Publication date: Available online 10 November 2025 Source: Journal of Proteomics Author(s): Xcanda Ixchel Herrera Lopez, Karina Martinez-Perez, Sanjukta Guha Thakurta, Benjamin Levi, Joao A. Paulo

ABSTRACT The total flavonoid extract of Lithocarpus litseifolius (Hance) Chun (TFLLC), a medicinal and edible plant of the Fagaceae family, has been developed as a preclinical drug candidate in China due to its hypoglycemic effect. The metabolism of TFLLC is closely associated with both its pharmacological activity and potential toxicity. This study investigated the in vitro and in vivo metabolism of TFLLC, which mainly comprises phlorizin and trilobatin. Ultrahigh-performance liquid chromatography coupled with UV spectrometry and high-resolution mass spectrometry (UHPLC–UV–HRMS) was used to detect and characterize TFLLC metabolites in rats following oral administration at 50 mg/kg. TFLLC underwent extensive metabolic transformation, and 13 metabolites were identified. The major drug-related substances included phloretin—derived from glycosidic cleavage of phlorizin and trilobatin—and its conjugated derivatives, such as glucuronidated metabolite M450, sulfated metabolite M354, and dual-conjugated metabolite M530. These metabolites were primarily excreted in bile and urine. Intestinal incubation studies confirmed that phloretin formation could occur in the gut. These findings provide a pharmacokinetic basis to support the pharmacodynamic interpretation and future drug development of TFLLC.

ABSTRACT Tegoprazan (TEG) is a commonly used drug in the treatment of gastroesophageal reflux disease. This work aimed to develop and validate an ultra-high-performance liquid chromatography–tandem mass Spectrometry (UPLC–MS/MS) method to determine the levels of TEG in rat plasma and to apply it for a rat pharmacokinetic study. Electrospray ionization source (ESI) positive and multiple reaction monitoring (MRM) mode were selected. The internal standard revaprazan (REV) and TEG were analyzed separately on a Waters ACQUITY UPLC BEH column. Gradient elution with a flow rate of 0.5 mL/min was used. Accuracy and precision were from −8.5% to 12.2%. Linearity was from 2 to 1000 ng/mL. IS-normalized recovery ranged from 109.3% to 113.6%. IS-normalized matrix effect was from 99.0% to 102.8%. The coefficient of variation of matrix effect was less than 10%. Dilution integrity showed that a tenfold dilution also met the guidelines. In the rat pharmacokinetic study, AUC (0 − ∞) and C max of TEG were 9129.6 ± 1823.3 μg/L*h and 2513.2 ± 707.0 ng/mL, respectively. T max and T 1/2 were 1.8 ± 1.1 h and 1.6 ± 0.8 h, respectively. We finally established a rapid and robust UPLC–MS/MS quantitative analysis of TEG in rat plasma. Rat pharmacokinetics indicated that TEG was absorbed quickly and fast reached the maximum concentration.

PROTEOMICS, EarlyView.

PROTEOMICS, EarlyView.

Hydrogen-Deuterium Exchange Mass-Spectrometry (HDX-MS) is a powerful method to study protein conformational dynamics, but peptide-level measurements obscure residue-level detail. The authors present ReX, a statistical approach that infers residue-level significance, outperforms existing methods on unseen data, and reveals distinct conformational signatures of ligand binding.

Sporadic Alzheimer's disease (sAD) lacks effective preventive therapies, underscoring the need to target pathogenic drivers. Aberrant calcium signaling is an established early event in sAD pathogenesis that is closely linked to neuroinflammation. Aged rhesus macaques are predominantly APOE-{epsilon}4 homozygotes and naturally exhibit cognitive decline, calcium dysregulation, amyloid deposition, and tau pathology, which allows for a translationally relevant animal model. We previously identified an evolutionarily expanded role for postsynaptic type 3 metabotropic glutamate receptors (mGluR3) in dorsolateral prefrontal and entorhinal cortex, where they regulate cAMP-calcium opening of K+ channels to sustain neuronal firing and working memory. mGluR3 signaling is driven by N-acetylaspartylglutamate (NAAG) and constrained by glutamate carboxypeptidase II (GCPII), whose expression rises with age and inflammation. In prior work, chronic inhibition of GCPII with the orally bioavailable inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) improved neuronal firing, working memory, and reduced pT217Tau pathology in aged macaques. Here, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to define the proteomic consequences of chronic 2-MPPA treatment in vulnerable (entorhinal cortex, dorsolateral prefrontal cortex) versus resilient (primary visual cortex) regions. We identified >2,400 proteins across experimental conditions, and label-free quantification revealed region-specific differential expression patterns paralleling known vulnerability gradients in sAD. Gene ontology enrichment of vulnerable regions implicated pathways governing protein deneddylation, amyloid and tau-associated processes, synaptic plasticity, mitochondrial homeostasis, and oxidative stress, revealing putative targets for therapeutic intervention in sAD. These findings demonstrate that GCPII inhibition engages distinct, region-selective molecular programs in the aging primate cortex, consistent with the protection of circuits most vulnerable to sAD. By mapping the proteomic shifts that occur with treatment, we reveal molecular signatures that not only serve as candidate biomarkers but also highlight novel mechanistic pathways contributing to calcium-driven degeneration in sAD. As such, more focused investigations into these pathways of therapeutic interest are warranted, in addition to the analysis of key post-translational modifications and their potential roles in sAD.

