Journal of the American Society for Mass Spectrometry最新文献

Recent advances in ion mobility spectrometry have enabled the measurement of rotationally averaged collisional cross-sectional area (CCS) for millions of peptides as part of routine proteomic mass spectrometry workflows. One of the most striking findings in recent large ion mobility data sets is that CCS exhibits two distinct modes, most notably for charge 3⁺ peptides, with peptides predominantly exhibiting CCS in either the high or low mode. Here, using classical machine learning approaches, we identify that basic site positioning is a key sequence feature determining a peptide’s CCS mode. Molecular dynamics simulations suggest that peptides in the high CCS mode tend to adopt more extended conformations and form charge-stabilized helical structures, whereas those in the low CCS mode adopt more compact, globular conformations. Further supporting this structural hypothesis, we provide evidence for preferential protonation near the C-terminus and uncover multiple position-dependent sequence determinants that all suggest the predominance of helix formation in the high CCS mode. Together, these findings will enable better integration of CCS measurements into protein identification and quantification pipelines, improving the performance of ion mobility-based proteomics.

Digital quadrupoles are driven with rectangular RF waveforms and, through duty cycle control, can access higher-order Mathieu space stability zones (HZs) using comparatively low voltages. Analytically, accessing these zones remains attractive as HZs exhibit higher resolving powers ((m/z)/(Δm/z)) compared to the conventional quadrupole operation in zone (1,1). Presented here is a modification of a commercial quadrupole time-of-flight (Q-TOF) equipped with a low-voltage digital waveform driver to navigate the filtering quadrupole in the HZs. We demonstrate that a digital quadrupole operating in zones (3,1) and (3,2) achieves a narrowed isolation window for analytes up to 690 and 1543 m/z, respectively, compared to conventional operational modes. Experimental trends suggest this performance to continue to approximately 900 m/z in zone (3,1) and 2500 m/z in zone (3,2). These narrowed isolation windows were utilized to generate MS/MS data from a mixture of m-hydroxybenzoylecgonine (306 m/z) and cocaine-d3 (307 m/z), which exhibited minimal chimeric nature without the use of deconvolution software. The development of hybrid systems capable of both sine and digital operation provides a mechanism to maximize selectivity of the HZs while maintaining the benefits of traditional modes of operation. Minimizing chimeric interferences is of particular importance, and the narrow isolation widths afforded by HZs are readily accessible using tandem instruments for analytes <2500 m/z.

Advances in liquid chromatography–mass spectrometry have significantly improved proteomic analyses of human plasma. However, information at the level of intact proteoforms remains limited due to the high dynamic range of protein abundance and the complexity of post-translational modifications. To address this challenge, we introduce soluble plasma proteoform analysis via acetonitrile precipitation (SPAP), a streamlined workflow for top-down mass spectrometry-based proteomics that isolates small, intact proteoforms from the acetonitrile-soluble plasma fraction, enabling direct measurement of proteoform diversity and post-translational modifications with high resolution. This simple and scalable method employs cold acetonitrile to precipitate abundant plasma proteins, thereby enriching the sample for lower-molecular-weight proteoforms. We first assessed the method’s performance using a reference plasma sample. To explore its clinical applicability, we applied SPAP to a cohort of 40 individuals, including 30 patients with liver cirrhosis and 10 healthy controls. In total, we report 3746 proteoforms from 255 proteins, including those with phosphorylation, truncation, and disulfide bond modifications. Reproducibility was confirmed with a coefficient of variation of <10% for the majority of enriched proteoforms, including those potentially associated with hemostasis, lipoprotein metabolism, cytoskeletal structure, and protease regulation. SPAP enabled effective stratification of the three cirrhosis stages, verifying previously published results and supporting the identification of candidate biomarkers. Although liver cirrhosis was used as a model system, the SPAP workflow is broadly applicable to human disease with proteoform-level resolution, offering a new path to stronger correlations in smaller cohorts and addressing key challenges in diagnostic and biomarker discovery.

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful technique for probing protein dynamics, stability, and interactions. However, multistate and nonequilibrium experiments currently do not have available analysis tools. We present HydroBot, a software designed for comprehensive and interactive HDX-MS data analysis and visualization. HydroBot supports rapid and automated uptake plotting, statistical testing of differences between protein states, and multiple interactive visualization modes including bar plots, Woods plots, and heatmaps. Statistical tools such as volcano plots and error distribution analyses are integrated to assess data robustness, enabling interactive exploration of labeling differences. Correlated structural dynamics are revealed by k-means or hierarchical clustering, facilitating pattern discovery within multistate HDX data for proteins at equilibrium and nonequilibrium. Additionally, scripts are created to enable visualization of HydroBot analyses on protein structures. This user-friendly tool streamlines HDX-MS validation and interpretation from processed data to biologically relevant insights.

