Journal of the American Society for Mass Spectrometry最新文献

Electrospray mass spectrometry has become indispensable in many disciplines including the classic "omics" techniques such as proteomics or lipidomics, as well as other life science applications in molecular, cellular, and structural biology. However, a limiting factor that often arises for the detection of biomolecular analytes is their poor ionization efficiency in the ion source. Here, we present an add-on device for the electrospray source, termed MS SIEVE (MS Spectral Impurity Eliminator & Value Enhancer), which is placed between the electrospray needle and the cone of the mass spectrometer. We probed the application of MS SIEVE for various biomolecules including proteins, peptides, lipids, glycans and DNA oligonucleotides and even synthetic polymers such as polyethylene glycol and found that MS SIEVE selectively improves the signal intensity, while suppressing the spectral contribution of contaminants such as NaCl. Importantly, MS SIEVE can, in principle, be adapted for any electrospray ion source and, therefore, represents a promising alternative for routine "omics" methods as well as special applications on challenging analytes.

The elucidation of structural motifs in extremely complex mixtures is very difficult since the standard methods for structural elucidation are not capable to provide significant information on a single molecule. The best method for the analysis of complex mixtures is ultrahigh resolution mass spectrometry, but the utilization of this method alone does not provide significant information about structural details. Here, a combination with a separation method is necessary. While chromatography is a well-established technique, it has some disadvantages in regard to the separation of complex mixtures, as often no separation of individual isomers is possible. Therefore, here the combination of an ion mobility separation with ultrahigh resolution mass spectrometry is evaluated. As a sample matrix, crude oil is used because it is an excellent matrix to develop new analytical techniques on complex samples. Crude oil is the most complex natural sample known, but only little information is available on the structural identity or functionalities due to a high number of structural isomers or isobars. A lab-built APPI/APLI-FAIMS source was revised to optimize ion transmission and used to follow up on the ion mobility of crude oil constituents after photoionization. An MS/MS approach using collision-induced dissociation (CID) was used to elucidate structural motifs of the transmitted isomers.

Native mass spectrometry can characterize a range of biomolecular features pertinent to structural biology, including intact mass, stoichiometry, ligand-bound states, and topology. However, when an instrument's ionization source is tuned to maximize signal intensity or adduct removal, it is possible that the biomolecular complex's tertiary and quaternary structures can be rearranged in a way that no longer reflect its native-like conformation. This could affect downstream ion activation experiments, leading to erroneous conclusions about the native-like structure. One activation strategy is surface-induced dissociation (SID), which generally causes native-like protein complexes to dissociate along the weakest subunit interfaces, revealing critical information about the complex's native-like topology and subunit connectivity. If the quaternary structure has been disturbed, then the SID fingerprint will shift as well. Thus, SID was used to diagnose source-induced quaternary structure rearrangement and help tune an instrument's source and other upstream transmission regions to strike the balance between signal intensity, adduct removal, and conserving the native-like structure. Complementary to SID, electron-capture dissociation (ECD) can also diagnose rearranged quaternary structures and was used after in-source activation to confirm that the subunit interfaces were rearranged, opening the structure to electron capture and subsequent dissociation. These results provide a valuable guide for new practitioners of native mass spectrometry and highlight the importance of using standard protein complexes when tuning new instrument platforms for optimal native mass spectrometry performance.

Electrospray ionization mass spectrometry (ESI-MS) can retain intact protein structures, but details about partially folded and unfolded protein structures during and after introduction to the gas phase are elusive. Here we use ESI-MS with chemical cross-linkers to compare denatured cytochrome c structures in both solution and gas phases. Solution phase cross-linking prior to ESI captures solution phase structures, while gas phase cross-linking through ion/ion reactions in the trap cell captures gas phase structures. Comparing the ECD fragmentation of the cross-linked products under both conditions shows very similar cross-linker identifications, alluding to no major structural dissimilarities between solution and gas structures. Molecular modeling of the denatured protein using the identified cross-linked sites as distant restraints allows for visualization of the denatured structures to pinpoint where unfolding begins. Our data suggest that cytochrome c likely begins to unfold due to interior hydrophobic expansion, followed by α helical unfolding. This localization of structural changes is more specific than using CCS measurements alone.

