Collision-induced Dissociation Research Articles

A spectroscopic test suggests that fragment ion structure annotations in MS/MS libraries are frequently incorrect

Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS2) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS2 spectra as reference. The ability of different in silico algorithms to correctly predict MS2 spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS2 spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSn product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS2 libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions.

New Full-Dimensional Reactive Potential Energy Surface for the H4 System.

As the most abundant molecule in the universe, collisions involving H2 have important implications in astrochemistry. Collisions between hydrogen molecules also represent a prototype for assessing various dynamic methods for understanding fundamental few-body processes. In this work, we develop a new and highly accurate full-dimensional potential energy surface (PES) covering all reactive channels of the H2 + H2 system, which extends our previously reported H2 + H2 nonreactive PES [J. Chem. Theory Comput., 2021, 17, 6747] by adding 39,538 additional ab initio points calculated at the MRCI/AV5Z level in the reactive channels. The global PES is represented with high fidelity (RMSE = 0.6 meV for a total of 79,000 points) by a permutation invariant polynomial neural network (PIP-NN) and is suitable for studying collision-induced dissociation, single-exchange, as well as four-center exchange reactions. Preliminary quasi-classical trajectory studies on the new PIP-NN PES reveal strong vibrational enhancement of all reaction channels.

A High-Throughput Workflow for Mass Spectrometry Analysis of Nucleic Acids by Nanoflow Desalting.

Broad interest in nucleic acids, both their therapeutic capabilities and understanding the nuances of their structure and resulting function, has increased in recent years. Post-transcriptional modifications, in particular, have become an important analysis target, as these covalent modifications to the sugars, nitrogenous bases, and phosphate backbone impart differential functionality to synthetic and biological nucleic acids. Characterizing these post-transcriptional modifications can be difficult with traditional sequencing workflows; however, advancements in top-down mass spectrometry address these challenges. Online desalting platforms have enabled facile sample cleanup and reliable ionization of increasingly large (100 nt) oligonucleotides, and application of existing tandem mass spectrometry techniques has yielded information-rich spectra which can be used to interrogate primary sequences. To extend the capabilities of top-down MS and its analysis of nucleic acids, we have developed a nanoflow desalting platform for high-throughput and low sample-use desalting coupled with collision-induced dissociation (CID), 213 nm ultraviolet photodissociation (UVPD), and activated-ion electron photodetachment dissociation (a-EPD) to yield high-quality MS/MS spectra. Fragments identified using an m/z-domain isotope matching strategy yielded high sequence coverage (>70%) of a yeast phenylalanine tRNA.

Competitive Radical Migrations and Ribose Ring Cleavage in Adenosine and 2'-Deoxyadenosine Cation Radicals.

We report a combined experimental and computational study of adenosine cation radicals that were protonated at adenine and furnished with a radical handle in the form of an acetoxyl radical, •CH2COO, that was attached to ribose 5'-O. Radicals were generated by collision-induced dissociation (CID) and characterized by tandem mass spectrometry and UV-vis photodissociation action spectroscopy. The acetoxyl radical was used to probe the kinetics of intramolecular hydrogen transfer from the ribose ring positions that were specifically labeled with deuterium at C1', C2', C3', C4', C5', and in the exchangeable hydroxyl groups. Hydrogen transfer was found to chiefly involve 3'-H with minor contributions by 5'-H and 2'-H, while 4'-H was nonreactive. The hydrogen transfer rates were affected by deuterium isotope effects. Hydrogen transfer triggered ribose ring cleavage by consecutive dissociations of the C4'-O and C1'-C2' bonds, resulting in expulsion of a C6H9O4 radical and forming a 9-formyladenine ion. Rice-Ramsperger-Kassel-Marcus (RRKM) and transition-state theory (TST) calculations of unimolecular constants were carried out using the effective CCSD(T)/6-311++G(3d,2p) and M06-2X/aug-cc-pVTZ potential energy surfaces for major isomerizations and dissociations. The kinetic analysis showed that hydrogen transfer to the acetoxyl radical was the rate-determining step, whereas the following ring-opening reactions in ribose radicals were fast. Using DFT-computed energies, a comparison was made between the thermochemistry of radical reactions in adenosine and 2'-deoxyadenosine cation radicals. The 2'-deoxyribose ring showed lower TS energies for both the rate-determining 3'-H transfer and ring cleavage reactions.

