RESUMEN
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.
RESUMEN
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 (I2MS) 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 â¼8 to â¼232 kDa.
Asunto(s)
Espectrometría de Movilidad Iónica , Proteínas , Espectrometría de Masas/métodos , Proteínas/química , Espectrometría de Movilidad Iónica/métodosRESUMEN
We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.
Asunto(s)
Péptidos , Proteómica , Proteómica/métodos , Espectrometría de Masas/métodos , Proteoma/metabolismo , Proteínas SanguíneasRESUMEN
We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific™ Orbitrap™ mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.
RESUMEN
Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instrumentsâthe Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS2) analysis of â¼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to â¼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a â¼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.
Asunto(s)
Proteínas , Proteómica , Humanos , Proteómica/métodos , Células HEK293 , Proteínas/química , Espectrometría de Masas en Tándem/métodos , FosfopéptidosRESUMEN
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 (I2MS). Until now, I2MS required manual adjustment of injection times to collect spectra in the individual ion regime. To increase sample adaptability, enable online separations, and reduce the barrier for entry, we report an automated method for adjusting ion injection times in I2MS for image current detectors like the Orbitrap. Automatic Ion Control (AIC) utilizes the density of signals in the m/z domain to adjust an ensemble of ions down to the individual ion regime in real-time. The AIC technique was applied to both denatured and native proteins yielding high quality data without human intervention directly in the mass domain.
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Proteínas , Humanos , Espectrometría de Masas/métodos , Iones/química , Proteínas/análisisRESUMEN
Structural characterization of novel metabolites in drug discovery or metabolomics is one of the most challenging tasks. Multilevel fragmentation (MSn) based approaches combined with various dissociation modes are frequently utilized for facilitating structure assignment of unknown compounds. As each of the MS precursors undergoes MSn, the instrument cycle time can limit the total number of precursors analyzed in a single LC run for complex samples. This necessitates splitting data acquisition into several analyses to target lower concentration analytes in successive experiments. Here we present a new LC/MS data acquisition strategy, termed Met-IQ, where the decision to perform an MSn acquisition is automatically made in real time based on the similarity between the experimental MS2 spectrum and a spectrum in a reference spectral library for the known compounds of interest. If similarity to a spectrum in the library is found, the instrument performs a decision-dependent event, such as an MS3 spectrum. Compared to an intensity-based, data-dependent MSn experiment, only a limited number of MS3 are triggered using Met-IQ, increasing the overall MS2 instrument sampling rate. We applied this strategy to an Amprenavir sample incubated with human liver microsomes. The number of MS2 spectra increased 2-fold compared to a data dependent experiment where MS3 was triggered for each precursor, resulting in identification of 14-34% more unique potential metabolites. Furthermore, the MS2 fragments were selected to focus likely sources of useful structural information, specifically higher mass fragments to maximize acquisition of MS3 data relevant for structure assignment. The described Met-IQ strategy is not limited to metabolism experiments and can be applied to analytical samples where the detection of unknown compounds structurally related to a known compound(s) is sought.
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Metabolómica , Cromatografía Liquida/métodos , Humanos , Espectrometría de Masas/métodos , Metabolómica/métodosRESUMEN
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 â¼150 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 ß-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.
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Procesamiento Proteico-Postraduccional , Proteínas , Iones , Espectrometría de MasasRESUMEN
Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 µM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.
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Filtración/instrumentación , Polietilenglicoles/aislamiento & purificación , Proteínas/química , Animales , Anticuerpos Monoclonales/química , Diseño de Equipo , Humanos , Espectrometría de Masas/instrumentación , Piruvato Quinasa/químicaRESUMEN
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 (I2MS) 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.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/química , Proteómica/métodos , HumanosRESUMEN
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 (I2MS) and spectral output directly into the mass domain. Here I2MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I2MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I2MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.
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Proteómica , Espectrometría de Masas en Tándem , IonesRESUMEN
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. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.
