Structural isomers are critical analytes in the biological and chemical arenas. Despite the ability of tandem mass spectrometry to provide fragment ion information, their high structural similarity impedes confident identification. To address this, we developed a novel method leveraging energy-resolved mass spectrometry (ER-MS) of fragment ions generated by electron activation dissociation (EAD). EAD initiated rapid radical chain dissociation via electron excitation and removal mechanisms, delivering superior isomer discrimination compared to conventional collision-induced dissociation (CID). Subsequent energy-resolved analysis further enhanced the distinction by integrating these dissociation mechanisms. Our strategy employed a cosine-based multidimensional spectral similarity algorithm to visualize and quantify subtle spectral differences across multiple energies. This method successfully distinguished many types of isomers, such as linkage, composition, and conformation isomers in disaccharides and flavonoid glycosides and achieved 93.8% top-1 identification accuracy against an in-house library. When applied to pomelo peel and commercial beverages for key metabolite characterization, it provided 44.4-50.0% top-1 annotation accuracy across all detected interest features. These results demonstrate that the multidimensional similarity algorithm that combines EAD and ER-MS significantly advances the depth and accuracy of compound annotation.

A quadrupole-ion-trap-integrated time-of-flight secondary ion mass spectrometry (QIT-ToF-SIMS) system was developed to enable detailed molecular surface analysis via tandem mass spectrometry combined with laser photodissociation spectroscopy. In this approach, secondary molecular ions were selectively isolated in the ion trap using the stored waveform inverse Fourier-transform technique and subsequently subjected to collision-induced dissociation or photoinduced dissociation. Using isomeric rhodamine dyes as model systems, structurally diagnostic fragment ions were observed, allowing for clear molecular identification despite identical nominal masses. The laser photodissociation spectra obtained for the isolated ions revealed a distinct wavelength-dependent fragmentation behavior, consistent with known gas-phase absorption profiles. Because of the integration of tandem mass spectrometry and laser spectroscopy with time-of-flight secondary ion mass spectrometry, the QIT-ToF-SIMS system serves as an effective tool for molecular-level surface characterization, which is particularly well suited for probing increasingly complex organic materials and interfaces.

Peptide-nucleic acids (PNAs) remain a promising therapeutic modality, although their success in the clinic remains modest compared to other nucleic acid-based products, such as thiophosphoryl and thiophosphoramide oligonucleotides. To address this, extensive efforts have been made in the past decade to optimize the structure of PNAs aiming at improving their manufacturability, in vivo stability, specificity and targeted delivery. Large sizes and complex structures of the newest-generation PNAs (featuring nonuniform distribution of backbone modifications to increase stability and enhance binding to therapeutic targets, and incorporating extended peptide segments serving as targeted delivery vectors) pose unique challenges vis-à-vis structural characterization of these molecular entities. We use a top-down MS/MS approach to sequence a 6 kDa hybrid PNA molecule comprising a short polybasic cell permeabilizing segment (CPS) and a long antisense sequence segment, and containing γ-hydroxymethyl groups distributed unevenly across the backbone to stabilize the helical structure. Localization of the positive charge within the CPS enables backbone cleavages only within this PNA segment when conventional ion fragmentation methods are used, while the non-natural γ-peptide backbone prevents the use of proteases to assist the structural analyses. However, high-energy collisional activation (implemented with MALDI TOF/TOF) provides complete sequence coverage of the entire PNA molecule, including localization of all γ-hydroxymethyl groups, thereby enabling de novo sequencing of this molecular entity. The use of the fragment ions mass spectrum of the reference PNA molecule enables "mass fingerprinting" analysis by allowing the isomeric PNA molecules with scrambled sequences to be readily distinguished from the reference PNA.

Structure elucidation and quantification of the active pharmaceutical ingredient in a non-approved drug and in cat serum using QTRAP-MS/MS and ZenoTOF-MS/MS.

