Journal of the American Society for Mass Spectrometry最新文献

Depsipeptides, a structurally diverse class of nonribosomal peptides with broad bioactivities, present significant challenges for systematic characterization due to their complex fragmentation patterns in mass spectrometry (MS). This study aims to develop a generic three-step strategy based on high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) to facilitate the rapid screening and identification of both linear and cyclic depsipeptides. The workflow is defined by three sequential, logic-driven steps: (1) filtering potential depsipeptide precursors via diagnostic and adduct ions along with depsipeptide classification; (2) identifying the structural residues by monitoring diagnostic product ions and neutral losses; and (3) sequencing the peptide backbone through comprehensive analysis of MS/MS spectra. To validate the strategy’s universality, it was applied to the analysis of depsipeptides in a complex biological matrix (extracts of Bombyx batryticatus). A total of 62 depsipeptides (encompassing octa-, hexa-, tetra-, and didepsipeptides, with both cyclic and linear topologies) were identified or tentatively characterized, including 34 potential novel analogs. Notably, the strategy successfully distinguished 10 methionine-containing depsipeptides with subtle redox modifications (native methionine, methionine sulfoxide, methionine sulfone), demonstrating its ability to resolve structurally similar derivatives. This three-step strategy offers a simple, rapid, and robust tool for comprehensive depsipeptide profiling. Its application to complex matrices highlights the strong potential for extending to other biological samples, advancing systematic analysis of depsipeptide families in natural products and biological systems.

Ambient Mass Spectrometry (AMS) has revolutionized forensic analysis by enabling rapid and direct detection of chemical compounds on complex surfaces with minimal or no sample preparation. Among AMS techniques, desorption electrospray ionization (DESI) and direct analysis in real time (DART) mass spectrometry have emerged as powerful analytical tools due to their speed, sensitivity, and versatility. This review examines the major applications of DESI and DART in forensic science, including the detection of explosives, gunshot residue, illicit drugs, inks, biological fluids, and latent fingerprints. The principles of each technique are briefly discussed, followed by a comparative analysis of their advantages, limitations, and performances in various forensic scenarios. Recent developments, practical considerations for implementation, and future perspectives are also addressed, highlighting the growing impact of DESI and DART in real-time forensic investigations and evidence processing.

The inherent heterogeneity of biological macromolecules offers a unique challenge for analysis. The combination of ion mobility (IM) and mass spectrometry (MS) is sensitive to the size, shape, and dynamics of, for example, proteins and their complexes. Combining multiple dimensions of ion mobility and mass spectrometry (IM-IM-MS) while leveraging unique gas-phase manipulations between dimensions has great potential for increasing the information content for challenging analytes. Here, we introduce an instrument, SLIMPHONY, which was built using the Structures for Lossless Ion Manipulations (SLIM) architecture. SLIMPHONY is unique in that eight independently controlled traveling-wave regions work in concert to enable complex, multidimensional separations. Single-dimension IM-MS experiments were used to separate a mixture of protein and protein-complex ions and demonstrate that the peak-to-peak resolution increases roughly with the square root of the separation length for a pair of hexakis(fluoroalkoxy)phosphazine ions. Ion selection and trapping between dimensions was then used to probe the gas-phase unfolding of a subpopulation of ubiquitin ions. Finally, by varying the guard potential used to confine ions, we demonstrate tunable activation of ubiquitin subpopulations, which we analyzed using IM separations of various lengths. With the ability to select and activate ions in multiple regions, to vary the number of dimensions of IM, and to control the length of IM separation, SLIMPHONY is a flexible platform for characterizing protein ions.

