Mass Spectrometry Experiments Research Articles

Magnaporthe oryzae infection triggers rice resistance to brown planthopper through the influence of jasmonic acid on the flavonoid biosynthesis pathway.

In agroecosystems, plants are constantly exposed to attack from diverse herbivorous insects and microbes, and infestation with one species may change the plant defense response to other species. In our investigation of the relationships among rice plants, the brown planthopper Nilaparvata lugens (Stål) and the rice blast fungus Magnaporthe oryzae, we observed a significant increase in the resistance of rice treated with rice blast to N.lugens, as evidenced by improved plant survival rates in a small population resistance study. Subsequent transcriptome data analysis revealed that the rice blast fungus can induce the expression of genes in the jasmonic acid (JA) and flavonoid pathways. Similar to the flavonoid pathway, the JA pathway also contains 2 types of genes that exhibit similar and opposite trends in response to N.lugens and rice blast. Among these genes, the osjaz1 mutant and the osmyc2 mutant were phenotypically confirmed to positively and negatively regulate rice resistance to N.lugens and rice blast, respectively. Subsequent mass spectrometry and quantification experiments showed that the exogenous application of methyl jasmonate (MeJA) can induce the accumulation of eriodictyol, naringenin and quercetin, as well as the expression of OsF3H, Os4CL5 and OsCHI in the flavonoid pathway. This suggests a close connection between the JA pathway and the flavonoid pathway. However, OsF3'H, which negatively regulates rice resistance to N.lugens and rice blast, did not show increased expression. Phenotypic and molecular experiments confirmed that OsMYC2 can bind to and inhibit the expression of OsF3'H, thus revealing the mechanism of rice resistance to N.lugens after treatment with rice blast. These findings will deepen our understanding of the interactions among rice, N.lugens and rice blast.

E3 ubiquitin ligase UBR5 modulates circadian rhythm by facilitating the ubiquitination and degradation of the key clock transcription factor BMAL1.

The circadian clock is the inner rhythm of life activities and is controlled by a self-sustained and endogenous molecular clock, which maintains a ~ 24 h internal oscillation. As the core element of the circadian clock, BMAL1 is susceptible to degradation through the ubiquitin-proteasome system (UPS). Nevertheless, scant information is available regarding the UPS enzymes that intricately modulate both the stability and transcriptional activity of BMAL1, affecting the cellular circadian rhythm. In this work, we identify and validate UBR5 as a new E3 ubiquitin ligase that interacts with BMAL1 by using affinity purification, mass spectrometry, and biochemical experiments. UBR5 overexpression induced BMAL1 ubiquitination, leading to diminished stability and reduced protein level of BMAL1, thereby attenuating its transcriptional activity. Consistent with this, UBR5 knockdown increases the BMAL1 protein. Domain mapping discloses that the C-terminus of BMAL1 interacts with the N-terminal domains of UBR5. Similarly, cell-line-based experiments discover that HYD, the UBR5 homolog in Drosophila, could interact with and downregulate CYCLE, the BMAL1 homolog in Drosophila. PER2-luciferase bioluminescence real-time reporting assay in a mammalian cell line and behavioral experiments in Drosophila reveal that UBR5 or hyd knockdown significantly reduces the period of the circadian clock. Therefore, our work discovers a new ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian rhythm and elucidates the underlying molecular mechanism. This work provides an additional layer of complexity to the regulatory network of the circadian clock at the post-translational modification, offering potential insights into the modulation of the dysregulated circadian rhythm.

Probing Glycerolipid Metabolism using a Caged Clickable Glycerol-3-Phosphate Probe.