Mass spectrometry approaches to natural and synthetic melanin characterization

AbstractCharacterization of synthetic and natural melanin nanoparticles (MNPs) has been challenging due to the variability of properties and chemical bonding. Although eumelanin was the first to be discovered, four additional subcategories of melanin now exist including pheomelanin, neuromelanin, allomelanin and pyomelanin. Mass spectrometry (MS) methods have provided crucial insights into the bonding, structure, and mechanism regarding MNP formation, but inconsistent findings in the literature have caused further confusion in the analysis and structure of MNPs. This mini‐review will focus on the types of MS experiments that have been used to analyze MNPs and what kind of information can be gained from different ionization sources, collisional activation experiments, and reaction monitoring quantitative methods when analyzing different subcategories of MNPs. Additionally, future directions regarding alternative applications of MNPs in MS experiments, mass defect analyses of complex mixtures, and a standardized nomenclature for isomeric redox states will all be addressed. © 2023 The Authors. Polymer International published by John Wiley & Sons Ltd on behalf of Society of Industrial Chemistry.

Mass spectrometric study of low energy Cs+ ion-induced sputtered fragmentation of PADC polymer.

In this study, low-energy cesium (Cs+ ) ion-induced sputtered fragmentation of poly allyl diglycol carbonate (PADC) was investigated using mass spectrometry. The collision-induced dissociation mechanism revealed emission of various fragments, including monoatomic (H- , C1 - , O1 - ), diatomic (C2 - ), and multiatomic (C3 - , CO2 - , C2 O2 - , C3 O2 - ) species within the Cs+ ion energy range of 1-5keV. The anion current of these fragments exhibited a linear increase with rising incident Cs+ ion energy, indicating a corresponding rise in fragment abundance. Analysis of normalized yield indicated that at 1keV incident energy, the dominant fragment was monoatomic hydrogen (H- ), followed by diatomic carbon (C2 - ), monoatomic carbon (C1 - ), and monoatomic oxygen (O1 - ). Although C2 - remained dominant up to 5keV, other fragments exhibited varying normalized yields at different ion energy steps. The sputter yield estimation revealed that monoatomic hydrogen (H- ) and diatomic carbon (C2 - ) exhibited the highest yields, increasing exponentially beyond 3keV, while multiatomic fragments like C3 - , CO2 - , C2 O2 - , and C3 O2 - displayed the lowest yields. The sputter dissociation mechanism pointed to dehydrogenation, chain scission, and bond breakage as the primary processes during low-energy Cs+ ion impact. Postsputtering Scanning Electron Mircoscope (SEM) micrographs show craters, pits, and micropores on the PADC surface, indicating significant surface degradation. X-ray Diffraction (XRD) spectra exhibited reduced diffraction intensity, while Fourier Transform Infrared Spectroscopy (FTIR) analysis indicated the absence of molecular bands in the IR spectrum, confirming extensive surface damage due to Cs+ ion-induced sputtering.

Proteins in Scalp Hair of Preschool Children

Background. Early childhood experiences have long-lasting effects on subsequent mental and physical health, education, and employment. The measurement of these effects relies on insensitive behavioral signs, subjective assessments by adult observers, neuroimaging or neurophysiological studies, or retrospective epidemiologic outcomes. Despite intensive research, the underlying mechanisms of these long-term changes in development and health status remain unknown. Methods. We analyzed scalp hair from healthy children and their mothers using an unbiased proteomics platform combining tandem mass spectrometry, ultra-performance liquid chromatography, and collision-induced dissociation to reveal commonly observed hair proteins with a spectral count of 3 or higher. Results. We observed 1368 non-structural hair proteins in children and 1438 non-structural hair proteins in mothers, with 1288 proteins showing individual variability. Mothers showed higher numbers of peptide spectral matches and hair proteins compared to children, with important age-related differences between mothers and children. Age-related differences were also observed in children, with differential protein expression patterns between younger (2 years and below) and older children (3–5 years). We observed greater similarity in hair protein patterns between mothers and their biological children compared with mothers and unrelated children. The top 5% of proteins driving population variability represented biological pathways associated with brain development, immune signaling, and stress response regulation. Conclusions. Non-structural proteins observed in scalp hair include promising biomarkers to investigate the long-term developmental changes and health status associated with early childhood experiences.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Direct Performance of Triple-Stage Tandem Mass Spectrometry Analysis Using Dual-Direction Dipolar Excitation in a Digital Linear Ion Trap.

