Journal of the American Society for Mass Spectrometry最新文献

The characterization of isomeric oligosaccharides, such as galactooligosaccharides (GOS), in complex mixtures is a long-standing analytical challenge. Due to the exponential increase in the number of possible structures with an increasing degree of polymerization (DP), it is difficult to obtain a complete set of standards. Cyclic ion mobility mass spectrometry (cIMS-MS) has the potential to overcome this challenge by comparing oligosaccharide fragment ions with their respective disaccharide standards, thus reducing the reliance on higher DP oligosaccharide standards. To achieve this, effective separation and recognition of disaccharide fragments using cIMS is a primary prerequisite. Therefore, we studied the ability of combined cIMS separation and collision-induced dissociation (CID) fragmentation to identify isomeric disaccharides consisting of galactose (Gal) and glucose (Glc) with varying compositions and linkage types. Comparison of the drift time selectivity and fragment yields of lithium, sodium, potassium, rubidium, and cesium adducts revealed that lithium overall performed best, by providing good cIMS resolution and far superior fragment yields compared to the other metal ions. cIMS drift times were influenced primarily by the composition of disaccharides and consistently followed the order Gal-Gal < Gal-Glc < Glc-Glc for disaccharides with the same linkage type. CID cross-ring fragmentation patterns were linkage-type specific and did not strongly depend on composition or anomeric configuration. Overall, unknown disaccharides can straightforwardly be identified using our cIMS-MS/MS approach, as monosaccharide compositions can be deduced from cIMS drift times and linkage types from CID fragmentation patterns. These findings are a stepping stone for the identification of larger oligosaccharides by cIMS-MS-based approaches.

Accurate quantification of Δ⁹-tetrahydrocannabinol (THC) and Δ⁹-tetrahydrocannabinolic acid (THCA) across diverse cannabis- and hemp-derived products remains challenging due to severe matrix effects, wide concentration variability, and the need for matrix-matched calibration in traditional LC–MS workflows. Here, we develop an in-sample calibration curve (ISCC) method based on multiple isotopologue reaction monitoring (MIRM) from stable-isotope-labeled (SIL) analytes to enable robust quantification of THC and THCA without external calibration curves. The approach leverages the theoretical relative isotopic abundances of SIL calibrators to generate multiple internal calibration points within each injection. By incorporating two SIL calibrators for THC (THC-D₃ and THC-D₉), applying a response-correction factor to harmonize labeled and unlabeled analytes, and utilizing native-analyte isotopologue transitions at high abundance, the method achieves a >600-fold dynamic range. The ISCC method demonstrated excellent linearity (R² > 0.999), precision (<10% RSD), and accuracy (±10%) in commercial CBD oils, gummies, creams, waxes, dietary supplements, and plant materials. Comparison with external calibration showed strong agreement across all matrices. Collectively, this work develops the ISCC–MIRM framework for heterogeneous consumer and forensic samples and establishes a practical, matrix-tolerant calibration strategy for routine cannabinoid analysis.

Mass spectrometry is an indispensable tool for the rapid and in-depth analysis of complex mixtures across diverse biologically important fields including metabolomics, lipidomics, and proteomics. These applications demand high speed instruments with subppm mass measurement accuracy over a wide dynamic range of sample concentrations. Here, we introduce an liquid chromatography-mass spectrometry/MS (LC-MS/MS) quadrupole time-of-flight mass spectrometer featuring a novel collision cell, a high dynamic range detector, and a compact multireflecting orthogonal time-of-flight analyzer. This innovative instrument achieves high analytical performance, acquiring full mass range spectra at 100,000 Full Width Half Maximum (FWHM) resolution up to 100 spectra/s acquisition speed. The instrument achieves excellent linearity within a dynamic range of 10⁵, with a correlation coefficient R² = 0.984. The speed, resolution and dynamic range are in excellent balance as demonstrated by the analysis of isotopically labeled lipids in human blood plasma.

