Fragment Ions Research Articles

Machine Learning Correlation of Electron Micrographs and ToF-SIMS for the Analysis of Organic Biomarkers in Mudstone.

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.

Reconstructing real-space geometries of polyatomic molecules undergoing strong field laser-induced Coulomb explosion

Coulomb explosion is an established momentum imaging technique, where the molecules are ionized multiple times on a femtosecond time scale before breaking up into ionized fragments. By measuring the momentum of all the ions, information about the initial molecular structure is theoretically available. However, significant geometric changes due to multiple ionizations occur before the explosion, posing a challenge in retrieving the ground-state structure of molecules from the measured momentum values of the fragments. In this work, we investigate theoretically and experimentally such a connection between the ground-state geometry of a polyatomic molecule (OCS) and the detected momenta of ionic fragments from the Coulomb explosion. By relying on time-dependent density functional theory (TDDFT), we can rigorously model the ionization dynamics of the molecule in the tunneling regime. We reproduce the energy release and the Newton plot momentum patterns of an experiment in which OCS is ionized to the 6+ charge state. Our results provide insight into the behavior of molecules during strong field multiple ionization, opening a way toward precision imaging of real-space molecular geometries using tabletop lasers.

Predicting Precursor Ions Combined with Fragmentation Pathway for Screening and Identification of Flavan-3-ol Oligomers in Tea (Camellia sinensis. var. assamica).

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.

Time-resolved Coulomb explosion imaging of vibrational wave packets in alkali dimers on helium nanodroplets.

Vibrational wave packets are created in the lowest triplet state 13Σu+ of K2 and Rb2 residing on the surface of helium nanodroplets, through non-resonant stimulated impulsive Raman scattering induced by a moderately intense near-infrared laser pulse. A delayed, intense 50-fs laser pulse doubly ionizes the alkali dimers via multiphoton absorption and thereby causes them to Coulomb explode into a pair of alkali ions Ak+. From the kinetic energy distribution P(Ekin) of the Ak+ fragment ions, measured at a large number of delays, we determine the time-dependent internuclear distribution P(R, t), which represents the modulus square of the wave packet within the accuracy of the experiment. For both K2 and Rb2, P(R, t) exhibits a periodic oscillatory structure throughout the respective 300 and 100ps observation times. The oscillatory structure is reflected in the time-dependent mean value of R, ⟨R⟩(t). The Fourier transformation of ⟨R⟩(t) shows that the wave packets are composed mainly of the vibrational ground state and the first excited vibrational state, in agreement with numerical simulations. In the case of K2, the oscillations are observed for 300ps, corresponding to more than 180 vibrational periods with an amplitude that decreases gradually from 0.035 to 0.020Å. Using time-resolved spectral analysis, we find that the decay time of the amplitude is ∼260ps. The decrease is ascribed to the weak coupling between the vibrating dimers and the droplet.

Dissociative triple ionization of argon dimers in elliptically polarized two-color laser fields.

Using ion-ion coincidence measurements, we experimentally investigate the dissociative triple ionization of argon dimers in relative phase controlled elliptically polarized two-color femtosecond laser fields. By examining the kinetic energy release-dependent momentum angular distribution of the ejected ionic fragments, two distinct pathways, each associated with different intermediates, are identified. Control over the emission directions of the ionic fragments is achieved by varying the relative phase of the elliptical two-color laser fields. Notably, the two pathways exhibit nearly opposite asymmetries in their dependence on the relative phase. Our findings demonstrate the critical role of intermediates in the dissociative triple ionization of dimers and provide a method to distinguish the intermediates involved in this process.