Flavan-3-ol oligomers (FLOs), including proanthocyanidins (PAs) and theasinensins (TSs), contribute greatly to the flavor and bioactivity of the tea beverage. Ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry has been widely used in profiling a wide range of compounds in tea. However, the detection and identification of FLOs with low concentration and high structural diversity remain meaningful yet challenging work. Herein, we propose a strategy that enables efficient discovery and annotation of FLOs, especially those with a relatively high degree of polymerization (DP, ≥3). Based on the known monomers and the specific polymerization pattern between them, the strategy predicted a theoretical list of precursor ions of FLO. Matching the predicted list against the experimental ion features screened out 490 features as the candidate of FLOs from over 10 000 raw features. Investigation of the fragmentation pathways of 17 known FLOs found that both PAs and TSs are easily subjected to RDA cleavage, which produced a series of characteristic fragmentation ions and neutral losses. Moreover, successive cleavage of the C₄ → C₈ bond between monomer units is observed for PAs, leading to the generation of characteristic fragmentation ions corresponding to monomeric flavan-3-ols. Assisted by the characteristic fragmentation pathways, 52 FLOs (DP: 2-6) were finally annotated from the 490 retained features. Their chemical structures were verified by depolymerization experiments using menthofuran as the nucleophilic trapping reagent. Among them, the pentamers and hexamers were detected in a Yunnan large leaf tea for the first time. Semiquantitation and multivariate statistical analysis indicate that PAs exhibit higher contents in green tea, and TSs show higher levels in black and white tea.

The spatial distribution of organics in geological samples can be used to determine when and how these organics were incorporated into the host rock. Mass spectrometry (MS) imaging can rapidly collect a large amount of data, but ions produced are mixed without discrimination, resulting in complex mass spectra that can be difficult to interpret. Here, we apply unsupervised and supervised machine learning (ML) to help interpret spectra from time-of-flight-secondary ion mass spectrometry (ToF-SIMS) of an organic-carbon-rich mudstone of the Middle Jurassic of England (UK). It was previously shown that the presence of sterane molecular biomarkers in this sample can be detected via ToF-SIMS (Pasterski, M. J. et al., Astrobiology 2023, 23, 936). We use unsupervised ML on scanning electron microscopy-electron dispersive spectroscopy (SEM-EDS) measurements to define compositional categories based on differences in elemental abundances. We then test the ability of four ML algorithms─k-nearest neighbors (KNN), recursive partitioning and regressive trees (RPART), eXtreme gradient boost (XGBoost), and random forest (RF)─to classify the ToF-SIM spectra using (1) the categories assigned via SEM-EDS, (2) organic and inorganic labels assigned via SEM-EDS, and (3) the presence or absence of detectable steranes in ToF-SIMS spectra. In terms of predictive accuracy and balanced accuracy, KNN was the best performing model and RPART the worst. The feature importance, or the specific features of the ToF-SIM spectra used by the models to make classifications, cannot be determined for KNN, preventing posthoc model interpretation. Nevertheless, the feature importance extracted from the other models was useful for interpreting spectra. We determined that some of the organic ions used to classify biomarker containing spectra may be fragment ions derived from kerogen which is abundant in this mudstone sample.