A three-dimensional gas-phase ion separation instrument platform that integrates high-sensitivity and high-resolution racetrack field asymmetric waveform ion mobility spectrometry (r-FAIMS) with quadrupole time-of-flight mass spectrometry (Agilent 6560 IMS-QTOF MS) was developed to probe the conformational diversity of cytochrome C ions. A charge state envelope of +8 to +19 for the cytochrome C ions was observed in the acquired MS spectrum. For the +8 to +11 charge states of cytochrome C ions, multiple peaks were clearly observed in the IMS spectra, while only one peak was observed in the corresponding FAIMS spectra. In contrast, well-separated multiple peaks were clearly observed for the +13 to +19 charge states of cytochrome C ions in the FAIMS spectra. However, the ions from different FAIMS peaks were shown to have very close drift times in the second dimension IMS measurements, implying that these ions had essentially the same collisional cross-sectional areas. To verify these FAIMS peaks, corresponding to different compensation voltages, for the +13 to +19 charge states of cytochrome C ions indeed represent different protein conformers. The CV-selected cytochrome C ions were further subjected to ion fragmentation (MS/MS) analysis in the third dimension QTOF MS. MS/MS analyses have clearly demonstrated that the product ion spectra for the same charge state of cytochrome C ions from different FAIMS CV peaks are highly different under the same MS/MS operation conditions (i.e. under the same collision energy). The results from the MS/MS analyses thus convincingly prove that the protein ions from different FAIMS peaks are different conformers. Gas-phase ion separations in FAIMS and IMS are, thus, highly orthogonal. Unique to any other alternatives, the proposed r-FAIMS-IMS-MS/MS technique allows for a highly efficient FAIMS separation of protein structural isomers, detailed IMS measurements of the collisional cross-sectional area for each isomeric protein, and direct confirmation of isomeric proteins via MS/MS.

Covalent conjugates of peptides containing aromatic amino acid residues Phe, Tyr, 3-nitro-Tyr, Trp, and 5-hydroxy-Trp with diaryltetrazole groups carrying 4-nitro and 4-methoxy substituents and linked to the peptide lysine were synthesized and used to study photodissociation of their gas-phase ions at 213 and 250–290 nm (UVPD). UVPD resulted in a competitive loss of N₂ from the tetrazole ring and peptide backbone cleavage that was wavelength dependent, showing >90% specificity of tetrazole dissociation at 250–280 nm. This wavelength region corresponded to π–π* excitations within the diaryltetrazole moiety, as established by time-dependent density functional theory calculations. The intermediates from the N₂ loss were analyzed by collision-induced dissociation (CID-MS³) to reveal high (74–99%) yields of cross-linked macrocyclic products that were distinguished by internal residue losses. The 4-methoxyphenyltetrazole conjugate was exceptional in that it underwent facile elimination of 4-methoxyaniline. High-resolution cyclic ion mobility measurements were employed that pointed out near conformational homogeneity of the conjugate ions. The measured collision cross sections (CCS_exp) were matched, typically within 1%, by theoretical CCS_calc that were obtained by combined Born–Oppenheimer molecular dynamics, density functional theory, and ion trajectory calculations. Ion mobility measurements of the N₂-loss intermediates allowed us to separate mixtures of several products that in each case were dominated by a single component that was identified as a cyclic isomer. The calculated fully optimized ion structures showed no π–π stacking of the amino acid aromatic rings and the diaryltetrazole moieties for most conjugate combinations. The 3-nitrotyrosine-diaryltetrazole conjugate was an exception in that it preferred stacked structures in low-energy conformers. The electronic properties of the conjugates in the ground and multiple excited electronic states were addressed by time-dependent density functional theory calculations that provided vibronic absorption spectra at 300 K. Electron transitions that were near resonant with the laser excitation lines occurred within the diaryltetrazole system (as in the Phe conjugate) or comprised electron transfer with the aromatic amino acid residue for the 3-nitro-Tyr, and Trp residues. The Trp-diaryltetrazole interactions occurred dynamically as a result of the conformational motion in thermal ions. The electron-transfer excitation in the 3-nitro-Tyr and Trp side-chain groups was associated with internal energy distribution throughout the peptide chains, driving backbone dissociations while lowering the yields of tetrazole UVPD.

The efficiency of many tandem mass spectrometry (MS/MS) techniques depends on the position of the trapped ion cloud and its overlap with a beam of photons or electrons. Here, we report photoactivated tomography profiles measured in a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to characterize the position and radial extent of the ion cloud. Tomography profiles generated by measuring single-photon processes, namely, fluorescence and ultraviolet photodissociation (UVPD), produced estimates of ion cloud full width at half-maximum (FWHM) of at least 1.7 mm under the experimental conditions employed. The effects of vertical and horizontal beam steering ion transfer optics were characterized using visible light photodissociation (VisPD) tomography to monitor ion cloud centroids, FWHM of the fitted tomography profiles, and maximum photodissociation levels. A robust alignment procedure is described to ensure proper overlap of the laser beam with the ion cloud. The ion cloud behavior characterized in this laser tomography study has made it possible for users to better control the ion cloud prior to optical excitation, thereby increasing the efficiency of in-cell MS/MS processes such as photodissociation and fluorescence. This method for rapid determination of the position of the ion cloud can be implemented on other types of mass spectrometers, which will be beneficial for photoactivation experiments.