In this study, we present the first comprehensive application of ion mobility mass spectrometry (IMS MS) combined with collision-induced dissociation (CID MS/MS) to the comparative analysis of the gangliosidome in human hippocampal tissue affected by temporal lobe epilepsy and corresponding healthy controls. Using nanoESI IMS MS, we profiled complex ganglioside mixtures extracted from the human hippocampus affected by temporal lobe epilepsy and the normal hippocampus. A total of 217 ions corresponding to 192 distinct ganglioside species were identified in the epileptic tissue, compared with 156 ions assigned to 137 species in the healthy hippocampus. The majority of these species were polysialylated and exhibited extensive structural diversity in both glycan and ceramide moieties, with important modifications including fucosylation, O-acetylation, GalNAc, and CH3COO- attachments. The gangliosidome associated with epilepsy was found characterized by a higher overall degree of sialylation than previously known, with the exclusive presence of highly sialylated GP, GS, and GO species reported here for the first time in relation to this disease. CID MS/MS experiments enabled the structural elucidation of several biologically relevant species, including O-Ac-GD1b (d18:1/20:2) and GT1b (d18:1/23:0), demonstrating the method's capacity to resolve complex structures and identify specific ganglioside isomers. Significant differences in the expression and modification patterns between pathological and control samples suggest disease-associated remodeling of membrane components. This study not only reveals novel molecular features of epilepsy-related ganglioside alterations but also establishes IMS CID MS/MS as a powerful analytical platform for advancing glycosphingolipidomics and exploring biomolecular signatures in neurological disorders.

Molecular characteristics of halogenated disinfection byproducts elucidated by Fourier transform ion cyclotron resonance mass spectrometry.

Theoretical Study Synchronized Reverse Scan Collision-Induced Dissociation in Digital Linear Ion Trap.

Ion mobility spectrometry (IMS) serves as a crucial analytical technique and has been extensively applied to the analysis of hazardous explosives. However, the detection of ions with the same or similar mobility remains a significant challenge for IMS. Just as tandem mass spectrometry plays a crucial role in analysis, the development of tandem ion mobility spectrometry has also greatly enhanced the qualitative capabilities; ion dissociation technology is one of the key components. This study creatively introduces a heat-induced dissociation (HID) technique and develops a HID module for ion dissociation. Primarily, the HID module was integrated into a homemade ion trap mass spectrometer to validate the feasibility of the HID technology. Subsequently, the thermal field distribution of the HID module was simulated, thereby highlighting the significance of the temperature in the dissociation process. Finally, a tandem drift tube ion mobility spectrometry (DTIMS) detection system incorporating a HID module was established. Five representative explosive samples were selected to validate the feasibility of the tandem DTIMS. Both atmospheric pressure dissociation mass spectra and mobility spectra were obtained, and their dissociation products were analyzed through comparison with collision-induced dissociation (CID). The results demonstrate that incorporating HID technology into the tandem DTIMS system enables ion dissociation and the acquisition of atmospheric pressure second-order ion mobility spectra. The combination of fragment peak information enhances the qualitative capabilities of ion mobility spectrometry. This advancement is anticipated to reduce the probability of false positives in future applications, thereby improving the accuracy and reliability of the system. This study provides critical technical support for explosive detection in the field of public safety.

Characterization and Elucidation of the Fragmentation Pathway of 17 Nitazenes by Liquid Chromatography High-Resolution Mass Spectrometry Using Collision-Induced Dissociation and Electron-Activated Dissociation.

Recent years have witnessed remarkable advances in ion resonance method design for ion trap mass spectrometers, especially in the context of instrument miniaturization. These developments aim to enhance ion selectivity, fragmentation efficiency, and scanning performance without altering the mechanical structure of the device. In the field of ion isolation, a series of waveform innovations-such as Grid-SWIFT, SWIFTSIN, and SAM-SFM-have been introduced to improve resolution, sensitivity, and computational efficiency. Real-time waveform synthesis strategies have further simplified implementation on portable platforms. For collision-induced dissociation (CID), techniques such as orthogonal excitation and repetitive frequency sweeps have enabled two-dimensional mass spectrometry on compact systems, allowing correlation between precursor and product ions and enriching the dimensionality of dissociation information in both time and frequency domains. In terms of ion scanning, innovations in voltage and frequency scan methods have significantly expanded the detectable mass range and improved mass resolution. Furthermore, the integration of ion mobility analysis with resonance excitation has enabled ultra-high-resolution separation of isomers and chiral compounds. These advancements collectively demonstrate the crucial role of ion resonance methods in overcoming the performance limitations of miniature ion trap mass spectrometers and open up new possibilities for their future development.

