Capillary zone electrophoresis (CZE)-mass spectrometry (MS) has been proposed as a powerful analytical tool for bottom-up, top-down, and native proteomics (multi-level proteomics) decades ago to analyze complex biological samples at the levels of peptides (bottom-up), proteoforms (top-down), and complexoforms (native). However, its broad adoption has been impeded by the limited robustness and reproducibility. Here, we present multi-level proteomics data from nearly 170 CZE-MS runs (~170 hours of instrument time), demonstrating qualitatively (i.e., the number of identified peptides and proteoforms, the number of detected complexoforms, and their migration time) and quantitatively (i.e., peptide, proteoform, and complexoform intensity) reproducible measurement of complex samples with varying levels of complexity, i.e., Escherichia coli cells, HeLa cells, and human plasma. CZE-MS-based native proteomics enabled the detection of hundreds of complexoforms up to 800 kDa from the complex systems via consuming only nanograms of protein material. The results indicate that CZE-MS is sensitive and reproducible enough for broad adoption for multi-level proteomics-based biomedical research.

Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid resolved isotope analysis in a proteomics compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single cell proteomics style sample preparation. Using human HeLa cells cultured in 13C-glucose enriched and control media, we show that reliable relative delta 13C measurements can be obtained from as few as 50 cells, corresponding to

A recent genome mining study identified class II lanthipeptides encoded in Nostoc punctiforme PCC73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we heterologously produced the nostolysamides by co-expression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in Escherichia coli. We structurally characterized the NpuA-derived product and established the position of the thioether crosslinks. All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfey's analysis. Tandem mass spectrometry as well as mutagenesis studies indicate an N-terminal non-overlapping methyllanthionine ring and three overlapping rings at the C-terminus for which the most likely ring pattern is proposed. After removal of the leader peptide, the resulting lanthipeptide exhibits antifungal activity against Candida species as well as antimicrobial activity against gram positive bacteria by disrupting cell membranes. The antibacterial activity is shown not to involve lipid II, consistent with the observed antifungal activity because fungi do not contain this bacterial cell wall precursor. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long chain acyl groups to the side chain of a Lys residue in position 1 of the precursor peptide. In vitro studies of NpuN shows relatively broad substrate specificity with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide showing that it is not required for these activities.

Base excision repair (BER) is the primary pathway that removes oxidatively-induced DNA base damage from the nuclear and mitochondrial genomes, with 8-oxoguanine DNA glycosylase (OGG1) initiating repair at the two most frequently-formed base lesions: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoGua) and 2,6-diamino-4-oxo-5-formamidopyrimidine (FapyGua). Humans expressing a catalytically-compromised variant of OGG1 (S326C) are at increased risk for type 2 diabetes, Alzheimers disease, and Parkinsons disease. To potentially enhance the overall catalytic efficiency of this variant, a prior medicinal chemistry screen discovered seven chemically distinct agonists of OGG1 that stimulated activity in vitro and attenuated a paraquat (PQ) challenge in cultured cells. Herein, we developed structure-activity relationships around one specific core structure, F01. Using fluorescence-based DNA cleavage assays, we assessed the abilities of these compounds to stimulate the overall rate of OGG1 catalysis. Multiple compounds were identified that increased OGG1 activity on DNAs containing a site-specific 8-oxoGua by 2-fold or greater, with 9 compounds showing EC50 concentrations lower than F01 and were specific for OGG1. Selected agonists were shown to enhance OGG1-catalyzed release of 8-oxoGua and FapyGua from {gamma}-irradiated high-molecular-weight DNA using gas chromatography tandem mass spectrometry analyses. Since these assays did not reveal which step in the overall reaction was stimulated, we used a separation of function OGG1 mutant that possessed glycosylase, but not abasic-site (AP) lyase activity to demonstrate that the glycosylase step was not enhanced. In contrast, all agonists stimulated the AP lyase activity to levels equal to or greater than the magnitude of stimulation observed for overall chemistry, implicating agonist-mediated turnover as a potential contributor to the overall rate stimulation. The biological activities of selected agonists were evaluated in OGG1-deficient Kasumi-1 cells under conditions of paraquat (PQ)-induced oxidative stress, with several compounds mitigating PQ challenge.

Protein function is governed by more than abundance alone. Here, the authors introduce a supercharging-enhanced nDIA-MS workflow to map drug-induced solubility changes across the proteome, enabling high-throughput analysis of protein state transitions.

The growing demand for rare earth elements (REEs) underscores the need for sustainable recovery methods and a reliable supply. Here the authors establish a platform using a yeast to produce bio-oxalic acid for REE recovery, achieving 99% REE recovery from low-grade ore.