The ion trap mass spectrometer offers a unique advantage over other mass spectrometers by enabling multistage tandem mass spectrometry analysis with a single mass analyzer. It is employed to generate fragment ions through collision-induced dissociation (CID) usually by applying alternating current (AC) signals to a pair of electrodes for dipole excitation. The process of achieving double-stage tandem mass spectrometry analysis (MS/MS) in the mass spectrometer involves successive stages of injection, cooling, isolation, excitation, and scanning. For triple-stage tandem mass spectrometry analysis (MS/MS/MS), additional stages of isolation, cooling, and excitation need to be added based on the MS/MS analysis, resulting in a complex and time-consuming mass spectrometry workflow. In this study, a digital ion trap technology with the method of simultaneously applying dipole excitation signals to two pairs of electrodes in the ion trap was developed. This allows fragmentation of the precursor ion in one direction while exciting the first-generation product ions in the other direction, enabling direct acquisition of MS/MS/MS spectra. This approach simplifies the process of tandem mass spectrometry, as demonstrated by experimental studies on methamphetamine, which show that dual-direction excitation effectively reduces workflow and enhances the intensity of product ions. Additionally, the method of direct MS/MS/MS spectra achieved through dual-direction excitation in a digital ion trap mass spectrometer allows for a lower q value of the precursor ion owing to a pseudopotential well depth that is 1.648 times greater than that of a traditional sinusoidal ion trap. The experiments of analyzing high concentration n-butyl acetate and isobutyl acetate have shown that the implementation of MS/MS/MS analysis using dual-direction excitation can provide more mass spectral information and effectively distinguish between the two isomeric samples. The results of direct triple-stage spectra obtained by this technique for several typical volatile hazardous chemicals demonstrate the method's capability for rapid analysis and detection of such substances. In summary, the developed method of dual-directional excitation coupled with digital ion trap technology enables direct performance of triple-stage tandem mass spectrometry analysis, improving fragment ion intensities and providing more valuable mass spectral information. It offers advantages such as simplified workflows, faster analysis, and enhanced accuracy for analyzing compounds with low mass fragment ions.

Two-Dimensional Tandem Mass Spectrometry for Biopolymer Structural Analysis.

Biopolymer analysis, including proteomics and glycomics, relies heavily on the use of mass spectrometry for structural elucidation, including sequence determination. Novel methods to improve sample workup, instrument performance, and data analysis continue to be developed to address shortcomings associated with sample preparation, analysis time, data quality, and data interpretation. Here, we present a new method that couples in-source collision-induced dissociation (IS-CID) with two-dimensional tandem mass spectrometry (2D MS/MS) as a way to simplify proteomics and glycomics workflows while also providing additional insight into analyte structures over traditional MS/MS experiments. Specifically, IS-CID is employed as a gas-phase digestion method, i.e., to break down intact full-length polysaccharide or peptide ions prior to mass analysis. The resulting mixtures of oligomeric ions are analyzed by 2D-MS/MS, a technique that allows association of product ions with their precursor ions without isolation of the latter. A novel data analysis strategy is introduced to leverage the second dimension of 2D MS/MS spectra, in which stairstep patterns, representing outputs of a molecule's MSn scans, are extracted for structural interconnectivity information on the oligomer. The results demonstrate the potential applicability of 2D MS/MS strategies to the modern omics workflow and structural analysis of various classes of biopolymers.

Self-Assembly of a Novel Pentapeptide into Hydrogelated Dendritic Architecture: Synthesis, Properties, Molecular Docking and Prospective Applications.

