Journal of the American Society for Mass Spectrometry最新文献

Mass spectrometry imaging (MSI) is a powerful technique for studying the spatial localization of N-linked glycans in biological tissues, which are important biomarkers for various diseases. Analyzing N-linked glycans requires the deposition of an enzyme onto a biological tissue section to release them from proteins. However, existing equipment for enzyme deposition is relatively expensive and may not be readily accessible to some laboratories. To address this challenge, we developed and evaluated a cost-effective approach for enzyme application onto tissue sections using a mini-humidifier. We demonstrate the capabilities of this approach by applying peptide N-glycosidase F (PNGase F) for MSI of N-linked glycans present within mouse brain tissue sections using nanospray desorption electrospray ionization (nano-DESI). The performance of the mini-humidifier was comparable to that of a widely used HTX-TM Sprayer, establishing it as a cost-effective and efficient alternative for enzyme deposition onto tissue samples in MSI experiments. This work offers an accessible approach for enzyme application on biological samples to study the spatial distribution of N-linked glycans using MSI.

Noncovalent interactions play an important role in the way protein structures and protein–protein interactions are stabilized. Mapping these interactions at a molecular level is crucial as peptide complexes serve as models for protein–protein interfaces. In this work, complementary analyses using trapped ion mobility spectrometry (TIMS), tandem ECD/UVPD MS fragmentation, and molecular dynamics were applied to the study of native peptide–peptide complexes. In particular, three complexes representing peptide–peptide intramolecular interactions of the intrinsically disordered high-mobility group AT-Hook2 (HMGA2) protein were described. AT-hook peptides, when complexed with the C-terminal tail (CTMP), showed a higher gas-phase stability for ATHP1–CTMP, followed by ATHP2–CTMP and ATHP3–CTMP. High sequence coverage was obtained by using ECD and UVPD fragmentation for the single peptides (∼100%) and the peptide–peptide complexes (∼75%). At least three peptide–peptide structures were separated in the mobility domain for the ATHP–CTMP complexes. All three complexes showed high structural diversity and the possibility of being aligned in forward and backward orientations.

Reliable mass accuracy is essential for the confident identification of diagnostic fragment ions in high-resolution mass spectrometry. During the analysis of perfluoroalkyl sulfonic acids (PFSAs) with a Q Exactive Orbitrap, we consistently observed the characteristic O₃Ṡ^– fragment, expected at a mass-to-charge ratio (m/z) of 79.9574, reported at m/z 79.9568, a systematic deviation of approximately −6 parts per million (ppm). Similar errors affected other ions below m/z 100, while precursors and higher-m/z fragments remained within the specification. Comparison with an Exploris 120 instrument, calibrated with anchors down to m/z 59, confirmed that the deviation is not intrinsic to the ion but originates from the limited calibration range of the Q Exactive standard calibration solution. We demonstrate that extending calibration to include additional low-m/z anchors generated in situ by in-source fragmentation fully corrects the error without affecting the accuracy at a higher m/z. This adjustment resolves systematic deviations for ions below m/z 100 in the Q Exactive instruments.

Stable and long-lived radioactive isotopes are ubiquitous in nature and serve as unique tracers across diverse fields such as nuclear astrophysics, atmosphere chemistry, hydrology, environmental chemistry, and the diagnosis of diseases in the human body. Over the past decades, accelerator mass spectrometry and spectroscopic methods have been used to measure the abundance of stable and long-lived radioactive isotopes. However, their accuracy has been constrained by the systematic uncertainties inherent in sophisticated instrumentation and limitations in the abundance sensitivity. Here, we present a novel approach based on the fundamental mechanism of molecular Coulomb explosion fragmentation (i.e., molecules breakup as a result of Coulomb repulsion between the positively charged nuclei within molecules that are rapidly stripped of their electrons), utilizing a two-dimensional coincidence time-of-flight spectrometer to detect fragmented isotopic ion pairs. The present method enables direct determination of the isotopic abundances of ¹³C and ¹⁸O with an accuracy better than 0.02%, significantly improving abundance sensitivity by powerful identification and eliminating systematic uncertainties. Our molecular Coulomb explosion spectrometry provides high-accuracy measurement of stable and long-lived radioactive isotope abundance, with significant potential to advance isotope tracer studies in the Earth environment, anthropology, archeology, global ecological cycles, fundamental nuclear physics, and biomedicine.

Chromatography combined with tandem mass spectrometry is a conventional strategy for metabolite differentiation and identification. However, coelution and overlapping fragmentation patterns often limit confident assignment of isomeric and isobaric species. Caffeine metabolites represent a particularly challenging case. Here, we demonstrate a high-field asymmetric waveform ion mobility spectrometry (FAIMS) approach coupled with infrared multiple photon dissociation (IRMPD) ion spectroscopy for the identification of the major caffeine metabolites paraxanthine (PX), theobromine (TB), and theophylline (TP). FAIMS provided baseline separation of these isomers into four distinct populations, including two distinct protomers of protonated TB. The nature of each FAIMS population was probed by IRMPD, which enabled structural assignment and revealed unique protomeric signatures for PX and TP. This combined FAIMS–IRMPD workflow not only resolves isomeric metabolites but also distinguishes protomeric and tautomeric forms, expanding the scope of ion mobility–spectroscopy approaches in metabolite analysis.

Covalent ligands are widely used to label, probe, and modulate proteins, but peptide-centric readouts obscure how modifications colocalize on intact proteoforms. This can limit insight into ligand mechanism, modification stoichiometry, and the architecture of multisite protein conjugates. We present a general native top-down mass spectrometry workflow that quantifies electrophile reactivity directly on intact proteins. Using NHS esters as a model electrophile class, we apply a deconvolution framework to infer differential reactivity at primary amines across promiscuous, multisite modification patterns. The approach preserves full modification connectivity, avoids sample-preparation artifacts associated with denaturation and digestion, and should extend to electrophiles with unknown reactivity. Overall, this framework provides a general platform for designing covalent therapeutics, bioconjugates, and activity-based probes with proteoform-level resolution.

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.