Journal of the American Society for Mass Spectrometry最新文献

Gas-phase ion conjugates containing a 4-(2-phenyl-2H-tetrazol-5-yl)benzoyl group that was tethered to the lysine side chain in peptides SAAAK and TAAAK, denoted here as (SAAA-tet-K + H)⁺ and (TAAA-tet-K + H)⁺, were found to undergo loss of N₂ and cyclization upon UV photodissociation (UVPD) at 213 and 250–300 nm, forming two distinct populations of photofragment ions. Ions of the first type underwent prompt dissociation following loss of N₂ that involved transfer of the N-terminal amide oxygen and two exchangeable hydrogen atoms onto the reactive nitrile-imine intermediate, followed by loss of an N-terminal HN = CH-R fragment, R = CH₂OH and CH(OH)CH₃ for Ser and Thr, respectively. This prompt dissociation was most abundant at 5.82 eV, depleting the population of the ions of the first type, but gradually decreased at lower excitation energies in the 4.96–4.13 eV range. The other types of photofragment ions were represented by stable denitrogenated intermediates that were formed competitively upon UVPD. These were probed by collision-induced dissociation, CID-MSⁿ, showing losses of internal alanine residues and indicating macrocyclic structures. Precursor ion structures were established by matching collision cross sections (CCS_exp), obtained by high-resolution cyclic ion mobility measurements, with theoretical CCS_calc that were obtained by density functional theory (DFT) calculations for low-Gibbs-energy ion conformers. The macrocyclic ring formation was found to involve the serine and threonine hydroxyl groups that attacked the nitrile-imine carbon atom, forming O-linked structures, as corroborated by ion mobility measurements and matching CCS_calc and CCS_exp. The cyclization energetics and kinetics was probed by Rice-Ramsperger-Kassel-Marcus (RRKM) calculations of rate constants and Born–Oppenheimer molecular dynamics at points along the C–O reaction coordinate that stressed the role of hydroxyl proton transfer onto the neighboring N-terminal amine group. Structures were also proposed for the products of UV-induced prompt dissociation for which matching CCS_calc and CCS_exp were obtained. The serine and threonine hydroxyl cyclization to nitrile imines upon UVPD represents a new reaction type in the formation of peptide macrocyclic structures.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been documented as a useful tool for top-down proteomics (TDP). However, CZE-MS/MS-based TDP typically has limited backbone cleavage coverage for identified proteoforms due to the use of traditional collision-based fragmentation methods (i.e., higher-energy collisional dissociation, HCD). Here, for the first time, we coupled CZE to an Orbitrap Ascend Tribrid mass spectrometer to investigate the performance of collision-, electron-, and photon-based fragmentation methods and their combinations for boosting the backbone cleavage coverage of proteoforms during the electrophoretic time scale using a standard protein mixture covering a mass range of about 10–70 kDa. CZE-MS achieved reproducible measurement of six proteins including three insulin-like growth factor (IGF) proteoforms with different modifications. Systematic investigations of HCD, electron-transfer dissociation (ETD), electron-transfer/HCD (EThcD), and ultraviolet photodissociation (UVPD) during CZE-MS/MS analysis revealed distinct yet complementary fragmentation characteristics. ETD, EThcD, and UVPD, in general, provided higher backbone cleavage coverage than HCD. The integration of HCD, ETD, EThcD, and UVPD data offered 67 and 98% sequence coverage for carbonic anhydrase (a 30 kDa protein) and thioredoxin (a 12 kDa protein), which is 158 and 100% higher than that produced by HCD alone. Adding internal fragments further boosted the backbone cleavage coverage substantially, for example, from 67 to 94% for 30 kDa carbonic anhydrase and from 21 to 82% for 50 kDa protein AG. The results demonstrate the capability of CZE-MS/MS with the integration of various fragmentation techniques for comprehensive characterization of proteoforms in a wide mass range.

In recent publications, we have demonstrated applications of multiplexed affinity selection electrospray mass spectrometry based on three different principles: size-exclusion chromatography, flow-induced dispersion analysis, and Taylor/non-Taylor dispersion. To enable this multiplexing─i.e., simultaneous measurement of pools of ligands, with their masses acting as selective labels─higher resolving power was required than is typically achievable in native MS; therefore, we worked under conditions that promoted gas-phase ejection of protein-bound ligands, allowing their detection with high mass accuracy in the low-m/z region of the spectrum. Subsequent data analysis required correlation of the extracted ion chromatograms (EICs) of candidate ligands with the EIC of the target protein. Even when relying on simple visual inspection, generating these EICs manually is laborious even for only a few dozen ligand candidates, and a quantitative correlation based on statistical tests quickly becomes very time-consuming. Performing such an experiment for a larger compound library or even without a defined target list, but by instead extracting the chromatogram for every low-m/z signal present, is entirely impractical. Here, we present CATALYST (Computer-Assisted Time Alignment for Ligand Yield and Screening Tool), an open-source software package that can perform this type of analysis─in either targeted or untargeted mode─in a matter of seconds. CATALYST performs several statistical tests to correlate EICs and identify protein-binding ligands and then visualizes the results, greatly accelerating affinity selection mass spectrometry workflows.