In this study, we present the probe SATE-G3P-N3 as a novel tool for metabolic labeling of glycerolipids (GLs) to investigate lipid metabolism in yeast cells. By introducing a clickable azide handle onto the glycerol backbone, this probe enables general labeling of glycerolipids. Additionally, this probe contains a caged phosphate moiety at the glycerol sn-3 position to not only facilitate probe uptake by masking negative charge but also to bypass the phosphorylation step crucial for initiating phospholipid synthesis, thereby enhancing phospholipid labeling. The metabolic labeling activity of the probe was thoroughly assessed through cellular fluorescence microscopy, mass spectrometry (MS), and thin-layer chromatography (TLC) experiments. Fluorescence microscopy analysis demonstrated successful incorporation of the probe into yeast cells, with labeling predominantly localized at the plasma membrane. LCMS analysis confirmed metabolic labeling of various phospholipid species (PC, PS, PA, PI, and PG) and neutral lipids (MAG, DAG, and TAG), and GL labeling was corroborated by TLC. These results showcased the potential of the SATE-G3P-N3 probe in studying GL metabolism, offering a versatile and valuable approach to explore the intricate dynamics of lipids in yeast cells.

Imaging with mass spectrometry: Which ionization technique is best?

The use of mass spectrometry (MS) to acquire molecular images of biological tissues and other substrates has developed into an indispensable analytical tool over the past 25 years. Imaging mass spectrometry technologies are widely used today to study the in situ spatial distributions for a variety of analytes. Early MS images were acquired using secondary ion mass spectrometry and matrix-assisted laser desorption/ionization. Researchers have also designed and developed other ionization techniques in recent years to probe surfaces and generate MS images, including desorption electrospray ionization (DESI), nanoDESI, laser ablation electrospray ionization, and infrared matrix-assisted laser desorption electrospray ionization. Investigators now have a plethora of ionization techniques to select from when performing imaging mass spectrometry experiments. This brief perspective will highlight the utility and relative figures of merit of these techniques within the context of their use in imaging mass spectrometry.

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5μM up to 2.5μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Observation of Solvent-Dependence in the Mechanism of Neutral-Catalyzed Isomerization of para-Aminobenzoic Acid Protomers.

Proton-transfer reactions are commonplace during electrospray ionization (ESI) mass spectrometry experiments and are often responsible for imparting charge to analyte molecules. Multiple protonation-site isomers (protomers) can arise for polyfunctional molecules and these isomers can interconvert via solvent-mediated proton transfer reactions during various stages of the ESI process. Studying the populations and interconversion of protonation isomers provides key insight into the ESI process, ion-molecule interactions, and ion dissociation mechanisms. An archetype molecule to study protomer interconversion fundamentals in this context is para-aminobenzoic acid (pABA), where both the amino and carboxylic acid protomers are typically formed under ESI and the mechanisms for interconversion are still under refinement. Using ion-trap mass spectrometry reaction kinetics (2.5 mTorr, 300 K), this study examines gas-phase interconversion catalysis of pABA protomers by seven neutral species, which are commen solvents and additives used for ESI: water, formic acid, methanol, ethanol, propanol, ammonia, and acetonitrile. Three distinct reaction cases are reported: (i) formic acid, methanol, ethanol, propanol, and ammonia each catalyze the interconversion between the amino and carboxylic acid protomers via a n = 1 solvent-molecule vehicle mechanism; (ii) for water, however, a n = 6 adduct complex is detected and this suggests that the observed protomer interconversion occurs through a Grotthuss mechanism, in accord with literature reports; (iii) acetonitrile inhibits proton transfer by the formation of particularly stable n = 1 and 2 adduct complexes. The second-order rate constants for the protomer interconversion are observed to increase in the following order: H2O < HCO2H < MeOH < EtOH < PrOH < NH3. Potential energy schemes are reported for all neutral-catalyzed proton transfer reactions using the DSD-PBEP86-D3(BJ)/aug-cc-pVDZ level of theory. A central transition state, which connects the protonation site adducts, is shown to be the key rate-limiting step. The energy of this transition state is sensitive to the proton affinity of the neutral solvent, and this is supported by the correlation between the reaction rate and the solvent proton affinity.

