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.
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.
Structural characterization of novel metabolites in drug discovery or metabolomics is one of the most challenging tasks. Multilevel fragmentation (MS n ) based approaches combined with various ...dissociation modes are frequently utilized for facilitating structure assignment of unknown compounds. As each of the MS precursors undergoes MS n , 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 MS n 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 MS n 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.
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.
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.
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.
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/Δm 50% 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.
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.
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.
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