A multimass velocity-map and covariance-map imaging study of the dissociative electron ionisation dynamics of carbonyl sulphide (OCS)

Abstract We present the results of a velocity-map imaging study into the dissociative electron ionisation dynamics of OCS at electron energies of 50, 75, and 100 eV, supported by new EOM-CCSD and CASSCF potential energy surface calculations for the OCS+ cation and OCS2+ dication. We report partial ionisation cross-sections and scattering distributions for the observed ionic fragments, and interpret these in terms of the dissociation dynamics of the singly and doubly charged OCS parent ions formed in the electron-molecule collisions. For many of the dissociation channels, consideration of the calculated potential energy surfaces supplemented by comparison with data from photoionisation studies provides insight into the electronic potential energy surfaces involved. For fragmentation products arising from dissociation of the dication, time-of-flight and recoil-frame covariance maps provide mechanistic insight into the stepwise dissociation mechanisms leading to these products and support previous conclusions by other authors.

Dimethyl labeling of N-terminal amines allows unambiguous identification of protein crosslinks

Protein crosslinks induced through either deliberate enzymatic oxidation or reactive oxidants (oxidative eustress/distress), are associated with multiple human pathologies including atherosclerosis, Alzheimer’s and Parkinson’s diseases. In many cases, the nature of the crosslinks, their position(s) either within (intramolecular) or between (intermolecular) polypeptide chains, and concentrations are unclear. Although limited data are available from specific antibodies, detailed characterization of protein crosslinks is often performed by mass spectrometric analysis of peptides from proteolytic digestion. Such analyses are challenging due to the low concentration of these species, and the complexity of their fragment ion spectra when compared to non-crosslinked species. We hypothesized that highly efficient and specific chemical amine labelling of the two N-termini in crosslinked peptides (compared to the single N-terminus of linear peptides), using “light” and “heavy” isotope-labeled reagents would facilitate identification, validation and quantification of crosslinks. This method was compared to a previous enzyme-catalyzed 18O C-terminal carboxylate labelling approach. N-terminal amine dimethyl labelling is shown to have major advantages over the 18O-approach including high labelling yields (92-100%) and well-defined mass spectrometric isotope distribution patterns. This approach has allowed identification of novel dityrosine crosslinks between pair of tyrosine (Tyr, Y) residues in photo-oxidized β-casein (Y195-Y195, Y195-Y208, Y208-Y208), and α-synuclein exposed to nitrosative stress (Y39-Y39, Y39-Y125, Y39-Y133, Y133-Y136). This approach is also applicable to disulfide bond mapping, with 15 of 17 disulfides in serum albumin readily detected. These data indicate that dimethyl labelling is a highly versatile and efficient approach for the site-specific identification of oxidation- and nitration-induced crosslinks in proteins.

Existing Forms of Notoginsenoside R1 in Rats and Their Potential Bioactivities.

Notoginsenoside R1 (NG-R1) is a primary active constituent in Panax notoginseng, a medicinal and edible plant. It is a saponin with protopanaxatriol (PPT) as its aglycone. UHPLC-ESI-Q-TOF-MS/MS was used to clarify the existing forms of NG-R1 and their distributions in rats. The nomenclature of the ESI MS fragmentation pathway and ions of PPT was proposed for the first time. Totally, 105 metabolites with 89 new metabolites were identified. In terms of their LC-MS data, 7 were accurately identified by comparison with reference compounds, and 41 were clearly identified. Polyhydroxylation, pentosylation, acetylation, glucuronidation, and amino acid conjugation are new metabolic reactions of NG-R1. In total, 69, 48, 47, 43, 24, 15, and 7 metabolites were detected in the large intestine, stomach, small intestine, liver, lungs, kidneys, and heart, respectively. Then, 48 metabolites were predicted to be effective by PharmMapper, and their mechanisms of action on three diseases were predicted by network pharmacology. Finally, the antitumor effects on cell proliferation and the anti-inflammatory effects of the eight compounds were verified by cellular experiments. These results help further elucidate the in vivo existing forms of dammarane-type triterpenoids and form the basis for discovering their effective forms in the future.