Publication date: Available online 9 November 2025 Source: Journal of Mass Spectrometry and Advances in the Clinical Lab Author(s): Chuangye Cai, Mengxue Zou, Mingxiao Chen, Peibo Yuan, Zhencheng Fang, Lanlan Zhong, Dingqiang Chen, Hongwei Zhou, Nianyi Zeng

ABSTRACT Triazole antifungals are clinically established agents for the prophylaxis and treatment of invasive fungal diseases in patients with hematologic malignancies. To address the requirements of clinical therapeutic drug monitoring, we developed and validated a novel ultra-high-performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of three triazole antifungals and their metabolites in human plasma: itraconazole (ICZ), hydroxy-itraconazole (ICZ-OH), voriconazole (VCZ), voriconazole N-oxide (VCZ N-oxide), and posaconazole. The five target analytes were successfully separated using a BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C, with gradient elution employing mobile phase A (5.0 mM ammonium acetate in water containing 0.1% formic acid) and mobile phase B (100% acetonitrile [ACN]) at a constant flow rate of 0.4 mL/min. A positive ion pattern was chosen for quantification under multiple reaction monitoring. Following the addition of 10 µL of internal standard, 50 µL of plasma underwent protein precipitation with ACN, and the resulting supernatant was diluted for subsequent analysis. Method validation followed Food and Drug Administration guidelines and Chinese Pharmacopoeia regulations, demonstrating acceptable accuracy, precision, matrix effects, recovery, and stability. The calibration curves exhibited excellent linearity over the range of 0.1–10 µg/mL for all five analytes, with correlation coefficients (r2) ≥ 0.9962, while the lower limit of quantification and limit of detection were established at 0.1 and 0.03 µg/mL, respectively. The intra- and inter-day coefficients of variation were below 8.7% at all concentration levels, and the accuracy was 90.0%–113.0%. This methodology was successfully applied for TDM of three triazole antifungals and their metabolites in 150 patients with hematologic malignancies. Therefore, the method demonstrates good analytical performance, establishing its reliability for clinical TDM of triazole antifungals.

Publication date: Available online 8 November 2025 Source: Journal of Chromatography A Author(s): Jiong Li, Jinhua Yan, Qingwen Cao, Dan Wu, Weijie Lin, Mengna Jin

Rapid Communications in Mass Spectrometry, EarlyView.

Publication date: 6 January 2026 Source: Journal of Proteomics, Volume 322 Author(s):

Publication date: Available online 8 November 2025 Source: Journal of Proteomics Author(s): Lin Wang, Wenmin Tian, Sen Wang, Yuhong Liu, Hongli Wang, Junjie Xiao, Zhongkuo Yu, Lixin Xie, Yang Chen

Background: A large number of microproteins (miPs) encoded by small open reading frames (smORFs) are expressed in endothelial cells, yet their function remains largely unknown. In this study, we characterized a novel 46-amino-acid miP encoded by a smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript that was upregulated under inflammatory conditions and we refer to as miP-PSTPIP2. Methods: Immunoprecipitation coupled with mass spectrometry-based proteomics, immunoblotting, immunofluorescence and proximity ligation assays were used to identify and validate miP-PSTPIP2 interacting proteins in human endothelial cells. The impact of adenovirus-mediated overexpression of miP-PSTPIP2 on endocytosis, cytoskeleton dynamics and abundance of proteins involved in these processes was investigated by confocal microscopy and immunoblotting. Live cell imaging was used to assess endothelial cell migration and vascular permeability. Results: miP-PSTPIP2 physically associated with caveolar proteins, proteins involved in the regulation of cytoskeleton dynamics, intracellular transport, clathrin adaptor activity, as well as nuclear proteins. Human endothelial cells overexpressing miP-PSTPIP2 demonstrated enhanced endocytosis and transcytosis of transferrin as well as low-density lipoprotein. Mechanistically, miP-PSTPIP2 modulated Arp2/3-mediated actin nucleation and branching, which are required for dynamic cytoskeleton rearrangements. Moreover, altered cytoskeleton dynamics in miP-PSTPIP2-expressing endothelial cells resulted in impaired cell migration as well as increased permeability and monocyte trans-endothelial migration. Conclusions: miP-PSTPIP2 is an inflammation-induced endothelial miP that regulates Arp2/3-dependent actin dynamics, thereby enhancing lipid uptake and leukocyte permeability. Its upregulation under inflammatory conditions suggests a contributory role in endothelial dysfunction and vascular inflammation.