The discovery of novel bioactive compounds remains a cornerstone of natural product (NP) chemistry. However, traditional NP discovery workflows are time- and resource-intensive, hindering sustainability and efficiency of multicondition screening projects. This study evaluates the application of the liquid microjunction surface sampling probe (LMJ-SSP) with partial least-squares discriminant analysis (PLS-DA) as a preliminary step in the NP discovery pipeline. By integrating ambient mass spectrometry with machine learning, we analyzed four strains of Penicillium fungi grown under 13 unique conditions in situ, without sample preparation. Using PLS-DA, we prioritized the growth conditions that maximized chemical diversity, offering insights into metabolite composition prior to resource-intensive steps in the NP discovery workflow. This LMJ-SSP-based approach achieved a significant reduction in sampling time (96%), overall cost (98%), and solvent consumption (98%)─streamlining the NP discovery pipeline through chemically informed prioritization and improved sustainability.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for studying protein structural dynamics. A critical step in the HDX-MS workflow is generating a peptide map from nondeuterated samples, which serves as the reference for identifying and monitoring peptides in subsequent deuterium-labeled experiments. Maximizing peptide identifications improves sequence coverage and redundancy, enhancing the information content and spatial resolution of the HDX-MS data. However, peptide identification is often limited by suboptimal peptide separation/fragmentation. In other proteomic workflows, longer liquid chromatography (LC) gradients are commonly used to improve the peptide identification by increasing resolution. However, in HDX-MS workflows, such gradients are generally incompatible due to time constraints imposed by deuterium/hydrogen back-exchange. To address this, we introduce a flexible workflow that uses long-gradients during initial peptide mapping, followed by retention time (RT) interpolation for application in subsequent short-gradient HDX-MS. By performing both long- and short-gradient peptide mapping, we used shared peptides to generate a regression model that predicts short-gradient RTs for all peptides identified in the long-gradient experiment. This enables the use of the richer peptide maps provided by long-gradient chromatography without compromising the deuterium retention. The method is implemented by RTinterpolator, a freely available R script compatible with widely used HDX analysis platforms that rely on reference RT values for peptide monitoring in deuterium-labeled data. By providing predicted RTs aligned to short gradients, RTinterpolator offers a practical, accessible, and instrument-independent way of increasing sequence coverage and redundancy in HDX-MS experiments, particularly for large or complex proteins susceptible to the limitations of short-gradient chromatography.

Tandem mass spectrometry (MS/MS) is frequently used in phospholipid characterization to determine lipid class, fatty acyl chain length, and degree of unsaturation. However, conventional MS/MS methods are limited in characterizing isomeric lipids resulting from variants in double bond and sn-positions within the fatty acyl chains. This limitation is due to the fact that conventional collision induced dissociation (CID) of even-electron lipid precursor ions does not generate highly efficient product ions from intrachain fragmentation of the fatty acyl substituents. Herein, we have developed a workflow that leverages a new lipid ion type to facilitate radical-directed dissociation (RDD). Briefly, low-energy CID of [M + Cu^II + anion]⁺ ion types results in a radical cation, and subsequent activation of the radical cation results in highly efficient intrachain fragmentation of fatty acyl chains. This method provides abundant diagnostic fragment ions that are associated with the double bond and fatty acyl chain positions on the glycerol backbone and thus can be used to differentiate isomeric phosphatidylcholines (PCs). The incorporation of an anionic ligand in the [PC + Cu^II + anion]⁺ ion type is key to this chemistry. Specifically, the major fragmentation channel for the [PC + Cu^I]⁺ ion type is neutral loss of phosphocholine headgroup but shifts to RDD for [PC + Cu^II + anion]⁺ ion types containing strongly electronegative anions.