Two novel fluorescent probes with different fluorescence signals based on phenothiazine for specific detection of ClO− and their applications

Hypochlorite plays an important role in life system, so it is necessary to develop an effective method to detect hypochlorite. In view of the advantages of fluorescent probes at present, we constructed two novel fluorescent probes (PTQ, PTN) based on phenothiazine, which can be used for specific detection of ClO−. Due to the introduction of nitrogen-containing heterocyclic quinazolinone, PTQ shows better optical properties: a great stokes shift (190 nm), excellent selectivity and anti-interference, low detection limit (0.054 μM) and fast response time (20 s). The color change of ClO− identified by the probe PTQ can be observed with naked eyes under sunlight. Through the calculation of density functional theory and mass spectrometry experiments, the mechanism of fluorescence detection of ClO− by PTQ was further verified. Furthermore, PTQ also realized the detection of ClO− in actual water samples and test strips, and achieved remarkable results. Due to its better optical properties and low cytotoxicity, probe PTQ has been successfully applied to the fluorescence detection of ClO− in living cells.

Consecutive Reduction of Five Carbon Dioxide Molecules by Gas-Phase Niobium Carbide Cluster Anions Nb3C4-: Unusual Mechanism for Enhanced Reactivity by the Carbon Ligands.

Studying the cleavage of the C═O bond during CO2 activation at room temperature is highly significant for comprehending the CO2 conversion processes. Herein, mass spectrometry experiments and density functional theory calculations indicate that the niobium carbide anions Nb3C4- can continuously convert five CO2 molecules to CO under thermal collision conditions, while the other clusters with less carbon ligands Nb3C1-3- reduce fewer CO2 molecules. Size-dependent reactivity of Nb3C1-4- cluster anions toward CO2 is observed. Interestingly, the carbon atoms in Nb3C4- not only act as highly active adsorption sites for CO2 but also serve as electron donors to reduce CO2. The stored electrons are released through a carbon-carbon coupling process. Our findings on the role of carbon ligands in enhancing transition metal carbide reactivity can offer new insights for designing active sites on catalysts with both high activity and selectivity.

Pyridine appended pyrimidine bis hydrazone: Zn2+/ATP detection, bioimaging and functional properties of its dinuclear Zn(II) complex

A pyridine functionalized pyrimidine-based system, H2P was successfully synthesized, characterized, and evaluated for its remarkable selective characteristics towards Zn2+ and ATP ions. The chemical sensing capabilities of H2P were demonstrated through absorption, fluorescence, and NMR spectroscopic techniques. The probe exhibited outstanding sensitivity when interacting with the ions, demonstrating relatively strong association constants and impressively low detection limits. The comprehensive binding mechanism of H2P with respect to Zn2+ and ATP ions was investigated using a combination of analytical methods, including Job's plot, NMR spectroscopy, mass spectrometry, and density functional theory (DFT) experiments. The interesting sensing ability of H2P for Zn2+/ATP ions was harnessed for live cell bioimaging and other diverse on-site detection purposes, including paper strips, cotton swabs, and applications involving mung bean sprouts. Further, the fluorescent probe demonstrated its effectiveness in detecting Zn2+ and ATP within live cells, indicating its significant potential in the realm of biological imaging applications. Moreover, the molecular configuration of the zinc complex (H2P–Zn2Cl4), derived from H2P, was elucidated using X-ray crystallography. This complex exhibited intriguing multifunctional attributes, encompassing its capability for detecting picric acid and for reversible acid/base sensing responses. The enhanced conducting behavior of the complex as well as its resistance properties were investigated by performing I–V characteristics and electrochemical impedance spectroscopic (EIS) experiments respectively.

Mitochondrial transcription factor A (TFAM) has 5′-deoxyribose phosphate lyase activity in vitro

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5′-deoxyribose phosphate (5′dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase β, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5′dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5′dRp residues at a rate comparable to that of DNA polymerase (pol) β, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

Identification and characterization of impurities in an insecticide, commercial chlorantraniliprole using liquid chromatography-tandem mass spectrometry.

