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PubMed2026

Proteomic insights into hepatic responses to high soybean meal inclusion with citric acid supplementation in hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) juveniles.

This study evaluated the growth performance and hepatic proteomic responses of juvenile hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) juveniles fed diets containing 30% or 40% soybean meal (SBM), with or without 3% citric acid supplementation, and compared with a fishmeal (FM) control diet over a 10-week feeding trial. Fish fed 30% SBM (LSBM) showed a final body weight comparable to the FM group, whereas fish fed 40% SBM (HSBM) recorded significantly reduced growth and net protein utilization (NPU) (p < 0.05). Supplementation of citric acid in the 40% SBM diet (HSBM + CA) significantly improved NPU and partially restored growth relative to HSBM. LC-MS/MS based quantitative proteomics identified 1544 hepatic proteins and showed clear dietary segregation. Differentially expressed proteins were predominantly associated with reduced abundance of proteins involved in lipid metabolism, amino acid metabolism, pyruvate metabolism, and the tricarboxylic cycle across all SBM diets, suggesting a shift in oxidative metabolic capacity. In contrast, LSBM induced upregulation of spliceosome-associated proteins and RNA processing components, suggesting adaptive post-transcriptional regulation under moderate SBM inclusion. In contrast, HSBM exhibited greater reduction in metabolic protein abundance without comparable upregulation of adaptive regulatory components, coinciding with reduced nutrient retention. Protein-protein interaction analysis further confirms the correlation between the regulation of spliceosomal clusters and mitochondrial lipid-catabolic modules. Citric acid supplementation did not markedly reverse hepatic metabolic protein changes but improved protein retention. Data are available via ProteomeXchange with identifier PXD081994. These findings suggest a tolerance limit between 30% and 40% SBM inclusion, with 30% inducing adaptive hepatic proteomic responses compatible with maintained growth performance.

Animals↗

PubMed2026

Distinct molecular responses to acute cold exposure revealed by comparative transcriptomic and metabolomic profiling in the bay scallop Argopecten irradians.

Acute cold stress can elicit distinct molecular responses even when bay scallop populations show similar phenotypic outcomes. We compared a seventh-generation fast-growing bay scallop line (BS) with a commercial control population (CC) during a 72-h acute cold exposure at -1 ± 0.3 °C. RNA-seq was used as the discovery layer, representative BS cold-responsive genes were evaluated by qRT-PCR, and paired LC-MS profiles provided a comparative metabolic layer. At baseline, 138 genes differed between BS and CC; after cold exposure, 134 of these baseline differences disappeared and 61 of 65 cold-state differences newly emerged. BS showed a larger transcriptomic response magnitude than CC, with 1129 cold-responsive genes compared with 28 genes in CC, and this ordering remained robust across multiple sensitivity analyses. Survival after 72 h was identical in BS and CC (83/90, 92.2% in each population). Biochemical responses were time-dependent and marker-specific: CAT, LZM, T-SOD and T-AOC showed population-by-time interactions, whereas GSH-Px and MDA did not, and the 72-h differences were not consistently favourable to BS. Metabolomic cold effects were strongly concordant between populations, and no feature showed a significant population-by-cold interaction. Features putatively assigned to arachidonic acid metabolism were enriched, but this provider-annotated pathway signal remains exploratory because authentic-standard confirmation was not performed. These findings indicate population-specific differences in molecular responsiveness but do not establish superior cold tolerance in BS.

Animals↗

PubMed2026

Comprehensive study on pesticide residues and mycotoxins in freeze-dried strawberries and raspberries.

Freeze-dried fruit has gained popularity because it preserves the flavour and nutritional value of fresh fruit while providing extended shelf life. Despite this, there are concerns regarding its chemical safety. This study evaluated 58 freeze-dried fruit products from the Czech retail market, focusing on potential contamination. Pesticide residues and mycotoxins were determined using LC-MS/MS and GC-MS/MS. Overall, 111 pesticide residues (or their metabolites) and 3 mycotoxins were quantified. After applying processing factors, 12 pesticide residues exceeded EU maximum residue limits. Prohibited substances, including carbofuran, omethoate, and haloxyfop, were detected. Tenuazonic acid was found in 71% of samples, while alternariol and tentoxin were detected less frequently. More than half (54%) of strawberry samples contained 10 or more pesticide residues, indicating potential cumulative exposure concerns, particularly for children with lower body weight. These findings highlight the need for continued monitoring of freeze-dried fruits and further assessment of dietary exposure.

Pesticide Residues↗

PubMed2026

Discovery and characterization of multifunctional bioactive peptides from Alaska Pollock (Gadus chalcogrammus) milt: hybrid in silico, in vitro, and proteomic approaches.