Molecular Orientation Resolved (e, 2e) Cross Sections for CF4 at 67 eV Impact Energy

In this investigation, the fully differential cross sections (FDCS) for ionization of electron impact at low energy (E<sub>0</sub> = 67 eV) were measured for the specific orientations of the molecules and around it axis. In the full perpendicular plane (FPP), the FDCS for CF<sub>4</sub> were experimentally figured out by using reaction microscope (REMI) for different kinematics such as two ejection electron energies of 5 eV and 8 eV, scattering angles of -25° and 30°, and two molecular axis directions of ϕ<sub>Mol</sub> = 0° and 45°, also their relative orientations such as 180° and 225°. The momentum vectors of the two ejected electrons (with energies E<sub>e1</sub>, and E<sub>e2</sub>) and the fragment ions (e, 2e+ ions) were detected by the triple coincidence method. For the duration of dissociation, the time of flight (TOF) of CF<sub>3</sub><sup>+</sup>, CF<sub>2</sub><sup>+</sup>, CF<sup>+</sup>, F<sup>+</sup>, and C<sup>+</sup> are observed in the TOF diagram. During the dissociation of the final fragment of CF<sub>3</sub><sup>+</sup> ion and its electronic ground state neutral F, the momentum vector of CF<sub>3</sub><sup>+</sup> allows us to draw conclusions about the orientation of the molecule in the course of the collision. Finally, it was remarked that concerning molecular orientation, electrons are highly sensitive at low energy and low scattering angle.

Exploration of Aflatoxin B1 Degradation Products via Kocuria rosea: Structure Elucidation and Toxicity Analysis

Aflatoxin B1, a natural mycotoxin produced by Aspergillus fungi with high toxicity and carcinogenicity to humans and animals, has attracted more and more attention in the past 40 years. In the study of the biological detoxification of aflatoxin B1—although it has been confirmed that Kocuria rosea has the ability to efficiently remove aflatoxin B1—the degradation products, degradation pathways, and toxicity of the degradation products of aflatoxin B1 have not been clarified. Therefore, in this study, it was found that the functional groups of aflatoxin B1 changed after being cultured with Kocuria rosea, indicating the production of aflatoxin B1 degradation products. Ten main AFB1 degradation products (including aflatoxicol, aflatoxin D1, and aflatoxin D2) were identified, and their structures and fragmentation mechanisms were further elucidated by the parent ions and fragment ions of the products. The possible degradation pathway of aflatoxin B1 was proposed according to the structure of the degradation products. Additionally, the toxicity of the degradation products was analyzed according to the quantitative structure–activity relationship theory, and cytotoxicity experiments and dead–live cell staining experiments showed that the toxicity of the degradation products was significantly less than that of aflatoxin B1. In this study, the mechanism of aflatoxin B1 degradation by Kocuria rosea was explored from several perspectives, indicating that aflatoxin B1 degradation by Kocuria rosea is a promising biological method.

Photochemical and Collision-Induced Cross-Linking of Asp, Glu, Asn, and Gln Residues in Peptide-Nitrile Imine Conjugate Ions in the Gas Phase.

Peptide conjugates furnished with a 2,5-diaryltetrazolecarbonyl tag at the C-terminal lysine, which we call peptide-tet-K, were found to undergo efficient cross-linking of Asp, Glu, Asn, and Gln residues to transient nitrile-imine intermediates produced by photodissociation and collision-induced dissociation (CID) of the tetrazole ring in gas-phase ions. UV photodissociation (UVPD) at 213 nm achieved cross-linking conversion yields of 37 and 61% for DAAAK-tet-K and EAAAK-tet-K, respectively. The yields for NAAAK-tet-K and QAAAK-tet-K were 29 and 57%, respectively. Even higher cross-link yields were found for CID-MS3 of stable denitrogenated ions, (peptide-tet-K-N2 + H)+, that were in the 69-83% range. Different types of cross-links were distinguished by CID-MSn that showed a distinct series of backbone fragment ions, loss of N-terminal groups, and loss of phenylhydrazine from the modified nitrile imines. The Asp and Glu side-chain carboxyl groups were major participants in cross-linking that resulted in proton and oxygen transfer to the nitrile imine group. Other types of cross-linking involved Asn and Gln CONH2 groups and backbone amides. Cyclic ion mobility-mass spectrometry was used to separate NAAAK-tet-K and QAAAK-tet-K conformers and products of their collision-induced denitrogenation. Linear nitrile-imine and cross-linked ion structures were identified by comparing the experimental collision cross sections (CCSexp) to those for structures obtained by combined Born-Oppenheimer molecular dynamics and density functional theory (DFT) calculations. The formation of cross-links was found to be energetically favorable and involved proton-facilitated nucleophilic attack at the nitrile-imine carbon atom.