Ensuring the global safety and effectiveness of agrochemicals has become imperative. An in-depth understanding of impurity profiles of products is crucial, especially for high-demand agrochemicals, where impurities may be more toxic and persistent than original agrochemicals. This study focuses on the detection and identification of impurities in a commercial chlorantraniliprole (CAP), an anthranilic diamide class broad-spectrum insecticide. Commercial CAP was collected from an agrochemical supplier in India and was analyzed using a high-performance liquid chromatography-photodiode array (HPLC-PDA) (Agilent 1260; wavelength, 220 nm) with a Zorbax RP SB-C18 (250 × 4.6mm, 5μm) column and liquid chromatography-mass spectrometry (LC-MS) (Agilent 6545 quadrupole time of flight (Q-TOF)) techniques to identify the impurities. The impurities were isolated by preparative HPLC using a Zorbax-DB C18 (250 × 9.4mm, 5μm) column. liquid chromatography- tandem mass spectrometry (LC-MS/MS) experiments (Q-TOF) were performed on CAP and its impurities to obtain their structural data. HPLC-PDA analysis of CAP showed four major impurities (IM-1 to IM-4) ranging from 0.76% to 4.1%. The positive ion electrospray ionization (ESI) mass spectra of CAP and its impurities showed dominant [M + H]+ ions in addition to [M + Na]+ , [M + K]+ , and [2M + Na]+ ions. High-resolution mass spectrometry (HRMS) data provided the elemental composition of the compounds, and isotopic distribution patterns revealed the number of Cl and/or Br atoms present in them. The structures of impurities were proposed based on the LC-MS/MS) data and further confirmed by nuclear magnetic resonance (NMR) data on isolated impurities/synthesis. The quality and impurities of CAP, a popular insecticide, must be assessed and described for its efficacy and safety. In this study, four impurities of CAP were detected using HPLC and successfully characterized using LC-HRMS, LC-MS/MS, and NMR data. The method is useful for verifying the purity of CAP as well as helping in the identification of its possible impurities.

Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp/protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

Gas-phase F-atom migration reaction of deprotonated N-fluoroarenesulfonamides.

Recently N-Fluoroarenesulfonamides (ArSO2 NHF) were found to be promising precursors for the preparation of N-fluorobenzenesulfonimide derivatives without applying F2 . However, very few studies have discussed the mass spectrometric behaviors of ArSO2 NHF with N-F structure. In this article, we applied high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) to study the effect on the mass spectrometric behaviors of ArSO2 NHF after the introduction of the F-atom to the N-atom of ArSO2 NH2 . High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) experiments showed that ArSO2 NHF produced only good signals in negative ion mode, and the dominating product ion SO2 F- at m/z 83 was observed in all HR-ESI-MS/MS of ArSO2 NF- with different substituents in the Ar group. The formation of the product ion SO2 F- was proof of the gas-phase F-atom migration rearrangement from the N-atom to the S-atom in ESI-MS/MS of ArSO2 NF- . To fully explain the gas-phase reaction mechanism from ArSO2 NF- to SO2 F- , we studied the HR-ESI-MS/MS of deprotonated ArSO2 NHF and also performed theoretical calculations. Both results confirmed that ArSO2 NF- first underwent Smiles rearrangement to yield intermediate I (INT1) ArNFSO2 - , and then the F-atom of ArNFSO2 - migrated from the N-atom to the S-atom to form intermediate II (INT2) ArN- SO2 F, which finally dissociated to SO2 F- at m/z 83 with loss of a neutral nitrene (ArN). All these results showed that the formation of the product ion SO2 F- from ArSO2 NF- was a common and intrinsic gas-phase reactivity of ArSO2 NF- .

The Effect of an Er:YAG Laser Combined with a 755 nm Picosecond Laser on Tattoo Removal in a Rat Model

Laser tattoo removal is an effective technique, but multiple rounds of treatment are required to eliminate tattoos, which can be a challenge for patients who do not have the time. Additionally, there is relatively limited research on the mechanisms of laser tattoo removal, necessitating further in-depth studies. This study aimed to investigate the efficacy of an Er:YAG laser combined with a 755-picosecond laser for tattoo removal and the changes in skin diffuse reflection, pigment particle size, macrophage recruitment, and inflammatory factor levels during combined laser treatment. Our experiments used three sets of lasers—an Er:YAG laser, a 755 nm picosecond laser, and an Er:YAG laser combined with a 755 nm picosecond laser—for tattoo removal. The effects of tattoo removal at different time points were evaluated. Moreover, the mechanisms were explored using HE staining, immunohistochemistry, diffuse reflectance measurements, in vitro gel tests, mass spectrometry, and ELISA experiments. The combined treatment was more effective for tattoo removal in rats. The combined laser treatment effectively reduced diffuse reflection from the skin, thereby increasing the effective laser power, reducing the size of the pigment particles, allowing for easier removal of the pigment, and increasing the recruitment of dermal macrophages and the release of inflammatory factors, thus increasing the rate of tattoo removal in vivo.