Analytical ChemistryDOI: 10.1021/acs.analchem.5c04063

ABSTRACT A simple, accurate, cost-effective, and sensitive analysis method for the determination of vancomycin in plasma samples has been reported using high-performance liquid chromatography-tandem mass spectrometry. UiO-66-NH2 metal–organic framework was synthesized through a hydrothermal method, 5 mg of this compound was introduced into 5 mL of model solution or pretreated plasma adjusted to a pH of 8. The mixture was vortexed for 1 min and then the sorbent particles were separated by centrifugation. The upper phase of sorbents was removed and the analytes adsorbed were eluted by a 250 µL mixture of deionized water (pH 2) and methanol at a 1:1 ratio, v/v, under vortexing for 4 min from the sorbent surface. After centrifugation, the eluent was analyzed by the determination system. The synthesized nanoparticles underwent characterization using X-ray diffraction, Fourier transform infrared spectrometry, and scanning electron microscopy. To achieve optimal efficacy, optimization was carried out at every stage of the project, encompassing adsorption, extraction, and desorption processes. Approved validation data consisting of method detection limit (22 and 225 ng/mL in deionized water and plasma, respectively) and limit of quantification (73 and 774 ng/mL in deionized water and plasma, respectively), a wide linear range of the calibration curve (73–250 ng/mL and 774–1250 ng/mL in deionized water and plasma, respectively), and low relative standard deviations for interday and intraday precisions (≤ 4.8%) were obtained by the method. The proposed method can be successfully applied in the analysis for the determination of vancomycin in plasma and deionized water samples.

AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate scope and role of ALKBH1, we used quantitative mass spectrometry to assess its activity in vitro and in human cell lines. To study ALKBH1 activity on defined substrates in vitro, we enzymatically generated tRNAs specifically carrying the m3C32 (tRNA-ThrUGU and tRNA-SerUCN) and i6A37 (tRNA-SerUCN) modifications. Here we show that ALKBH1 reduces m3C, m1A and m5C in vitro in total extracts of tRNAs, but does not impact on rRNA modifications in human cell lines. However, upon overexpression or siRNA-mediated knock-down of alkbh1 in human cell lines no impact on the modification of total tRNA extracts or specifically enriched RNAs could be observed. In addition, varying the glucose and fetal bovine serum (FBS) concentration in the growth medium of HEK293T cells, in combination with alkbh1 siRNA-mediated knock-down, also shows no impact on the tRNA modification spectra. Based on our data, we conclude that in human cells lines grown under optimal conditions ALKBH1 does not play an important role in the demethylation of tRNAs.

ABSTRACT Rationale Flavonoids are phenolic compounds with many health-benefiting properties. However, differentiating different types of flavonoids and their isomers is challenging due to their highly similar structures of various subtypes and different numbers and sites of substituents. Timely quality evaluation of flavonoid-based products is currently almost impossible. Methods An ambient ionization method of direct analysis in real time (DART) ion source and tandem mass spectrometry (MS) was used to characterize the fine structures of flavonoids. Different flavonoid subtypes and their isomers with varied numbers and sites of substituents were subjected to DART ionization and collision-induced fragmentation MS analysis. Results Seven classes of flavonoids, including methoxy-substituted compounds, exhibited distinctive fragmentation pathways such as retro-Diels-Alder reactions, cross-ring cleavages, and neutral losses. Many flavonoid isomers produced diagnostic MS2 and MS3 fragments through DART-tandem MS, enabling direct identification of various isomers within mixtures. An identification workflow was developed, culminating in the creation of a computational tool called FlavoFinder, which automatically determines flavonoid aglycone subtypes and their isomeric structures. Conclusions The method and the structural elucidation program were successfully used for the qualitative and quantitative analysis of different flavonoid isomers from real samples. The analysis procedure is high-throughput and is capable of characterizing complex flavonoid structures without extensive sample pretreatment and front-end chromatographic separations.

