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1. Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

   https://doi.org/10.1016/j.mcpro.2024.100814  

   Mikawy, Neven N; Rojas_Ramírez, Carolina; DeFiglia, Steven A; Szot, Carson W; Le, Jessie; Lantz, Carter; Wei, Benqian; Zenaidee, Muhammad A; Blakney, Greg T; Nesvizhskii, Alexey I; et al (September 2024, Molecular & Cellular Proteomics)

   Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions. 

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2. Analysis of plant metabolomics data using identification‐free approaches

   https://doi.org/10.1002/aps3.70001  

   Yuan, Xinyu; Smith, Nathaniel_S S; Moghe, Gaurav D (March 2025, Applications in Plant Sciences)

   Abstract Plant metabolomes are structurally diverse. One of the most popular techniques for sampling this diversity is liquid chromatography–mass spectrometry (LC‐MS), which typically detects thousands of peaks from single organ extracts, many representing true metabolites. These peaks are usually annotated using in‐house retention time or spectral libraries, in silico fragmentation libraries, and increasingly through computational techniques such as machine learning. Despite these advances, over 85% of LC‐MS peaks remain unidentified, posing a major challenge for data analysis and biological interpretation. This bottleneck limits our ability to fully understand the diversity, functions, and evolution of plant metabolites. In this review, we first summarize current approaches for metabolite identification, highlighting their challenges and limitations. We further focus on alternative strategies that bypass the need for metabolite identification, allowing researchers to interpret global metabolic patterns and pinpoint key metabolite signals. These methods include molecular networking, distance‐based approaches, information theory–based metrics, and discriminant analysis. Additionally, we explore their practical applications in plant science and highlight a set of useful tools to support researchers in analyzing complex plant metabolomics data. By adopting these approaches, researchers can enhance their ability to uncover new insights into plant metabolism. 

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3. Metabolomics of bacterial–fungal pairwise interactions reveal conserved molecular mechanisms

   https://doi.org/10.1039/D3AN00408B  

   Luu, Gordon T.; Little, Jessica C.; Pierce, Emily C.; Morin, Manon; Ertekin, Celine A.; Wolfe, Benjamin E.; Baars, Oliver; Dutton, Rachel J.; Sanchez, Laura M. (June 2023, The Analyst)

   Bacterial–fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial–fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles ( i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority. 

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4. microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data

   https://doi.org/10.1038/s41564-023-01575-9  

   Zuffa, Simone; Schmid, Robin; Bauermeister, Anelize; P. Gomes, Paulo Wender; Caraballo-Rodriguez, Andres M.; El Abiead, Yasin; Aron, Allegra T.; Gentry, Emily C.; Zemlin, Jasmine; Meehan, Michael J.; et al (February 2024, Nature Microbiology)

   Abstract microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms’ role in ecology and human health. 

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5. Mass Spectrometry Imaging of Diclofenac and Its Metabolites in Tissues Using Nanospray Desorption Electrospray Ionization

   https://doi.org/10.26434/chemrxiv.13194422  

   Brown, H.; Mesa Sanchez, D.; Yin, R.; Chen, B.; Vavrek, M.; Cancilla, M.; Zhong, W.; Shyong, B.; Zhang, R.; Li, F.; et al (May 2020, ChemRxiv)

   null (Ed.)

   Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns of toxicity as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, little is known about their localization in tissues. Herein, we used nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI-MSI) to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. Nano-DESI allows for label-free imaging with high spatial resolution and sensitivity without special sample pretreatment. Using nano-DESI-MSI, ion images for diclofenac and its major metabolites were produced. MSI data acquired over a broad m/z range showed fairly low signals of the drug and its metabolites. At least an order of magnitude improvement in the signals was obtained using selected ion monitoring (SIM), with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide is localized to the inner medulla and hydroxydiclofenac to the cortex of the kidney. The distributions observed for both metabolites closely match the previously reported localization of enzymes that process diclofenac into its respective metabolites. The localization of diclofenac acyl glucuronide to medulla likely indicates that the toxic metabolite is being excreted from the tissue. In contrast, a uniform distribution of diclofenac, hydroxydiclofenac and the diclofenac acyl glucuronide metabolite was observed in the liver tissue. Semiquantitative analysis found the metabolite to diclofenac ratios calculated from nano-DESI in agreement to those calculated from liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments. Collectively, our results demonstrate nano-DESI-MSI can be successfully used to image diclofenac and its primary metabolites in dosed liver and kidney tissues from mice and derive semi-quantitative data from localized tissue regions. 

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