Gas‐phase Smiles rearrangement of ortho‐nitro‐substituted diaryliodonium cations and their mechanistic insights

RationaleDiaryliodonium salts are useful electrophilic reagents in organic chemistry, finding extensive applications in arylations and photo‐induced polymerizations. However, comprehensive mechanistic investigations, particularly concerning the mass spectrometric behaviors of diaryliodonium salts, are relatively scarce in the literature.MethodsDiaryliodonium salts could be readily ionized using electrospray ionization mass spectrometry (ESI‐MS) to give [Ar1‐I+‐Ar2], and high‐resolution ESI‐tandem mass spectrometry (MS/MS) experiments were conducted to investigate their gas‐phase chemical reactions.ResultsInvestigations on ESI‐MS/MS of [Ar1‐I+‐Ar2] revealed two major fragmentation patterns: (1) reductive elimination resulting in the diaryl coupling product ion [Ar1‐Ar2]+• by the loss of I; (2) generation of aryl cations [Ar1]+ or [Ar2]+ through cleavage of the C–I bonds. We revealed that the introduction of NO2 into Ar2 of [Ar1‐I+‐Ar2] could lead to an unexpected fragmentation ion [Ar1O]+ in MS/MS, arising from an O‐atom transfer process from NO2 to Ar1. Particularly, when NO2 was ortho‐positioned to the iodine in Ar2, the [Ar1O]+ sometimes exhibited dominant behavior.ConclusionsComprehensive ESI‐MS/MS studies and theoretical calculations provided strong support for the O‐atom transfer mechanistic pathway: [Ar1‐I+‐(o‐NO2‐Ar2)] initially underwent a Smiles rearrangement to the intermediate [Ar1‐O‐(o‐NO‐Ar2I)]+, which subsequently dissociated to [Ar1O]+ or [o‐NO‐Ar2I]+•. Herein, we proposed an unexpected “ortho effect” in the gas‐phase fragmentation reaction of [Ar1‐I+‐(o‐NO2‐Ar2)], in which the crucial determinant factor for the aryl migration was identified as the Smiles rearrangement reaction.

AutonoMS: Automated Ion Mobility Metabolomic Fingerprinting.

Automation is dramatically changing the nature of laboratory life science. Robotic lab hardware that can perform manual operations with greater speed, endurance, and reproducibility opens an avenue for faster scientific discovery with less time spent on laborious repetitive tasks. A major bottleneck remains in integrating cutting-edge laboratory equipment into automated workflows, notably specialized analytical equipment, which is designed for human usage. Here we present AutonoMS, a platform for automatically running, processing, and analyzing high-throughput mass spectrometry experiments. AutonoMS is currently written around an ion mobility mass spectrometry (IM-MS) platform and can be adapted to additional analytical instruments and data processing flows. AutonoMS enables automated software agent-controlled end-to-end measurement and analysis runs from experimental specification files that can be produced by human users or upstream software processes. We demonstrate the use and abilities of AutonoMS in a high-throughput flow-injection ion mobility configuration with 5 s sample analysis time, processing robotically prepared chemical standards and cultured yeast samples in targeted and untargeted metabolomics applications. The platform exhibited consistency, reliability, and ease of use while eliminating the need for human intervention in the process of sample injection, data processing, and analysis. The platform paves the way toward a more fully automated mass spectrometry analysis and ultimately closed-loop laboratory workflows involving automated experimentation and analysis coupled to AI-driven experimentation utilizing cutting-edge analytical instrumentation. AutonoMS documentation is available at https://autonoms.readthedocs.io.

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 &amp; Sons Ltd on behalf of Society of Industrial Chemistry.