular waveforms is investigated experimentally by mapping the stability diagram, tracing secular frequencies, and exploring different isolation techniques. All fragmentation methods incorporated in the Omnitrap platform involving radical chemistry are shown to provide complete sequence coverage for partially unfolded ubiquitin. Three-stage (MS3) tandem mass spectrometry experiments combining collision-induced dissociation of radical ions produced by electron meta-ionization and further involving two intermediate steps of ion isolation and accumulation are performed with high efficiency, producing information rich spectra with signal-to-noise levels comparable to those obtained in a two-stage (MS2) experiment. The advanced capabilities of the Omnitrap platform to provide in-depth top-down MSn characterization of proteins are portrayed. Performance is further enhanced by connecting the Omnitrap platform to an Orbitrap mass analyzer, while successful integration with time-of-flight analyzers has already been demonstrated.
Subject(s)
Proteins , Tandem Mass Spectrometry , Electrons , Ions/chemistry , Proteins/chemistry , Tandem Mass Spectrometry/methods , UbiquitinABSTRACT
Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles in the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS)-based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near-cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultrahigh mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissues using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a considerable increase in duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps toward high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.
Subject(s)
Histones , Proteomics , Fourier Analysis , Histones/metabolism , Humans , Kidney/metabolism , Proteomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential.
Subject(s)
Inflammatory Bowel Diseases , Proteins , Humans , Ions , Mass Spectrometry/methods , Proteins/chemistry , Reproducibility of ResultsABSTRACT
Native mass spectrometry can provide insight into the structure of macromolecular biological systems. As analytes under investigation get larger and more complex, instrument capabilities need to be advanced. Herein, modifications to an Orbitrap Q Exactive Plus mass spectrometer that increase signal intensity, mass resolution, and maximum m/z measurable are described.
ABSTRACT
Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.
Subject(s)
Mass Spectrometry/methods , Membrane Proteins/analysis , Membrane Proteins/genetics , Nanostructures/chemistry , Mutation , Protein EngineeringABSTRACT
Investigation of the structure, assembly and function of protein-nucleic acid macromolecular machines requires multidimensional molecular and structural biology approaches. We describe modifications to an Orbitrap mass spectrometer, enabling high-resolution native MS analysis of 0.8- to 2.3-MDa prokaryotic 30S, 50S and 70S ribosome particles and the 9-MDa Flock House virus. The instrument's improved mass range and sensitivity readily exposes unexpected binding of the ribosome-associated protein SRA.