We report a study of internal covalent cross-linking with photolytically generated diarylnitrile imines of N-terminal arginine, lysine, and histidine residues in peptide conjugates. Conjugates in which a 4-(2-phenyltetrazol-5-yl)benzoyl group was attached to C-terminal lysine, that we call RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K, were ionized by electrospray and subjected to UV photodissociation (UVPD) at 213 nm. UVPD triggered loss of N₂ and proceeded by covalent cross-linking to nitrile imine intermediates that involved the side chains of N-terminal arginine, lysine, and histidine, as well as the peptide amide groups. Cross-linking yields were determined from UVPD-MS² measurements as 67%, 66%, and 84% for RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K ions, respectively. CID-MS³ of the denitrogenated ion intermediates from RAAA-tet-K, KAAA-tet-K, and HAAA-tet-K indicated overall cross-linking yields of 80%, 89%, and 80%, respectively. The nature of the cross-linking reactions and cross-link structures were investigated for RAAA-tet-K by high-resolution cyclic ion mobility mass spectrometry that identified precursor ion conformers and multiple dissociation products. All sequences were subjected to conformational analysis by Born-Oppenheimer molecular dynamics, and energy analysis by density functional theory calculations with M06-2X/def2qzvpp that provided relative and dissociation energies for several cross-link structural types. The cross-linking reactions were substantially exothermic, driving the efficient conversion of nitrile-imine intermediates to cyclic products. The principal steps in covalent cross-linking involved proton transfer onto the nitrile imine group accompanied by nucleophilic attack by the peptide side-chain and amide groups. Blocking the proton transfer and nucleophile resulted in a loss of cross-linking abilities.

Sulfatides are abundant components of the brain, and dysregulation of these molecules has been linked to several diseases. In sulfatide structures, a sugar is linked to a sphingoid backbone via an α-glycosidic or β-glycosidic linkage. While sulfatides are readily generated in negative ion mode imaging mass spectrometry experiments, resolving sulfatide diastereomers is challenging; therefore, identifications are usually reported as a single sulfatide. Herein, a gas-phase charge inversion ion/ion reaction between sulfatides and a strontium tris-phenanthroline [Sr(Phen)₃]²⁺ reagent is performed to separate the diastereomers, as they form complexes containing different numbers of phenanthroline ligands. The ability to separate these diastereomers using the reaction alone, without the need for any further dissociation, allows for the workflow to be readily implemented in an imaging mass spectrometry experiment. Imaging mass spectrometry was performed on sulfatides generated directly from rat brain tissue, and both the α- and β-linked sulfatide images were obtained.

Anatomical representation of site-specific clustering of biomolecules is a powerful way of predicting a potential interaction among signaling cascades and orchestrating molecular functions in cells and organs. The greater the number of molecules visualized simultaneously, the deeper we can understand each molecule's role in cellular metabolism and function. In the present study, we investigated site-specific localization of small biomolecules in the slug using Space and Time Coherent Mapping (STCM), a key technology in matrix-assisted laser desorption ionization time-of-flight imaging mass spectrometry. We acquired mass measurements and mass-based molecular images simultaneously under the microscope-mode instrumentation developed specifically in our laboratory. Mass images were generated in the increment of 0.2 in the mass-to-charge ratio (m/z) with spatial resolution of 2 μm. Resultant images were unique in each mass increment and allowed us to predict anatomical site-specific clustering of bioactive signaling molecules. We suggest that STCM is a useful tool to promote the compilation of comprehensive molecular maps and understand the role of individual molecules and their interactive mechanisms in situ.

A year has gone by, and it is still hard to believe that Dr. Shuying Liu had left us on November 22, 2023. Most her colleagues and friends remember vividly her passionate images captured in the talk she filmed for the Chinese Mass Spectrometry Conference 2020-2023 in June 2023. In that video, Dr. Liu reviewed the progresses made in China's mass spectrometry field over the last 40 years and urged young mass spectrometrists to continue the work and forge an even more luminous future. Dr. Liu dedicated nearly 60 years to mass spectrometry research since 1965. As a renowned scholar, a professor of the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, and Changchun University of Chinese Medicine and the founder of Jilin Ginseng Academy, Dr. Liu was a frequent and inspiring presence at academic conferences. Dr. Liu's talk, as always, resonated deeply with the audience, but nobody knew that would be the very last talk presented by her. Her demeanor was brimming with the same zest that had always characterized her presence, which made the disheartening news of her passing particularly jarring. Coming to terms with her departure is no easy feat; we find solace in envisioning her still in our midst, tirelessly dedicating herself to the mass spectrometry research she so deeply cherished.