Amyloid-beta (Aβ) oligomers are key contributors to the pathology and progression of Alzheimer’s disease (AD), making their characterization essential for understanding aggregation processes and developing potential therapeutic strategies. This study provides a systematic framework for analyzing Aβ(1–42) oligomers in vitro using cyclic ion mobility-mass spectrometry (cIMS). Compared to previous generations of traveling wave ion mobility (TWIM) devices, the cIMS platform offers superior resolution through its scalable ion mobility path length. However, the multistage character of the cIMS platform requires thorough investigation of parameters affecting oligomer transmission and activation to ensure reliable analysis of labile and dynamic systems such as Aβ oligomers. Our findings highlight the critical influence of cone voltage (CV) on in-source ion activation, subsequent structural changes, and oligomer detection. By balancing CV, we achieved detection of a broad range of oligomeric species while limiting their activation and maximizing signal intensity. Moreover, we present the first comprehensive set of optimized ion optics and ion mobility parameters that enable effective transmission and separation of oligomer Aβ(1–42) ions. Using the optimized method, we successfully detected a spectrum of Aβ(1–42) oligomers ranging from dimers to dodecamers. Additionally, the method was applied in collision-induced unfolding experiments, revealing size-dependent conformational transitions proving its applicability. This optimized cIMS methodology establishes a foundation for future studies on Aβ(1–42) aggregation mechanisms, AD pathogenesis, and therapeutic applications. Furthermore, our results offer valuable insights into cIMS instrument tuning, with potential applications in the analysis of other complex biological systems. Graphical Abstract

A one-pot enzymatic platform enables the biosynthesis and site-specific incorporation of chalcogen-containing tyrosine analogues (O, S, Se) into proteins in E. coli. Engineered tyrosine phenol lyase (TPL) variants and orthogonal synthetases are combined to expand the genetic code with redox-active residues, paving the way for designer proteins with tunable electronic and catalytic properties. ABSTRACT Expanding the genetic code with unnatural amino acids (UAAs) offers powerful opportunities to engineer proteins with novel redox and catalytic functions, but is often limited by the need for multistep UAA synthesis and inefficient cellular uptake. Here, we report an integrated biosynthetic–genetic incorporation strategy for chalcogen-containing proteins from the respective phenols. Structure-guided engineering of tyrosine phenol lyase (TPL) enabled the enzymatic production of 3-methoxy-, 3-methylthio-, and 3-methylseleno-L-tyrosine (MeSeY) directly in living cells. Using evolved orthogonal aminoacyl-tRNA synthetases, these analogues were site-specifically incorporated into green fluorescent protein (GFP), as confirmed by fluorescence assays, spectroscopy, and mass spectrometry. We further established a one-pot in vivo system that unifies analogue biosynthesis with translation, reducing precursor requirements and cellular toxicity. This work introduces selenium as a genetically encoded handle for protein engineering and establishes a scalable strategy that couples biocatalysis with genetic code expansion to access redox-active designer proteins. Importantly, installation of MeSeY at the GFP chromophore residue Tyr66 provides redox-responsive fluorescence. In a circularly permuted GFP (cpGFP) scaffold, improved chromophore accessibility enables reversible redox switching under H2O2/thiol cycling.

Stressful events are a leading factor in development of depression. The medial prefrontal cortex (mPFC) is strongly associated with depression etiology and exposure to uncontrollable stressors results in synaptic dysfunction and loss. Learned helplessness is a behavioral paradigm that measures effects of repeated exposure to uncontrollable, inescapable stress on later responses to escapable stress. We therefore performed a proteomic analysis of mPFC synaptosomes in a mouse learned helplessness model to identify molecular changes that could contribute to functional consequences of inescapable stress. Male and female mice were evaluated at baseline and following exposure to escapable or inescapable stress followed by an active avoidance test. Label-free mass spectrometry followed by pathway and protein-protein interaction network analyses identified alterations in signaling pathways involved in energy metabolism, neurotransmitter signaling, and protein shuttling. Furthermore, phosphoproteomics revealed alterations related to synaptic function, neurotransmitter signaling and protein internalization, as well as changes in activity of kinases previously identified as mediators of antidepressant efficacy (GSK3B) and receptor internalization (ADRBK1). We more deeply examined alterations in the Acetylcholine Receptor Signaling Pathway, and identified muscarinic receptor proteins (Chrm1, Chrm2, Chrm4) and key proteins involved in their translocation to and from the membrane. These results identify substantial changes in the mPFC proteome following exposure to inescapable stressors. In addition, mPFC muscarinic cholinergic signaling is well placed to mediate responses to an inescapable stressor. This proteomic study will be useful in guiding studies of human mPFC relevant to depression. Data are available via ProteomeXchange with identifier PXD073765.