Electron-induced dissociation methods, particularly electron impact excitation of ions from organics (EIEIO), offer enhanced capabilities for lipid structural elucidation over traditional collision-induced dissociation (CID). Despite their analytical promise, the practicality of EIEIO within routine liquid chromatography-mass spectrometry (LC-MS) workflows remains largely unexplored. In this study, we optimised LC-EIEIO-MS analysis for the rapid and detailed structural annotation of glycerides and phospholipids. We evaluated the effects of reaction times, accumulation times, and electron kinetic energies using lipid standards from multiple classes and at varying concentrations. Our results revealed that short reaction times of 30 ms consistently yielded stronger diagnostic signals crucial for lipid class identification and sn-position discrimination at concentrations as low as 200 pg on column. To systematically infer the position of double bonds from EIEIO spectra, we introduced LipidOracle, a software that tests all possible isomers and correctly accounts for missing data, noise, and crowded spectra. We demonstrated that longer accumulation times of 200 ms were most effective for determining carbon-carbon double bond (CC) positions, particularly in polyunsaturated lipids. Finally, we evaluated the performance of EIEIO with liver and plasma extracts. Overall, we demonstrate that comprehensive lipid structural characterisation, including sn-position and double bond locations in fatty acyl chains, is achievable within typical LC-MS timescales (∼0.2 s). Our findings outline practical guidelines for high-throughput analysis of complex lipid samples by EIEIO.

Prediction of Collision-Induced Dissociation Spectra of Deprotonated Oligonucleotides.

The interactions between d(CpoxG) (oxG = 8-oxo-guanine), a major form of the oxidatively damaged CpG island motif, and a photoactivatable anticancer Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2] (1), were investigated using electrospray ionization mass spectrometry (ESI-MS). Surprisingly, the primary MS analysis showed that the major photooxidative products were the platinum-free dinucleotides d(CpGh) (2a)/d(CpIa) (2b) (possibly a mixture of the two isomers) and d(CpDGh) (3), in which the guanine was oxidized to 5-guanidino-hydantoin (Gh) or iminoallantoin (Ia) and 5-guanidino-dehydrohydantoin (DGh), respectively. Moreover, two mono-platinated adducts, {[CpGh] + 1'}+ (4) and {[CpDGh] + 1'}+ (5) (1' = [PtII(N3)(py)2]+), and three Pt-crosslinked dinucleotide adducts, {[CpGh]2 + 1''}2+ (6), {[CpGh] + [CpDGh] + 1''}2+ (7) and {[CpDGh]2 + 1''}2+ (8) (1'' = [PtII(py)2]2+), were observed as the main platinated adducts. Tandem mass spectrometry with collision induced dissociation (CID) demonstrated that 1' bound at Gh or DGh in 4 and 5, while the inter-dinucleotide crosslinks by 1'' between Ghs, Gh and DGh, or DGhs in 6, 7 and 8 were implicated. Unexpectedly, the proposed platinated d(CpoxG) adducts were not observed, indicating that oxG preferentially undergoes further oxidation by the reactive oxygen species released during the photodecomposition of complex 1 rather than coordination with the reduced Pt(II). These results revealed the greater complexity of the photo-interaction of complex 1 with d(CpoxG) than with d(CpG), with the implication that oxG-containing DNA, in particular, the oxidative CpG island, might play a vital role in the mechanism of action of photoactivatable Pt(IV) prodrugs, which merits further exploration.