Analyzing cultural heritage (CH) materials, particularly organic substances, is challenging due to their complex chemical composition. A key requirement in such analyses is the use of analytical techniques that cause minimal damage to the artifact while providing the maximum amount of chemical information about the materials. Chromatographic and mass spectrometric (MS) techniques give valuable information about components of the organic materials, but these typically require a microsample from the object, along with specific preparation and instrumental conditions. For CH artifacts, techniques that work directly on the surface, thereby causing minimal damage, are far more desirable. We have developed a 355 nm optical fiber-coupled laser ablation (LA) atmospheric pressure chemical ionization (APCI)-MS system that enables the analysis of organic material directly from the solid surface of the artifact under ambient conditions with minimal surface damage. In this study, we coupled LA with APCI-Fourier transform ion cyclotron resonance (FT-ICR)-MS. The main aim was to evaluate the effectiveness of the developed LA-APCI high-resolution (HR)MS system for the analysis of five handmade mock-up materials of different paint and varnish layers and one real-life sample. The results demonstrate the analytical potential of the LA-APCI-HRMS technique, as high-quality and identifiable mass spectra were obtained for most of the analyzed materials. In the future, the developed LA-APCI-HRMS technique could be applied not only to cultural heritage but also to other fields (e.g., forensics, material science, etc.).

This article presents two programs to help users construct mass spectral libraries for use with the NIST/NIJ DART-MS Data Interpretation Tool (version 3.22). The Full Database Builder program─which is a modification of the original database building script published through NIST─generates libraries that follow the exact specification of the NIST DART-MS Library builder. The Basic Database Builder program requires less information from the user and has fewer computational dependencies, making it an ideal program for users building small in-house libraries for research and testing purposes. The programs are available at https://github.com/asm3-trentu/CRAFTS-DBBuilder.

This study examines the effects of radial asymmetry in a linear quadrupole mass filter with circular rods, introduced either by a change in the electrode radii or by displacement of a diametrically opposite pair. A radial potential model is developed to account for the resulting geometric deviations, enabling the analysis of the first stability region specific to the quadrupole component. The transmission characteristics of such asymmetric configurations are systematically examined, revealing a linear shift in the transmission peak along the q-axis as a function of the asymmetry parameter. This behavior is interpreted through modifications observed in the stability diagram. Furthermore, increased asymmetry is shown to broaden the transmission contours and generally reduce the resolution. Notably, a ‘magic’ asymmetry parameter of −0.02 corresponding to electrode displacement yields enhanced resolution compared to the symmetric case for a fixed rod-to-field radius ratio. An empirical relationship is established between the resolution and the combined contribution of the coefficients of octupole and dodecapole potential components, highlighting the critical role of higher-order field effects in performance optimization.

Linear ion traps (LITs) with simplified geometries have shown potential for miniaturized mass spectrometry systems, though structural simplifications often introduce nonlinear higher-order fields that compromise performance. This study investigates asymmetric hyperbolic electrode modifications to address the inherent 50% detection efficiency limitation in miniaturized LITs caused by bidirectional ion ejection. The research employed numerical simulations to evaluate a geometrically modified LIT design. Electrode asymmetry was introduced through independent adjustments of vertex curvature radii and bending angles in opposing x-direction electrodes, creating an asymmetric trapping field. Structural optimization involved initial x-axis elongation of a centrosymmetric ion trap followed by controlled geometric modifications. Field analysis confirmed the incorporation of odd-order multipole components through these adjustments. Experimental results demonstrated approximately 90% ion ejection efficiency from a single trap side when combining higher-order odd and even multipole fields. This work provides valuable insights into the design of asymmetric ion traps, offering a practical solution for high-performance portable MS development.

For tandem mass spectrometry, photoactivation capabilities enable a host of useful dissociation strategies. Herein we present the first implementation of an IR laser system on a next generation, quadrupole-Orbitrap-quadrupole linear ion trap hybrid MS system (Thermo Scientific Orbitrap Ascend). In addition, we establish xenon as an efficient source of radical cations for negative electron transfer dissociation (NETD) reactions. First, we take advantage of the instrument’s linear architecture to include a home-built photon detector to improve ease of use and laser alignment, along with straightforward introduction of xenon into the instrument. Second, we assess the instrument performance through infrared-activated NETD (IR-NETD) of a simple, unmodified 6-mer RNA molecule. Finally, we assessed the performance of IR-NETD for characterizing a synthetically complex small interfering RNA molecule, evaluating the effects of parameters including precursor charge state and IR laser power, demonstrating the benefits of concurrent IR photoactivation during NETD reactions. This straightforward instrumental approach represents a powerful and versatile tool for the characterization of complex biopharmaceutical molecules.

Field-induced ion activation in medium to high pressure regions of a mass spectrometer or ion mobility spectrometer can lead to changes in the ion structure, namely unfolding, tautomerization, or fragmentation. To either prevent mislabeling of spectra or utilize these effects efficiently, the underlying ion dynamics need to be understood. Hydroxyl-containing compounds in particular show significant fragmentation (loss of H₂O), yet the energetics and mechanisms are not well studied. This is particularly true for primary hydroxyl groups, as the presumably formed primary carbocations are highly instable. In this study, we investigate the dynamics of the field-induced fragmentation of protonated primary and secondary alcohols using a combined theoretical and experimental approach. Specifically, we combine density functional theory and reaction kinetics modeling with fragmentation measurements using a HiKE-IMS-MS and tandem IMS device. We find that the fragmentation mechanism of both primary and secondary protonated alcohols proceeds via a protonated cyclopropane (PCP⁺) moiety. Especially for primary alcohols, this moiety enables an intramolecular S_N2 reaction where the neutral H₂O at the terminal carbon is substituted by an H-shift, directly yielding a secondary carbocation. Our results suggest quite high fragmentation rates, even at moderate ion activations, rendering protonated alcohols very unstable. However, we also find that neutral background water can form ion–solvent clusters with the protonated alcohols that effectively prevent the fragmentation. This could also help stabilize other labile ions in the future.