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction, although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co-immunoprecipitation followed by phosphopeptide enrichment and high-resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co-immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insights into the posttranscriptional control of gene expression.

Reactive oxygen species (ROS) are central regulators of plant growth and stress responses. Cellular ROS levels are tightly controlled by antioxidant systems, including the evolutionarily conserved catalases that detoxify hydrogen peroxide (H2O2) predominantly within peroxisomes. Despite their importance, substantial gaps remain in our understanding of catalase biogenesis, regulation, subcellular targeting, and potential extra-peroxisomal functions. Using affinity purification of the UV-B photoreceptor UVR8 coupled with mass spectrometry, we identified a REGULATOR OF CHROMATIN CONDENSATION 1--like protein in Arabidopsis, which we named CATALASE-INTERACTING RCC1-LIKE 1 (CAIR1). CAIR1 interacts with all three catalase isoforms (CAT1--CAT3) as well as their chaperone NO CATALASE ACTIVITY 1 (NCA1). Loss-of-function cair1 mutants partially phenocopy cat2 and nca1, with reduced catalase activity, enhanced sensitivity to oxidative stress and alkaline growth conditions, and impaired primary root elongation. Mechanistically, cytosolic interaction between CAIR1 and CAT2 enhances total cellular catalase activity by facilitating peroxisomal import and proper subcellular localization of CAT2. In the absence of CAIR1, CAT2 forms aggregates, likely accounting for the observed loss of catalase activity. Notably, CAIR1 undergoes reversible, redox-dependent oligomerization that enhances its interaction with catalases. Mutation of CAIR1 at Cys-356 and Cys-545 compromises this interaction under elevated ROS conditions and fails to rescue the oxidative stress sensitivity of cair1 mutants. Moreover, UV-B exposure suppresses catalase activity by weakening the interaction between CAIR1 and catalases, thus linking environmental light signalling to cellular redox regulation. Together, our findings reveal CAIR1 as a dynamic redox-responsive regulator of catalase activity that maintains cellular redox homeostasis by coordinating catalase localization and function through reversible oligomerization.

Publication date: Available online 29 January 2026 Source: Journal of Chromatography B Author(s): Christof Manuel Schönenberger, Aurelie Berthet, Matthias Briel, Alain Amstutz, Matthias Cavassini, Loïc Sartori, Camille Rime, Davide Staedler, Fiorella Lucarini

Chemistry – A European Journal, EarlyView.

Journal of Mass Spectrometry, Volume 61, Issue 2, February 2026.

Journal of Mass Spectrometry, Volume 61, Issue 2, February 2026.

Polygenic scores (PGS) summarize the combined effects of common single-nucleotide polymorphisms and contribute to predictions of disease severity, but biological consequences linked to these common variants remain poorly defined. Here, we focused on polygenic liability for a measurable electrophysiologic trait (the QT interval). Prolonged QT interval, measured on patient electrocardiograms, is associated with an increased risk for cardiac arrhythmia. We investigated human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from donors with extreme PGS (i.e., high and low) related to QT interval duration. We paired global proteomics with multiplexed affinity purification mass spectrometry (AP-MS) centered on Kv11.1 (hERG), a major determinant of QT-interval repolarization. Global proteomics indicated increased mitochondrial protein abundance in high-PGS cardiomyocytes, but this did not explain the Kv11.1 interactome. In high-PGS cells, Kv11.1 showed increased associations with myosin motor proteins and endosomal recycling machinery, consistent with altered (and potentially increased) recycling/trafficking dynamics rather than trafficking deficiency observed with most pathogenic Kv11.1 variants. This proof-of-concept study underscores a framework for linking polygenic factors to tractable biological consequences by combining patient-specific hiPSCs, proteomics and affinity-purification. Linking polygenic scores to changes in protein networks provides testable mechanisms that can be applied across many diseases.