Publication date: Available online 7 November 2025 Source: Journal of Proteomics Author(s): Orlando Morales-Tarré, Xitlally Popa Navarro, Sergio Encarnación-Guevara

Publication date: Available online 7 November 2025 Source: Journal of Mass Spectrometry and Advances in the Clinical Lab Author(s): J. Brandsma, J.W. Thompson, K.L. Schully, J.G. Chenoweth, P. Genzor, S. Krishnan, D.A. Striegel, L.St. John-Williams, A. Moseley, G. Oduro, N. Adams, T. Vantha, E.L. Tsalik, C.W. Woods, D.V. Clark

Formalin-fixed paraffin-embedded (FFPE) samples are essential for clinical research and spatial proteomics (SP) but are technically challenging to analyze by liquid-chromatography coupled mass-spectrometry (LC-MS) due to losses during protein extraction, evaporation during decrosslinking and low-throughput sample preparation methods. Here, we present a streamlined micro-FFPE proteomics sample preparation protocol for laser-capture micro-dissection (LMD) that enables scalable and high-throughput processing in 96-well format, completing the entire process from tissue lysis to tryptic peptide mixtures on LC-MS-ready Evotips in just 2-hours. Our method integrates whole-slide imaging, LMD, and ultra-sensitive narrow-window data-independent acquisition (nDIA)-MS and facilitates enhanced lysis, decrosslinking, and proteolytic digestion directly within Teflon-based EVO96 chips. By utilizing nano-wells and the hanging droplet concept, our approach significantly reduces surface adsorption, thereby improving processing efficiency for both higher- and very low-input FFPE samples. This enables quantification of >5,500 protein groups (PGs) by nDIA-MS from 50,000 um2 (~250 cells) with robust performance maintained down to 1000 um2 (~5 cells) with ~2,500 PGs identified. Application to a panel of breast cancer (BC) tissues precisely discerned BC subtypes and their tumor micro-environment (TME) based on differential MS-based protein abundance of biomarkers including TP53, HER2 and PGR. Individually excised subcellular regions (~60 um2) yielded ~1,500 PGs, demonstrating reliable micro-scale spatial proteomics profiling approaching near-organelle resolution.

Objective: Primary liver cancer is a leading cause of cancer-related mortality and harbors recurrent mutations in chromatin regulators such as BRCA1-associated protein 1 (BAP1), yet their functional impact remains unclear. We investigated how BAP1 deficiency affects liver homeostasis and tumorigenesis to clarify its functional role. Design: We employed inducible, liver-specific BAP1 knockdown in mice subjected to diet-induced metabolic stress (including rescue experiments), alongside autochthonous hydrodynamic CRISPR models, and profiled livers by RNA-seq, immunohistochemistry, and mass spectrometry-based lipidomics. Complementary mechanistic assays in liver cancer cells examined the unfolded protein response (UPR) under endoplasmic reticulum (ER) stress; findings were supported by immunohistochemical and transcriptomic analyses of BAP1-mutant patient samples. Results: BAP1 safeguards liver homeostasis under diet-induced metabolic stress, as its loss triggers ER stress, hepatocyte death, and acute liver failure. Lipidomics revealed a shift toward ER-stress-associated dyslipidemia, and transcriptomics showed negative enrichment of fatty-acid metabolism and positive enrichment of UPR pathways. In contrast, BAP1 loss synergizes with oncogenic drivers to accelerate tumorigenesis in autochthonous liver cancer models, underscoring its context-dependent tumor suppressor function. Mechanistically, BAP1 directly regulates the ER stress mediator DDIT3 (CHOP) through chromatin remodeling, linking BAP1 loss to maladaptive stress responses. Consistently, elevated CHOP expression was observed in BAP1-mutant human liver cancers and other tumor types. Conclusion: These findings establish BAP1 as a key chromatin regulator that connects stress adaptation to both liver homeostasis and tumorigenesis, highlighting the BAP1-UPR axis for future translational assessment.

Publication date: Available online 7 November 2025 Source: TrAC Trends in Analytical Chemistry Author(s): David Fabregat-Safont, Francisco Madrid-Gambin, Alex Gomez-Gomez, Élida Alechaga, Gabriel Gil-Gómez, Óscar J. Pozo

Analytical ChemistryDOI: 10.1021/acs.analchem.5c04075

Analytical ChemistryDOI: 10.1021/acs.analchem.5c05632