The ability to characterize closely related lipids is clinically important, requiring the development of analytical tools to differentiate species responsible for metabolic disorders from those needed for metabolic homeostasis. Herein, we report a new liquid chromatographic (LC) method that utilizes online microdroplet-based epoxidation reactions during electrospray to enable C═C bond localization on conventional tandem mass spectrometry (MS/MS). Through a coaxial spray mechanism, charged microdroplets derived from the LC column and containing the lipid analyte were fused with nonthermal plasma, which facilitated (i) positive ion mode detection of various lipid classes and (ii) instantaneous C═C bond epoxidation via reaction with reactive oxygen species in the nonthermal plasma. Consequently, conventional low-energy MS/MS based on collision-induced dissociation was effective in characterizing the positional isomers of various lipids. Our ability to modify electrosprayed microdroplets post-column allowed independent optimization of the LC mobile phase, which in turn enabled both polar and nonpolar lipids to be separated on a C12 reverse-phase column. A data-dependent acquisition (DDA) method was created to enable the automated characterization of epoxide products in a 17-component lipid mixture. The DDA method was applied to characterize new triacylglycerol previously not detected in extra virgin olive oil.

Although the miniaturization of mass spectrometry (MS) frequently compromises analytical performance due to size and power limitations, the direct on-site analysis of complex samples requires a miniature mass spectrometer (mini-MS) to have enhanced instrument capabilities. To resolve this challenge, we have developed our "Brick" mini-MS into a next-generation system incorporating a differential-pressure dual-trap configuration. Each trap functions at distinct pressures, enabling parallel and optimized operations: ion accumulation/cooling and dissociation at higher pressures, in conjunction with ion isolation and MS analysis at lower pressure. Efficient ion transfer between the two traps enables parallel ion manipulation and diverse fragmentation modes. The parallel ion accumulation mode boosted the sensitivity of the miniature instrument by ∼20-fold, down to 50 pg/mL. In addition to conventional in-trap collision induced dissociation (CID), transfer dissociation during the ion accelerating and shuttling process and high-pressure collisional dissociation (HpCD) in a higher-pressure trap were also investigated. The results demonstrate that HpCD can generate more extensive ion fragments, which are typically observed in beam-type collisional activation dissociation methods. This study significantly advances the capabilities of mini-MS for high-performance, field-deployable analytical applications.

Trimethylaminoethyl ester derivatization and stable isotope derivatization for enhanced analysis of fatty acids in biological samples by electrospray ionization tandem mass spectrometry.

Vitamin B12 is one of nature's most structurally complex small molecules and an essential coenzyme that is only produced by prokaryotes. Due to its pivotal role in nerve function, metabolism, and the immune system in humans, vitamin B12 deficiency is considered a major public health concern. Numerous mass spectrometric analytical methods exist to identify and quantify vitamin B12 from a wide array of matrices, including food, diet supplements, tissues, and plasma. Nevertheless, structural elucidation of vitamin B12 through tandem mass spectrometry (MS/MS) fragmentation remains a challenge due to its intricate structure, versatility of derivatives, and isobaric impurities. This work combined native and force-degraded samples with synthetic impurity standards to elucidate the structures of collision-induced dissociation (CID) fragment ions of vitamin B12 over the full m/z range with a high mass accuracy. Our findings show that the complexity of vitamin B12 MS/MS fragments can be deduced through a "simple" combination of four (4) major CID cleavage sites. These results enabled us to propose a general vitamin B12 CID fragmentation pathway that allows for the calculation of the theoretical m/z for fragment ions of unknown cobalamin species and impurities. This work aims to provide a novel, universal analytical platform to identify and quantify vitamin B12 and its derivatives through mass spectrometry with high mass accuracy.