Analytical ChemistryDOI: 10.1021/acs.analchem.5c04150

Analytical ChemistryDOI: 10.1021/acs.analchem.5c05700

Loss-of-function mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive subtype of metastatic castration-resistant prostate cancer (mCRPC) characterized by genomic instability, and depletion of CDK12 activity downregulates the expression of long DNA repair genes. While these transcriptional defects are well-documented, potential alterations in chromatin that may topologically affect this silencing and the subsequent adaptive survival mechanisms of tumor cells remain poorly understood. Here, we employ a dynamic multi-omics strategy to map the remodeling of the RNA Polymerase II (RNA Pol II) complex and chromatin landscape. We utilized 5-ethynyl uridine (5-EU) metabolic labeling combined with mass spectrometry (MS) to specifically capture the transcriptionally active RNA Pol II complex, distinguishing active elongation complexes from static background, and map how its interactome changes when the activity of the major transcriptional kinases CDK7, CDK9 or CDK12/13 is inhibited. These data are then integrated with ATAC-seq, transcriptomics, and targeted synthetic lethality screen to validate findings from the RNA Pol II capture. We show that, unlike CDK9 inhibition, CDK12/13 inhibition triggers a progressive chromatin compaction. This is characterized by the decrease in the binding activity of CTCF to chromosomes closing, which acts as a physical blockade specifically silencing long genes when CDK12 activity is lost. In response to this topological remodeling, cells launch an adaptive survival program by recruiting the transcription elongation factor PAF1 and the DNA damage sensor DDB1 to the stalled RNA Pol II. We identify PAF1 as a critical vulnerability in this context. Collectively, we identify CDK12 as a regulator of chromatin topology rather than merely an elongation kinase. The chromatin compaction upon CDK12/13 inhibition creates a unique dependency on the PAF1-mediated adaptive remodeling. Specifically, targeting PAF1 converts this stress response into a lethal trap, establishing a potent, kinase-selective combination lethal strategy for CDK12-deficient prostate cancer.

Environmental Science & TechnologyDOI: 10.1021/acs.est.5c08824

Microbial metabolites play a critical role in regulating ecosystems, including the human body and its microbiota. However, understanding the physiologically relevant role of these molecules, especially through liquid chromatography tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics, poses significant challenges and often requires manual parsing of a large amount of literature, databases, and webpages. To address this gap, we established the Collaborative Microbial Metabolite Center knowledgebase (CMMC-KB), a platform that fosters collaborative efforts within the scientific community to curate knowledge about microbial metabolites. The CMMC-KB aims to collect comprehensive information about microbial molecules originating from microbial biosynthesis, drug metabolism, exposure-related molecules, food, host-derived molecules, and, whenever available, their known activities. Molecules from other sources, including host-produced, dietary, and pharmaceutical compounds, are also included. By enabling direct integration of this knowledgebase with downstream analytical tools, including molecular networking, we can deepen insights into microbiota and their metabolites, ultimately advancing our understanding of microbial ecosystems.

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00395

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00356

ABSTRACT Nitrooxidative stress, driven by excess reactive nitrogen species like peroxynitrite, contributes to the pathogenesis of many chronic diseases. Among its molecular footprints, 3-nitrotyrosine (3NT) has emerged as a biologically relevant marker of protein nitration. Its accumulation reflects oxidative damage and altered protein function, positioning it as a promising biomarker. Proteomics has advanced our understanding of nitrooxidative stress and its clinical implications. The integration of high-resolution MS with immunoaffinity and structural modeling enables precise mapping of nitration sites and functional interpretation. However, limitations such as low stoichiometry, ion suppression, and antibody cross-reactivity still constrain the field. Emerging computational predictors and miniaturized platforms offer promising avenues for expanding the clinical utility of 3NT. Future efforts should focus on standardizing workflows, validating site-specific modifications, and translating proteomic insights into diagnostic and therapeutic strategies. This review outlines the biochemical mechanisms of 3NT formation, emphasizing peroxynitrite-dependent and heme peroxidase-mediated pathways. Proteomic strategies for detecting and quantifying nitrated proteins are discussed, including mass spectrometry workflows, enrichment techniques, and immunodetection. Challenges in site-specific identification, antibody specificity, and ionization-induced fragmentation are addressed. Disease-specific patterns of 3NT accumulation in neurodegenerative, cardiovascular, and oncologic contexts are highlighted, along with in silico prediction of nitration sites. Despite significant methodological advances, key limitations such as low nitration stoichiometry, antibody cross-reactivity, and ionization-dependent artifacts continue to challenge confident site-specific analysis of 3-nitrotyrosine. Future progress will depend on improved enrichment strategies, standardized mass spectrometry workflows, and the integration of computational prediction tools with experimental validation. Addressing these gaps will be essential for translating nitrotyrosine profiling into robust mechanistic and clinical applications.

ABSTRACT Modified Xihuang Pills (MXP), a widely used traditional Chinese patent medicine in China, has been clinically applied for treating inflammatory and infectious diseases. However, there are only a few reports on its chemical components at present, which hinders its further research, development, and application. In this study, an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed firstly to investigate the major components in MXP. A total of 105 compounds were identified or characterized, among which 12 compounds were clearly confirmed by comparing the retention time and mass spectrometry data with those of reference standards. Based on the retention time, mass spectrometry data, and relevant literature information, the remaining 93 compounds were preliminarily characterized. Results showed that bile acids, bufadienolides, and terpenoids were the primary chemical components of MXP. The established analytical method has been proven to effectively characterize the chemical constituents of MXP and also provides an important reference for further in vivo pharmacokinetics and drug metabolism research on MXP.