Accurate structural identification of lipids in mass spectrometry is essential for advancing lipidomics and achieving a holistic understanding of complex cellular systems. Gas-phase charge inversion ion/ion reactions, which allow for alteration of the ion type before dissociation, have been shown to improve lipid identification. The products observed from these reactions arise from competing and consecutive pathways, but limited studies have been performed to characterize the mechanisms of these interactions. Specifically, we have used a charge inversion ion/ion reaction between 1,4-phenylenedipropionic acid (PDPA) and phosphatidylcholines (PCs) to provide structural information on fatty acyl sn-positions and enable separation of isobaric and isomeric lipids. Upon reaction with PDPA, [PC + H]⁺, [PC + Na]⁺, and [PC + K]⁺ analyte ion types each demonstrate differences in partitioning between two major product ion channels: successful lipid charge inversion resulting in a demethylated lipid anion, which can then be subjected to collision induced dissociation (CID) to reveal fatty acyl sn-positions, and single-particle transfer from PC to PDPA resulting in a neutral lipid and charge reduced PDPA, which provides no information on the lipid structure. In this work, density functional theory (DFT) calculations were performed to characterize relevant potential energy barriers for the competing processes, which enables insights into the factors that affect the relative product ion partitioning. These calculations provided detailed insights into the structural dynamics and potential energy barriers associated with proton transfer, methyl group migration, and other competing interactions. Our results revealed that specific transition states differ significantly depending on the ion type and reaction environment, suggesting that the energetic landscape of these processes is influenced by both the size and the coordination state of the cation. Understanding the roles of the energy barriers in these competing reaction processes within the ion–ion reaction complex is crucial to increasing reaction efficiency and designing next-generation reagents to enable improved lipid structural elucidation by gas-phase reactions. This research provides a fundamental perspective of ion/ion reaction mechanisms and illustrates the importance of the ion type and ion structure on product ion partitioning. This deeper mechanistic understanding highlights the nuanced balance between thermodynamics and kinetics in determining product distribution, and these factors can be used to intelligently select new reagents to precisely tune and control desired reaction products.

Ensuring the quality of pharmaceutical products, particularly for complex recombinant protein drugs such as TNF receptor 2-Fc fusion proteins (TNFR2-Fc, Etanercept), poses significant public health challenges. These products, including biosimilars and follow-on versions, exhibit intricate glycosylation patterns and heterogeneous post-translational modifications, complicating their analytical assessment. The Chinese market, hosting four different TNFR2-Fc products, presents a unique regulatory challenge for rapid differentiation and quality control. This study developed a novel mass spectrometry-based multiattribute method (MAM) to address this challenge, enabling simultaneous monitoring of multiple quality attributes and effective differentiation among products from various manufacturers. Conventional techniques initially indicated high purity across all products, but these methods provided limited capabilities for differentiation. The improved MAM approach, involving desialylation, partial deglycosylation, and digestion steps, minimizes heterogeneity and simplifies analysis. This method successfully indicates differences in primary amino acid sequences and specific quality attributes, allowing for a clear differentiation among manufacturers. Notably, products from manufacturers A and B, as well as C and D, despite their high similarity, could be differentiated by their O-glycan profiles. Further activity evaluations revealed that the products from manufacturers C and D exhibited lower binding and biological activity, potentially due to differences in primary amino acid sequences or disulfide bond mismatches. Additionally, all products demonstrated similar Fc-effector functions. In conclusion, this study underscores the variability among TNFR2-Fc products in the Chinese market and the necessity for robust regulatory oversight. The MAM method developed herein serves as a rapid, accurate, and technologically advanced platform for quality control with significant implications for regulatory authorities, healthcare providers, and patients in ensuring access to safe and effective TNFR2-Fc products.

The selective ionization of cyclopentadiene-derived polycyclic aromatic hydrocarbons (PAHs) in complex matrices remains a persistent challenge in direct ionization mass spectrometry, particularly under atmospheric pressure ionization (API) conditions. In this study, tetramethylammonium hydroxide (TMAH) was evaluated as a solvent modifier to enhance the ionization efficiency of PAHs in negative-ion mode using high-resolution Orbitrap MS. A systematic assessment with equimolar mixtures of neutral and acidic model compounds was performed to elucidate the fundamental effects of TMAH on ion suppression, deprotonation mechanisms, and gas-phase ion chemistry. The incorporation of TMAH significantly improved the detection of weakly acidic and neutral PAHs by promoting efficient gas-phase deprotonation, thus overcoming conventional ESI limitations. To demonstrate the method’s analytical robustness, TMAH-assisted ESI was subsequently applied to crude oil samples, enabling the detection of hydrocarbon species otherwise inaccessible under standard conditions, with a particular focus on low-alkylated fluorene derivatives. These results establish a simple and effective strategy for expanding the analytical scope of ESI-MS toward nonpolar and weakly acidic hydrocarbons, offering a valuable tool for the advanced molecular characterization of complex organic mixtures.