Here, we report on combining Random Forest (RF) classification with nanoprojectile secondary ion mass spectrometry (NP-SIMS) to analyze single extracellular vesicles (EVs) isolated from human liver cancer (HEPG2) and Normal liver cell lines. EVs were tagged with antibody-lanthanide (Ln) tags specific to marker proteins and dispersed on a surface, enabling NP-SIMS to produce millions of individual EV mass spectra. Previously, EVs were manually classified based on Ln-tag signals, and as a result, only 5% could be confidently classified as either from cancer or normal cells. Using a random forest model, optimizing data preprocessing, and expanding the spectral features resulted in a 60-fold increase in classification efficiency over manual analysis. We also performed untargeted RF classification, where both supervised and untargeted RF analyses resulted in consistent outcomes, showing the compatibility of RF with the NP-SIMS data for individual EV classification. The results from the untargeted analysis suggest that NP-SIMS with RF could aid in marker discovery in systems where limited sample quantities are available. Overall, using RF and NP-SIMS enables single-EV classification and provides a promising pathway for EV-based disease diagnostics.

A solid-state deep ultraviolet (DUV) optical parametric oscillator (OPO) at 206 nm wavelength was utilized for laser ablation electrospray postionization of peptides and proteins coupled with ion mobility mass spectrometry (IM-MS) analysis. Peptide and protein standard solutions were spray deposited on quartz microscope slides to obtain thin, surface-coated dry films. The pulsed laser irradiated the solid sample biomolecules in transmission geometry, and the ablated surface material merged with the electrospray plume for ionization before entering the IM-MS instrument. Laser ablated biomolecules remained intact, and no fragmentation was observed from the peptide or protein standards. The efficiency of ionization was estimated at approximately 1% for instantaneous ion signal; however, the signal was not stable over time. Mass spectra of laser ablation electrospray for peptide and protein standards revealed multiply charged ions similar to those observed in direct electrospray ionization (ESI) MS. The ion mobility drift times for proteins from laser ablation electrospray (LA-ESI) experiments were indistinguishable from those observed in direct ESI.

Apart from its role as a nutrient in mammalian milk, lactoferrin (LF) is also an important part of the innate immune system, where it functions as a potent microbicidal molecular factor. An important structural feature of LF, which makes it distinct from its next-of-kin serum transferrin (TF, an iron transporter that is not involved in the innate immune response), is the presence of an extended patch of positive charge on the surface of LF. While the relevance of this structural feature to the protein’s immunoprotective properties is indisputable, the specific molecular mechanism that governs its involvement in protection against a wide array of pathogens remains poorly understood. We use native mass spectrometry (MS) and molecular modeling to study LF interaction with glycosaminoglycans, whose structure approximates that of the highly sulfated segments of heparan sulfate (HS), a major component of the extracellular matrix. Even the shortest highly sulfated HS segments readily associate with LF under near-native conditions, suggesting that this interaction plays an important role in concentrating the microbicidal agent in the vicinity of the point of its release from the activated neutrophil, thereby preventing its removal from the infection site by diffusion or blood flow. No such properties are exhibited by TF, consistent with its role as an iron transporter that needs to be freely circulated until its encounter with a receptor on the surface of a cell that requires a supply of iron for its growth or specific function(s). We also examine the interaction of LF with heparin, a highly sulfated glycosaminoglycan that is released from the mast cells upon their activation. Native MS reveals the ability of a single heparin chain to accumulate a significant number of LF molecules (up to five), consistent with its proposed role as an antagonist of the heparin-associated tryptases (which are deactivated upon their dissociation from heparin). This provides a molecular basis for the immunomodulatory properties of LF as a factor limiting the harm to the host inflicted by tryptases in the course of the mast cells’ coactivation with neutrophils.

We introduced continuous flow paper spray ionization mass spectrometry (CFPSI MS) for the rapid detection and characterization of anticancer drugs in solid tissue samples. CFPSI is a paper spray-based semiquantitative method using continuous flow of an internal standard to quantify the amounts of drugs released from the tissue samples. Using patient-derived xenograft (PDX) mouse model tissue samples, we observed differential absorption of three anticancer drugs, palbociclib, copanlisib, and olaparib. Palbociclib was found to be bioabsorbed in the tissue samples to the largest extent. Tandem mass spectrometric analysis explored the in-source chemical reactivity of these drugs, leading to significant spectral complexity. Our findings highlight the importance of careful spectral interpretation in complex biological matrices and support the development of future rapid quantitative CFPSI analysis of these drugs in tissue samples.