Molecular Oxygen (O2) Artifacts in Tandem Mass Spectra.

Peak annotation plays an important role in mass spectral evaluation of the NIST 2023 tandem mass spectral library. While most fragment ions are formed by neutral losses, there are peaks that represent adduct ions from these fragments. Previously, we have reported two main types of addition reactions in the collision cell, namely addition of H2O and N2. Here we report a different reaction in the collision cell, with addition of O2 leading to a small peak that could only be assigned to a peroxyl radical ion. For example, some protonated iodoaromatics lose an iodine atom to form a radical cation [M+H-I]+•, which reacts with O2 to generate a peroxyl radical ion [M+H-I+O2]+•. Higher concentrations of O2 result in higher peroxyl radical peaks, which become dominant while the precursor ions are consumed, as examined by five compounds under different concentrations of O2. The correlation of the peroxyl radical peak intensities to the concentration of O2 provides a tool to estimate trace amounts of O2 within the instrument. In the NIST 2023 tandem mass spectral library, the peaks for [M+H-X+O2]+· are most abundant in numbers and in intensity for X = NO2 or I, are much less abundant for X = Br, and are rare for X = Cl. Other leaving groups in this library are SO3H, SO2NH2, CSNH2, CO2C6F5, SO2CH3, and COCH3. The O2 addition reaction is also observed with negative ions in this library. While adducts of H2O and N2 often constitute major peaks, the peaks of the peroxyl radicals under standard conditions are mostly very small and may be mistaken for noise, but their correct annotation improves the quality of the spectra and is important when comparing spectra from different instruments or conditions.

Unlocking the Mysteries of the Desorption-Ionization Mechanism via Separate Thermal and Charge Strategies.

Herein, a new strategy is employed to build a controllable thermal-coupled charge ionization (TCCI) device to elucidate the desorption-ionization mechanism of plasma ion sources. Efficient synergistic desorption and ionization are achieved within the TCCI device by independently controlling the desorption temperature and plasma charges. The TCCI device efficiently ionizes samples using abundant free electrons, charges, and active species from arc plasma. The coexistence of free electrons and hydroxide radicals confers redox capability to the TCCI system, implying the presence of a unified redox mechanism even when the arc plasma is transmitted through a metal conductor over a distance. In addition, molecular ions of the analytes facilitate the differentiation between primary and secondary amines during their analysis. Notably, the TCCI device enables a switch between hard and soft ionization by adjusting the thermal desorption temperature. At high temperatures (>400 °C), the TCCI device exhibits hard ionization characteristics, producing fragment ions beneficial for isomer discrimination. The TCCI mass spectrometry exhibits robust performance in terms of sensitivity and accuracy for detecting antibiotics and sterols in saline solutions, achieving linearity with correlation coefficients ≥0.99 and excellent reproducibility. The successful analysis of seven pharmaceuticals and four sterols in complex matrices using the TCCI device demonstrates its excellent salt and matrix tolerance. Overall, the TCCI device, with its independent control over thermal desorption and arc plasma, achieves efficient synergistic desorption and ionization, overcoming limitations in existing ionization technologies and contributing to the study of gas-phase ion dynamics and mechanisms.

Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography

To better understand host cell protein (HCP) retention in adeno-associated virus (AAV) downstream processes, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to quantitatively profile residual HCPs for four AAV serotypes (AAV2, -5, -8, and -9) produced with HEK293 cells and purified using POROS CaptureSelect AAVX affinity chromatography. A broad range of residual HCPs were detected in affinity eluates after purification (N total = 2,746), and HCP profiles showed universally present species (N universal = 1,117) and species unique to one or more AAV serotype. SWATH-MS revealed that HCP persistence was dominated by high-abundance conserved species (HACS), which appeared across all serotype conditions studied. Due to the notable contribution of these species to overall residual HCP levels, physical and functional characteristics of HACS were examined to determine trends that coincide with persistence. Subnetwork interaction mapping and Gene Ontology function enrichment analysis revealed extensive physical interactions between these proteins and significant enrichment for biological processes, molecular functions, and reactome pathways related to protein folding, nucleic acid binding, and cellular stress. The abundant and conserved nature of these HCPs and their functions offers a new perspective for mechanistic evaluations of impurity retention for AAV downstream processes.

Investigation of the mechanism of [M-H]+ ion formation in photoionized N-alkyl-substituted thieno[3,4-c]-pyrrole-4,6-dione derivatives during higher order MSn high-resolution mass spectrometry.

The mechanism underlying dopant-assisted atmospheric pressure photoionization's (APPI) formation of ions is unclear and still under debate for many chemical classes. In this study, we reexamined the gas-phase reaction mechanisms responsible for the generation of [M-H]+ precursor ions, resulting from the loss of a single hydrogen atom, in a series of N-alkyl-substituted thieno[3,4-c]-pyrrole-4,6-dione (TPD) derivatives. Atmospheric pressure photoionization combined with higher order MS/MSn using high-resolution mass spectrometry (APPI-HR-CID-MSn) and electronic structure calculations using density functional theory were used to determine the chemical structure of observed [M-H]+ ions. As a result, the higher order MSn (n = 3) experiments revealed a reversed Diels-Alder fragmentation mechanism, leading to a common fragment ion at m/z 322 from the studied [M1-5-H]+ ion species. In addition, the calculation for two chemical structure models (N-alkyl-TPD1 and N-alkyl-TPD5) showed that the fragment structure, resulting from the removal of the hydrogen atom connected to the third carbon atom of the N-alkyl side chain, has a more stable cyclic form compared with the linear one. The proposed chemical structure of the N-alkyl TPD ion species, following the loss of a single hydrogen atom, was revealed during APPI-HR-CID-MSn (n= 3) experiments on the [M-H]+ species. Hydrogen radical (H•) abstraction from the alkyl side chain (e.g., hexyl, heptyl, octyl, 2-ethylhexyl, and nonyl) triggered a rearrangement in the radical cation structure of the N-alkyl-TPD derivatives, initiating cyclization and forming a six-membered ring that connects the oxygen atom to the third carbon atom in the alkyl chain. In addition, theoretical calculations supported the APPI-HR-CID-MSn (n = 3) experiments by demonstrating that the proposed chemical structure, resulting from the intramolecular cyclization of the N-alkyl-TPD ion species, was stable in the presence of chlorobenzene. These findings will aid the structural determination and elucidation of molecules with similar core structures.

Deciphering supramolecular arrangements, micellization patterns, and antimicrobial potential of bacterial rhamnolipids under extreme treatments of temperature and electrolyte.