Currently, ultrashort oligopeptides consisting of fewer than eight amino acids represent a cutting-edge frontier in materials science, particularly in the realm of hydrogel formation. By employing solid-phase synthesis with the Fmoc/tBu approach, a novel pentapeptide, FEYNF-NH2, was designed, inspired by a previously studied sequence chosen from hen egg-white lysozyme (FESNF-NH2). Qualitative peptide analysis was based on reverse-phase high performance liquid chromatography (RP-HPLC), while further purification was accomplished using solid-phase extraction (SPE). Exact molecular ion confirmation was achieved by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-ToF MS) using two different matrices (HCCA and DHB). Additionally, the molecular ion of interest was subjected to tandem mass spectrometry (MS/MS) employing collision-induced dissociation (CID) to confirm the synthesized peptide structure. A combination of research techniques, including Fourier-transform infrared spectroscopy (FTIR), fluorescence analysis, transmission electron microscopy, polarized light microscopy, and Congo red staining assay, were carefully employed to glean valuable insights into the self-assembly phenomena and gelation process of the modified FEYNF-NH2 peptide. Furthermore, molecular docking simulations were conducted to deepen our understanding of the mechanisms underlying the pentapeptide's supramolecular assembly formation and intermolecular interactions. Our study provides potential insights into amyloid research and proposes a novel peptide for advancements in materials science. In this regard, in silico studies were performed to explore the FEYNF peptide's ability to form polyplexes.

Separation and identification of the synthetic cathinone isomers dipentylone and N-ethylpentylone using chromatographic and mass spectral characteristics

Synthetic cathinone isomers exhibit very similar structure and physicochemical properties, which makes them unable to be effectively separated and identified, therefore increasing the likelihood of misidentification. In this study, the chromatographic and mass spectral characteristics of a sample by gas chromatography-mass spectrometry (GC–MS) were highly like those of N-ethylpentylone. The powder, however, was finally identified as dipentylone (also known as N, N-dimethylpentylone) by high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS) and nuclear magnetic resonance (NMR). Because of the possibility of misidentification, the collision-induced dissociation (CID) and electron ionization (EI) pathways of dipentylone and its isomer N-ethylpentylone were further examined to assist forensic laboratories in identifying the synthetic cathinone isomers in their case work. The separation of the two isomers was successfully achieved through ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). These results demonstrate that the methods developed in this study can be used to separate and identify the two isomers.

Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is widely used in untargeted metabolomics, but large-scale and high-accuracy metabolite annotation remains a challenge due to the complex nature of biological samples. Recently introduced electron impact excitation of ions from organics (EIEIO) fragmentation can generate information-rich fragment ions. However, effective utilization of EIEIO tandem mass spectrometry (MS/MS) is hindered by the lack of reference spectral databases. Molecular networking (MN) shows great promise in large-scale metabolome annotation, but enhancing the correlation between spectral and structural similarity is essential to fully exploring the benefits of MN annotation. In this study, a novel approach was proposed to enhance metabolite annotation in untargeted metabolomics using EIEIO and MN. MS/MS spectra were acquired in EIEIO and collision-induced dissociation (CID) modes for over 400 reference metabolites. The study revealed a stronger correlation between the EIEIO spectra and metabolite structure. Moreover, the EIEIO spectral network outperformed the CID spectral network in capturing structural analogues. The annotation performance of the structural similarity network for untargeted LC-MS/MS was evaluated. For the spiked NIST SRM 1950 human plasma, the annotation coverage and accuracy were 72.94 and 74.19%, respectively. A total of 2337 metabolite features were successfully annotated in NIST SRM 1950 human plasma, which was twice that of LC-CID MS/MS. Finally, the developed method was applied to investigate prostate cancer. A total of 87 significantly differential metabolites were annotated. This study combining EIEIO and MN makes a valuable contribution to improving metabolome annotation.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

UV-A-Induced Photoisomerization and Photodimerization of Curcumin: An Ion Mobility Mass Spectrometry Study.