RESUMEN
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 677â¯000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only â¼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/análisis , Animales , Anhidrasas Carbónicas/análisis , Análisis de Fourier , Humanos , Iones/análisis , Mioglobina/análisis , Fosfopiruvato Hidratasa/análisis , Transferrina/análisis , Ubiquitina/análisisRESUMEN
Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Espectrometría de Masas/métodos , Mieloma Múltiple/genética , Proteoma/metabolismo , Proteínas Represoras/genética , Línea Celular Tumoral , Epigénesis Genética , Humanos , Lisina/metabolismo , Metilación , Mieloma Múltiple/metabolismo , Regulación hacia ArribaRESUMEN
Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.
Asunto(s)
Proteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Algoritmos , Anhidrasas Carbónicas/química , Electrones , Mioglobina/química , Espectrometría de Masas en Tándem/métodos , Ubiquitina/químicaRESUMEN
Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.
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Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Espectrometría de Masas/instrumentación , Péptidos/química , Proteómica/instrumentación , Factores de TiempoRESUMEN
A novel dual cell linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) and its performance characteristics are reported. A linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer has been modified to incorporate a LTQ-Velos mass spectrometer. This modified instrument features efficient ion accumulation and fast MS/MS acquisition capabilities of dual cell linear RF ion trap instruments coupled to the high mass accuracy, resolution, and dynamic range of a FT-ICR for improved proteomic coverage. The ion accumulation efficiency is demonstrated to be an order of magnitude greater than that observed with LTQ-FT Ultra instrumentation. The proteome coverage with yeast was shown to increase over the previous instrument generation by 50% (100% increase on the peptide level). In addition, many lower abundance level yeast proteins were only detected with this modified instrument. This novel configuration also enables beam type CID fragmentation using a dual cell RF ion trap mass spectrometer. This technique involves accelerating ions between traps while applying an elevated DC offset to one of the traps to accelerate ions and induce fragmentation. This instrument design may serve as a useful option for labs currently considering purchasing new instrumentation or upgrading existing instruments. BIOLOGICAL SIGNIFICANCE: A novel hybrid mass spectrometer that allows increased MS/MS acquisition rates with high mass measurement accuracy and new ion fragmentation methods greatly improves the number of proteins, posttranslational modifications and protein-protein interactions that can be identified from cells.
Asunto(s)
Ciclotrones , Análisis de Fourier , Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Iones/análisis , Iones/química , Péptidos/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
We describe the design and current performance of a 14.5 T hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer. Ion masses are routinely determined at 4-fold better mass accuracy and 2-fold higher resolving power than similar 7 T systems at the same scan rate. The combination of high magnetic field and strict control of the number of trapped ions results in external calibration broadband mass accuracy typically less than 300 ppb rms, and a resolving power of 200,000 (m/Delta m50% at m/z 400) is achieved at greater than 1 mass spectrum per second. Novel ion storage optics and methodology increase the maximum number of ions that can be delivered to the FTICR cell, thereby improving dynamic range for tandem mass spectrometry and complex mixture applications.
RESUMEN
An automated top-down approach including data-dependent MS(3) experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS(2) spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified. Subsequent MS(3) analyses pinpointed the location of these modifications. In addition, data-dependent MS(3) experiments were performed on standard proteins and wild-type yeast proteins using the stand alone linear trap mass spectrometer. Initially, the most abundant molecular ions underwent collisionally activated dissociation, followed by data-dependent dissociation of only those MS(2) fragment ions for which a charge state could be automatically determined. The resulting spectra were processed to identify amino acid sequence tags in a robust fashion. New hybrid search modes utilized the MS(3) sequence tag and the absolute mass values of the MS(2) fragment ions to collectively provide unambiguous identification of the standard and wild-type yeast proteins from custom databases harboring a large number of post-translational modifications populated in a combinatorial fashion.
Asunto(s)
Cromatografía Liquida/métodos , Mapeo Peptídico/métodos , Robótica/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Sistemas en Línea , Análisis de Secuencia de Proteína/métodosRESUMEN
We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.