The micellization properties of rhamnolipids (RLs) in extreme electrolyte concentrations and temperatures have gained considerable attention due to their broad industrial applications. In this study, the aggregation behavior, specifically the micellization pattern (critical micelle concentration (CMC)) of RLs produced from a newly isolated thermophilic strain of Pseudomonas aeruginosa from a harsh environment of an oil field, was investigated by a spectrophotometric method at various temperatures (293-393 K) and electrolyte concentrations (NaCl: 2-20%). The result indicated that the CMC values (0.267-0.140 mM⋅dm-3) were both electrolyte- and temperature-dependent exhibiting a U-shaped trend as temperature and NaCl concentration increased. Variations in NaCl concentration and temperature also affected the standard Gibbs free energy (ΔGo mic ), enthalpy (ΔHo mic ), and entropy (ΔSo mic ) of micellization. The molecule also showed stability at a broad range of temperatures, pH, and NaCl concentrations. Thin-layer chromatography (TLC) and Fourier-transform infrared (FTIR) analysis confirmed the similarity in composition between the crude extract and the commercial RL with Rf values of 0.72 for mono-rhamnolipids and 0.28 for di-rhamnolipids. FTIR analysis confirmed the chemical nature particularly key aliphatic functional groups present in the fatty acid tail of RLs and the -COC- bond in the structure of the rhamnose moiety. Additionally, LC-ESI-QTOF analysis confirmed corresponding ionic fragments of mono- and di-rhamnolipids congeners. Furthermore, the antimicrobial potential was determined against different human pathogens in the absence and presence of NaCl by measuring zones of inhibition. The result revealed enhanced inhibitory effects against Gram-positive pathogens (S. aureus, S. epidermidis, and L. monocytogene), with zones of inhibition of 26, 30, and 20 mm in the presence of NaCl. These findings underline the role of NaCl in the micellization of RL molecules and highlight their importance in environmental applications, pharmaceuticals, and various life science sectors.

CNSC-08. PROGNOSTIC BIOMARKER DISCOVERY THROUGH PROTEOMIC ANALYSIS OF GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor known for its high recurrence rates and dismal survival outcomes. Despite advancements in surgical and therapeutic strategies, the prognosis for GBM patients remains poor. This study applies proteomic analysis to postoperative GBM tumor specimens to identify molecular markers that could enhance prognosis prediction. Through the analysis of protein expression profiles before and after tumor recurrence, we identified significant alterations in protein pathways via Gene Set Enrichment Analysis (GSEA), highlighting potential biomarkers for disease outcome prediction. METHODS We employed Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH-MS) proteomics to analyze protein samples from both primary and recurrent GBM patients. Gene Set Enrichment Analysis (GSEA) was utilized to compare protein expression profiles and discern significantly altered pathways. RESULTS Analysis revealed 778 differentially expressed proteins (DEPs), with notable alterations in pathways such as glycolysis, interleukin signaling, and MAPK signaling. Proteins such as TOLLIP, ENO1, and ITGA2B emerged as potential prognostic markers. Validation through an online tumor prognostic analysis platform (ToPP), integrated with The Cancer Genome Atlas (TCGA) data, facilitated the development of a prognostic risk score model. This model effectively categorized patients into high and low-risk groups based on their protein expression profiles, enhancing the prediction of patient outcomes. The proteins and pathways identified offer new insights into the molecular dynamics of GBM postoperatively. The association between protein expression profiles and patient prognosis underscores the potential of these markers in developing customized therapeutic strategies. Additionally, the prognostic risk score model serves as a valuable tool for improving prognostic accuracy and could guide personalized treatment plans. CONCLUSION Proteomic profiling of GBM tumors significantly contributes to uncovering the molecular mechanisms driving this aggressive cancer and facilitates the development of dependable prognostic tools. These findings not only affirm the critical role of proteomics in cancer research but also advance personalized medicine strategies in GBM management.

A fragmentation study of disaccharide flavonoid C-glycosides using triple quadrupole mass spectrometry and its application for identification of flavonoid C-glycosides in Odontosoria chinensis.