Curcumin, the bioactive compound present in spice plant turmeric, has been shown to exhibit selective phototoxic activities toward mammalian cancer cells, and it is being used extensively as a photosensitizer (PS) in photodynamic therapies (PDT). However, so far, the fate of curcumin toward photochemical transformations is not well understood. Here we report our findings of a number of novel photochemical reaction channels of curcumin in water-methanol mixture, like photoisomerization, photodimerization, and photooxidation (H2-loss). The reaction was performed by irradiating the curcumin solution with ultraviolet (UV) light of wavelength 350 nm, which is abundant in the earth's troposphere. Product identification and structure elucidation are done by employing an integrated method of drift tube ion mobility mass spectrometry (DTIMS) in combination with high-performance liquid chromatography (HPLC) and collision-induced dissociation (CID) of the mass-selected molecular ions. Two photoisomers of curcumin produced as a result of trans-cis configurational changes about C═C double bonds in the excited state have been identified, and it has been shown that they could serve as the precursors for formation of isomeric dimers via [2 + 2] cycloaddition and H2-loss products. Comparisons of the experimentally measured collision cross-section (CCS) values of the reactant and product ions obtained by the DTIMS method with those predicted by the electronic structure theory are found to be very effective for the discrimination of the produced photoisomers. The observed photochemical reaction channels are potentially significant toward uses of curcumin as a photosensitizer in photodynamic therapy.

Enrichment of nitrogen compounds in heavy oils by complexation with ZrCl4 and detailed characterization using FT-ICR MS and ion mobility spectrometry

Nitrogen compounds in heavy oils have significant deleterious impacts on the petroleum products and processes. It is of great significance to characterize the structures of nitrogen compounds during hydrodenitrogenation (HDN) in heavy oils, but the low content of nitrogen compounds after HDN is not conducive to structural analysis. Zirconium tetrachloride (ZrCl4) was found as an efficient complexing agent to enrich both basic and non-basic nitrogen compounds in heavy oils. The molecular weight ranges of the basic and non-basic nitrogen compounds were similar before and after ZrCl4 enrichment. The distribution of carbon number and double bond equivalent (DBE) of basic N1 species in the enriched feedstock was very close to that of the original feedstock, while the basic N1 species in the enriched product behaved a higher DBE distribution. The non-basic nitrogen compounds with higher DBE in the feedstock and product were also more likely to be enriched. Moreover, multi-heteroatomic non-basic nitrogen compounds were more inclined to be enriched than the non-basic N1 species due to the possible synergism of multi-heteroatoms. The numbers of detected multi-heteroatomic non-basic N2, N1O1, and N1O2 compounds obtained by high resolution mass spectrometry increased by 40 to nearly 70 times. The double bond equivalent (DBE) of these refractory multi-heteroatomic non-basic nitrogen compounds in the deep HDN product was much lower than those in the feedstock, due to the hydrogenation saturation reaction during HDN. The representative core structures of refractory basic nitrogen compounds were deduced by ESI+ FT-ICR MS coupled with collision-induced dissociation (CID), and the side chains were proposed by ion mobility spectrometry (IMS). Two typical kinds of refractory basic N1 compounds were suggested in the HDN product. One was a compact structure with multiple naphthenic rings and short side chains. The other had a large molecular size with aromatic rings and long side chains. ZrCl4 as a complexing agent can effectively enrich nitrogen compounds in heavy oil, especially for the hydrotreated heavy oils. This enrichment method facilitates the structural characterization of refractory nitrogen compounds in heavy oil HDN process by FT-ICR MS and IMS. These results provide valuable information for the improvement of HDN processes and catalysts.

Ultra-high-performance liquid chromatography with tandem mass spectrometry method for cellular toxicity and pharmacokinetic study of PEG1K polymers.

Polyethylene glycol (PEG) is one of the most commonly used polymers in drug delivery systems. The investigation of the pharmacokinetic behavior of PEG is important for revealing the toxicity and efficiency of PEG-related Nano-drug delivery systems. A high through-put and selective ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method coupled with collision-induced dissociation (CID) in source technique was developed and validated to determine PEG1K polymers in cellular samples in this study. The countless precursor ions of PEG1K are dissociated in the source to generate numerous product ions which have different numbers of subunits. The transition of [M+H]+ precursor ions → product ions at m/z 177.1 (four subunits)→89.1 (two subunits) was selected to determine PEG1K due to its high sensitivity. The UHPLC-MS/MS method coupled with CID in the source showed good linearity over the range of 0.1-10μg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within±12.39%. The assay was successfully applied to a cellular pharmacokinetic study of PEG1K in human breast cancer cells. The cytotoxicity of PEG1K polymers was also studied and the results indicated that the cytotoxicity of PEG1K was not significant in the range of 5-1200μg/mL.

Ion mobility mass spectrometry uncovers regioselectivity in the carboxylate-assisted C-H activation of palladium N-heterocyclic carbene complexes.