Distinguishing metabolite isomers often relies on comparing relative data, such as relative chromatographic retention times and ion mobility arrival time orders, or relative product ion abundances. These approaches necessitate the need for quality reference data and/or chemical standards. An ideal method for differentiating isomers would leverage one of the absolute physiochemical properties of the isomers, and would have no reliance on instrument vendor, chromatographic column chemistry, or external reference data. For example, the pK_a of an aromatic hydroxy hydrogen changes according to ring position across isomers (e.g., 4- vs 5-hydroxyindole). Herein, we leverage the difference in pK_a to resolve 4- and 5-hydroxy positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine), the structural moiety of compounds with profound effects on the serotonergic system. We first use hydrogen–deuterium exchange (HDX) to rapidly exchange the indole amine hydrogen and gradually exchange the indole hydroxy hydrogen atoms to deuterium atoms. We then back-exchange the indole amine deuterium atom back to a hydrogen atom on the LC column and monitor the kinetic exchange rates of the retained aromatic hydroxy deuterium atom using high resolution mass spectrometry (HRMS). HDX kinetics allow for facile differentiation of the two isomers, with only 4-hydroxy-N,N-dimethyltryptamine exchanging at an appreciable amount within hours. These results could ultimately be used to characterize a variety of unknown structural isomers.

Here, we introduce Spectral Cruncher, an interactive extension to the PatternLab for Proteomics platform, designed to bridge the gap between manual curation and state-of-the-art computational analysis of proteomic tandem mass spectra. Spectral Cruncher integrates de novo sequence tag extraction, automated spectral annotation, targeted tag search, and a customized transformer-based fragment-ion intensity predictor (SpecFormer) within a unified graphical environment, designed for interactive and instrument-specific visualization. Central to this workflow is SpecFormer, a compact transformer architecture trained on multiple data sets, providing independent ion intensity models for Q-Exactive + bulk, Astral bulk, and Astral single-cell proteomics data, enabling accurate and instrument-specific intensity prediction even under conditions of sparse fragmentation and low signal-to-noise ratios. Evaluation of SpecFormer demonstrates high predictive performance, with average cosine similarities of approximately 0.98 for bulk Q-Exactive + data sets, 0.91 for bulk Astral, and 0.87 for Astral single-cell data. These tools enable researchers to interrogate ambiguous spectra, validate peptide identifications, and develop intuition for algorithmic limitations. The tools are freely available within PatternLab 5.1, lowering technical barriers and promoting broader adoption of interactive, expert-driven workflows as well as providing a learning environment. A video of our tool in action is available at https://youtu.be/tc2sPiqJkLA.

The increasing adoption of high-resolution ion mobility (HRIM) in untargeted omics workflows underscores the need for precise collision cross-section (CCS) measurements which are highly reproducible across various laboratories and instrumentation. To evaluate the reproducibility of a high-resolution ion mobility platform, an interlaboratory study was undertaken using structures for lossless ion manipulation-based traveling wave ion mobility spectrometry (TWSLIM) in nitrogen drift gas. Across 250 lipid features spanning glycerophospholipids, glycerolipids, and sphingolipids detected from a lipidomic extract of human plasma standard reference material, the platform demonstrated high CCS measurement reproducibility, with an average relative standard deviation (%RSD) of ∼0.1%. Triglycerides in general were found to exhibit multiple IM features that served as an illustrative example where analysis via HRIM and data-independent, mobility-aligned fragmentation (MAF) provides critical insights into their chemical structures. To support the future development of HRIM in lipidomic workflows, a large (n = 250) number of lipid consensus ^TWSLIMCCS_N2 values was compiled from the interlaboratory study into a HRIM database for community use.

Ultrahigh-resolution mass spectrometry (UHRMS) is a well-established analytical method for characterizing complex molecular mixtures. It is usually performed with Fourier transform techniques, based either on ion cyclotron resonance (FTICR-MS) or mass-dependent oscillations in an ion trap (FT-Orbitrap-MS). In spite of the high technical level of these instruments, often spectral interpretation remains difficult, in particular in a nontargeted approach of complex samples. Here, we introduce a Diophantine method for molecular formula assignment. Taking the ubiquitous Gaussian distribution as an example, we first show how knowledge about random mass error can be used to assign molecular formulas in a statistically consistent way. By considering all possible attributions within a large mass error range, we show how the systematic error stemming from suboptimal calibration can be distinguished from the random mass error in peak position. Correcting for systematic mass error leaves us with a quantifiable, Lorentzian random mass error as expected for Fourier transform-based instruments with long transients. This indicates that our method is self-consistent, assigning molecular formulas close to the theoretical limit of achievable accuracy.

Histological and immunofluorescence imaging techniques are widely used for studying protein localization in biological tissues, from the identification of disease to the development of drugs with greater target specificity and lower toxicity. However, the complexity of such samples often requires the use of chemical labels and targeted analysis, thereby limiting proteome coverage. In contrast, mass spectrometry imaging (MSI) enables label-free, highly multiplexed analysis of tissue proteomes with cellular spatial resolution. The complexity of protein mixtures analyzed in MSI often leads to mass spectra at each pixel in the image with extensive signal overlap and a pronounced, curved baseline, which complicates data analysis and interpretation. Here, we introduce a parallelized Gábor-transform-based batch deconvolution workflow (“iFAMS Imager”) that resolves strongly overlapping protein signals in MSI and offers flexible options for baseline correction. Together, these advantages facilitate the removal of interferent signals strongly overlapped in m/z with those of the target analyte, resulting in high-fidelity images. Use of this open-source, publicly available software is demonstrated for imaging of common proteins in rat brain tissue.

Depsipeptides are peptides that contain both amino acid and hydroxy acid residues. In this study, we sought to investigate how terminal hydroxyl groups and/or backbone ester linkages from hydroxy acid residues in depsipeptides affected collision-induced dissociation (CID). The canonical tripeptide glycine-alanine-glycine (GAG) was compared to all three of its depsipeptide analogues: glycolic acid-AG (gAG), G-lactic acid-G (GaG), and GA-glycolic acid (GAg). Experimental data was supported by density functional theory (DFT) calculations to gain insight into which sites on the molecules have sufficient proton affinity (PA) to localize the proton and the resulting charge-directed fragmentation processes.