Flavonoid C-glycosides have a wide range of pharmacological activities. However, there are few mass spectrometric research on C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides. Their low-energy collision-induced dissociation (ESI-CID-MS/MS) fragmentation pattern and differences have not been reported, and the fragment ion library is incomplete. Therefore, it was essential to elucidate the fragmentation patterns of disaccharide flavonoid C-glycosides, which is described in this study. Four disaccharide flavonoid C-glycosides such as vitexin-4″-O-glucoside were analyzed by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometer (UPLC-MS/MS) using electrospray ionization (ESI) in both positive and negative ion modes. Each ion and its proposed fragmentation pathways of the four disaccharide flavonoid C-glycosides were analyzed comprehensively. Finally, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and the established fragmentation patterns have been used to identify disaccharide flavonoid C-glycosides in Odontosoria Chinensis. The fragmentation pathways of C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides are similar. They both have the mass spectrometric characteristics of O-glycoside and C-glycoside. Product ions after mixed pathways of neutral fragments such as saccharide ring fragment, O-glycosides, H2O, and CH2O elimination appeared in both types of flavonoid C-glycosides, but their relative abundances are significantly different. According to the established fragmentation patterns, di-C,O-saccharide flavonoid glycosides were also found in Odontosoria chinensis. The fragment ions at m/z 431, 413, 341, 311, 293, and 282 in negative ion mode and m/z 293, 282, 577, 559, 541, 523, 529, and 499 in positive ion mode can serves as the main characteristics for identifying C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides.

The Proteomics Standards Initiative Standardized Formats for Spectral Libraries and Fragment Ion Peak Annotations: mzSpecLib and mzPAF.

Mass spectral libraries are collections of reference spectra, usually associated with specific analytes from which the spectra were generated, that are used for further downstream analysis of new spectra. There are many different formats used for encoding spectral libraries, but none have undergone a standardization process to ensure broad applicability to many applications. As part of the Human Proteome Organization Proteomics Standards Initiative (PSI), we have developed a standardized format for encoding spectral libraries, called mzSpecLib (https://psidev.info/mzSpecLib). It is primarily a data model that flexibly encodes metadata about the library entries using the extensible PSI-MS controlled vocabulary and can be encoded in and converted between different serialization formats. We have also developed a standardized data model and serialization for fragment ion peak annotations, called mzPAF (https://psidev.info/mzPAF). It is defined as a separate standard, since it may be used for other applications besides spectral libraries. The mzSpecLib and mzPAF standards are compatible with existing PSI standards such as ProForma 2.0 and the Universal Spectrum Identifier. The mzSpecLib and mzPAF standards have been primarily defined for peptides in proteomics applications with basic small molecule support. They could be extended in the future to other fields that need to encode spectral libraries for nonpeptidic analytes.

Enhanced Payload Localization in Antibody-Drug Conjugates Using a Middle-Down Mass Spectrometry Approach with Proton Transfer Charge Reduction.

Antibody-drug conjugates (ADCs) represent a novel class of immunoconjugates with growing therapeutic relevance, since they combine the efficacy of cytotoxic drugs with the specificity of antibodies. However, by design, ADCs introduce structural features into the monoclonal antibody scaffold that complicate their analysis. Payload attachment to cysteine or lysine residues can often result in product heterogeneity, regarding both the number of attached drug molecules and their conjugation site, necessitating the use of state-of-the-art MS instrumentation to elucidate their complexity. In middle-down mass spectrometry (MD MS), the gas-phase sequencing of ∼25 kDa ADC subunits with different ion activation techniques generally produces rich fragmentation mass spectra; however, spectral congestion can cause some fragment ions to go undetected, including those that can pinpoint the exact location of payload conjugation sites. Proton transfer charge reduction (PTCR) can substantially simplify fragment ion spectra, thereby unveiling the presence of product ions whose signals were previously suppressed. Herein, we present an MD MS strategy relying on the use of PTCR to investigate a cysteine-based ADC mimic with a variable drug-to-antibody ratio, targeting the unambiguous localization of payload conjugation sites. Unlike traditional tandem MS experiments (MS2), which could not provide a complete map of conjugation sites, a single PTCR-based experiment (MS3) proved to be sufficient to achieve this goal across all variably modified ADC subunits, including isomeric ones. Combining the results obtained from orthogonal ion activation techniques followed by PTCR further strengthened the confidence in the assignments.