Carboxylate-assisted Pd-catalyzed C-H bond activation constitutes a mild and versatile synthetic tool to efficiently and selectively cleave inert C-H bonds. Herein, we demonstrate a simple method to experimentally evaluate both reactivity and selectivity in such systems using mass spectrometry (MS) methods. The N-heterocyclic carbene (NHC) cations [(NHC)PdX]+, bearing as X- ligand bases commonly used to promote the C-H activation (carboxylates and bicarbonate), are generated in the gas-phase by ESI-MS. Their C-H bond activation at the N-bound groups of the NHC is then studied using Collision Induced Dissociation (CID) experiments. Ion Mobility Spectrometry (IM)-MS is exploited to identify a number of regioisomers associated with the distinctive site selective C-H activations. It is demonstrated that such C-H activation concomitant with acetic acid release occurs from a mixture of activated [(NHC-H)Pd(CH3CO2H)]+ and non-activated [(NHC)Pd(CH3CO2)]+ complexes. The identity of the X-type ligands (X = Cl-, carboxylates and bicarbonate) has a significant impact on the regioisomer branching ratio upon CID conditions. IM-MS in conjunction with a DFT mechanistic study is presented for the acetate-assisted C-H activation of the [(NHC)Pd(CH3CO2)]+ cation featuring butyl and aryl as N-donor groups.

Generation and reactivity of the fragment ion [B12I8S(CN)]- in the gas phase and on surfaces.

Gaseous fragment ions generated in mass spectrometers may be employed as "building blocks" for the synthesis of novel molecules on surfaces using ion soft-landing. A fundamental understanding of the reactivity of the fragment ions is required to control bond formation of deposited fragments in surface layers. The fragment ion [B12X11]- (X = halogen) is formed by collision-induced dissociation (CID) from the precursor [B12X12]2- dianion. [B12X11]- is highly reactive and ion soft-landing experiments have shown that this ion binds to the alkyl chains of organic molecules on surfaces. In this work we investigate whether specific modifications of the precursor ion affect the chemical properties of the fragment ions to such an extent that attachment to functional groups of organic molecules on surfaces occurs and binding of alkyl chains is prevented. Therefore, a halogen substituent was replaced by a thiocyanate substituent. CID of the precursor [B12I11(SCN)]2- ion preferentially yields the fragment ion [B12I8S(CN)]-, which shows significantly altered reactivity compared to the fragment ions of [B12I12]2-. [B12I8S(CN)]- has a previously unknown structural element, wherein a sulfur atom bridges three boron atoms. Gas-phase reactions with different neutral reactants (cyclohexane, dimethyl sulfide, and dimethyl amine) accompanied by theoretical studies indicate that [B12I8S(CN)]- binds with higher selectivity to functional groups of organic molecules than fragment ions of [B12I12]2- (e.g., [B12I11]- and [B12I9]-). These findings were further confirmed by ion soft-landing experiments, which showed that [B12I8S(CN)]- ions attacked ester groups of adipates and phthalates, whereas [B12I11]- ions only bound to alkyl chains of the same reagents.

Unveiling the intricacy of gapmer oligonucleotides through advanced tandem mass spectrometry approaches and scan accumulation for 2DMS.

Antisense oligonucleotides (ASOs) are crucial for biological applications as they bind to complementary RNA sequences, modulating protein expression. ASOs undergo synthetic modifications like phosphorothioate (PS) backbone and locked nucleic acid (LNA) to enhance stability and specificity. Tandem mass spectrometry (MS) techniques were employed to study gapmer ASOs, which feature a DNA chain within RNA segments at both termini, revealing enhanced cleavages with ultraviolet photodissociation (UVPD) and complementary fragment ions from collision-induced dissociation (CID) and electron detachment dissociation (EDD). 2DMS, a data-independent analysis technique, allowed for comprehensive coverage and identification of shared fragments across multiple precursor ions. EDD fragmentation efficiency correlated with precursor ion charge states, with higher charges facilitating dissociation due to intramolecular repulsions. An electron energy of 22.8 eV enabled electron capture and radical-based cleavage. Accumulating multiple scans and generating average spectra improved signal intensity, aided by denoising algorithms. Data analysis utilised a custom Python script capable of handling modifications and generating unique mass lists.