Ionic liquids (ILs) have been gaining increasing focus in a variety of applications including emerging electric-propulsion concepts. A quantitative understanding of how IL ions fragment during high-energy collisions with background gases is therefore essential for interpreting mass spectra, predicting ion lifetimes in plasma and vacuum environments, and designing IL-based technologies. This work uses molecular dynamics (MD) simulations with a reactive force field to numerically model the collision-induced dissociation (CID) of isolated ions (both positive and negative) and ion clusters (2:1 and 1:2 clusters) of the prototypical ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIM-BF4), colliding with a nitrogen (N2) molecule, exploring all possible fragmentation channels arising from the breaking of both ionic and covalent bonds at collision energies ranging from 10 electron volts (eV) to 100 electron volts (eV) in the laboratory frame. The molecular dynamics results are compared with the observations from tandem mass spectrometry (MS2) experiments to assess the reliability of the MD results. The MD modeling predicts the dissociation onset collision energy of the [EMIM]+ ion to be 20 eV (lab frame), while the [BF4]- ion requires a collision energy of at least 40 eV (lab frame) to undergo dissociation. The primary fragmentation product of the [EMIM]+ ion is found to be the 3-methylimidazolium cation, [C4H7N2]+, with the cyanide anion, [CN]-, being the major fragment ion at higher collision energies (≥60 eV, lab frame). The [BF4]- ion, on the other hand, dissociates to form the fluoride ion, [F]-, and the neutral BF3 molecule, with the [BF2]+ ion being formed at higher collision energies (≥60 eV, lab frame). Both the 2:1 and 1:2 ion clusters are found to fragment at the lowest simulated collision energy of 10 eV (lab frame), with the fluoride ion, [F]-, being formed with rising abundance as the collision energy is increased. When compared, the mass spectra from MD modeling and experiments demonstrate a reasonable agreement, which suggests that reactive MD can be a reliable surrogate for CID studies of complex IL ions.

Membrane proteins (MPs) challenge biophysical and structural biology methods. Native mass spectrometry (MS) has emerged as a powerful tool to study MP structures and their modulation by lipids. We examine the pathways by which three MPs, tetrameric Aquaporin Z (AqpZ), trimeric ammonium transporter (AmtB), and pentameric mechanosensitive channel of large conductance (MscL) decompose following collisionally activated dissociation (CAD) or electron capture dissociation (ECD) in native top-down MS. MPs subjected to CAD typically decompose along well-characterized pathways releasing highly charged monomers and low-charge state complementary subunits, but a lesser pathway accessed by low pressure collisions cleaves the backbone into fragments covering much of the sequence. Collisions also rearrange structures, e.g., AmtB subunits rearrange to form novel interactions and/or salt bridges that surprisingly retain a formerly surface-exposed segment despite ejecting the binding interface. MscL dissociation pathways depend on precursor charge-state, a behavior observed in a few soluble complexes, yet unaddressed mechanistically. Salt bridges in low charge state complexes stabilize subunits from ejection while facilitating only smaller, local rearrangements that release covalently cleaved products from transmembrane regions. With fewer opposite charges, on average, higher charge state molecules can rearrange intersubunit salt bridges on the experimental time scale to partition charge asymmetrically and free a subunit. ECD with supplemental activation can retain higher order structures of proteins and inform about the strongly interacting regions that preclude product ion release. With extensive regions lacking ionizable residues, MPs enable key interactions that guide the structure and dynamics of gas phase protein assemblies to be probed.

Sphingoid bases (SPHs) serve as the core structural backbone of all sphingolipid classes, with their diversity arising from intrachain modifications such as carbon-carbon double bonds (CC), hydroxyl groups, and methyl branching. Traditional tandem mass spectrometry (MS/MS) relying on collision-induced dissociation (CID) often fails to yield diagnostic fragmentation patterns for precise localization of these modifications, underscoring the need for advanced dissociation techniques. In this work, we present a novel analytical strategy combining carnitine derivatization of sphingoid amines with electron-activated dissociation (EAD) in MS2 to enable in-depth structural characterization. This technique uniquely suppresses intrachain fragmentation while generating diagnostic ions at modification sites, resulting in simplified mass spectra that facilitate unambiguous identification of subtle structural variations. This method was integrated into a reversed-phase liquid chromatography-mass spectrometry workflow and further applied for in-depth profiling of total SPHs in Astragalus and Escherichia coli (E. coli). Our analysis uncovered two pairs of regioisomeric SPHs: CC positional isomers of SPH d18:1 in Astragalus and hydroxylation positional isomers of SPH t18:1 in E. coli, demonstrating the method's utility in resolving structurally complex sphingolipid bases.