The growing demand for multifunctional bioactive peptides has sparked interest in underutilized marine by-products as sustainable bioresources. This study explored Alaska Pollock (Gadus chalcogrammus) milt protein as a novel source of peptides with anti-inflammatory, anti-hypertensive, and anti-diabetic effects. Protein composition was analyzed via LC-MS, followed by in silico digestion and bioactivity prediction. Molecular docking identified peptides targeting DPP-IV, α-glucosidase, ACE, GLP-1 receptor, COX-2, MuRF1, and the 20S proteasome. Among the candidates, a promising peptide (CLPPH) was synthesized and validated in vitro, demonstrating inhibitory effects on nitric oxide production, DPP-IV, ACE, and α-glucosidase. These results highlight CLPPH's potential as a multifunctional bioactive peptide and support the valorization of Alaska Pollock milt as a sustainable source for functional foods and nutraceutical applications.

Animals↗

PubMed2026

Biocontrol efficacy of Bacillus albus SSR3 for controlling postharvest fungal pathogens and mycotoxin contamination.

Sweetpotato black rot, caused by Ceratocystis fimbriata, is a major postharvest disease that leads to substantial storage losses worldwide. In this study, a salt-tolerant rhizobacterial strain, Bacillus albus SSR3, was isolated from the rhizosphere of sweetpotato grown in saline-alkali soil, with broad-spectrum antagonistic activity against postharvest fungal pathogens. LC-MS/MS analysis revealed diverse bioactive metabolites associated with its antifungal activity. Integrated transcriptomic and metabolomic analyses showed that SSR3 bioactive metabolites extensively reprogrammed fungal metabolism, particularly pathways involved in carbohydrate and amino acid metabolism, antioxidant defense, and energy production. These alterations were accompanied by disruption of cell wall and membrane integrity, excessive reactive oxygen species accumulation, and mitochondrial dysfunction, ultimately inhibiting fungal growth. Here, we also found that SSR3 bioactive metabolites effectively inhibited aflatoxin B1 production by Aspergillus flavus and deoxynivalenol accumulation in Fusarium graminearum. In vivo assays further demonstrated that SSR3 bioactive metabolites significantly reduced sweetpotato black rot severity and effectively limited fungal colonization and mycotoxin contamination in stored agricultural commodities. Collectively, our findings demonstrate that B. albus SSR3 suppresses postharvest fungal pathogens through coordinated metabolic reprogramming, oxidative stress induction, and cellular integrity disruption, highlighting its potential as a sustainable biocontrol agent for postharvest disease management.

Bacillus albus↗

PubMed2026

Towards microplastic bioremediation: Fungal degradation of pristine and pretreated high-density polyethylene and polystyrene.

Microplastic (MP) contamination has become a significant ecological issue because of its enduring existence in the ecosystem and its possible negative impacts. Therefore, using degrading strategies to eliminate these stubborn polymers has been a subject of scientific research. However, the currently used degradation methods are relatively inefficient. Given the pervasiveness of High-Density Polyethylene (HDPE) and Polystyrene (PS) and their resistance to biodegradability, disposal strategies are critical and must be addressed. This manuscript examines the biodegradation of pristine and UV-treated HDPE and PS MPs by Aspergillus flavus species in minimal growth media over 70 days. The maximum weight loss observed at 70 days for pristine HDPE and PS in sole carbon source (SCS) media was (29.33 ± 0.28) % and (17.67 ± 0.35) %, respectively. Whereas, for UV-treated HDPE and PS MPs, the % weight reduction was (33 ± 0.21) % and (25 ± 0.19) %, respectively. UV-treated MPs exhibited greater weight reduction, as UV induced oxygenated functional groups enhance polymer susceptibility to enzymes, thereby promoting biodegradation. HDPE MPs typically show a higher proportion of particles in the lower size range compared to PS MPs. This assertion was based on the weight loss, particle size distribution, and SEM analysis. Furthermore, chemical changes were evaluated using Fourier transform Infrared Spectroscopy (FTIR) analysis, which also displayed chemical oxidation occurring during biodegradation. Liquid Chromatography-Mass Spectrometry (LC-MS) results indicate that UV pretreatment enhances biodegradability by promoting chain scission. These findings further suggest that this fungus's natural and ubiquitous occurrence in terrestrial and marine environments may actively contribute to MP biodegradation while requiring few nutrients.

Microplastics↗

PubMed2026

Antivenomic and Proteomic Assessment of Inter- and Intrapopulation Venom Variations in Nikolsky's Adder Vipera nikolskii: Comparison to Common Adder Vipera berus.

Snake venom variation has important clinical implications, yet individual-level venomics remains limited. We investigated inter- and intrapopulation variability in forest-steppe adder Vipera nikolskii and its recognition by commercial V. berus antivenom using proteomic and immunological approaches. Venoms from 12 individual V. nikolskii specimens representing two geographically distinct populations (BG and KM), together with three pooled V. nikolskii and one pooled V. berus samples were analyzed by LC-MS/MS, ELISA, Western blot, and pull-down assays. Multivariate analysis revealed relative homogeneity in BG and pronounced heterogeneity in KM venoms. Area-based proteomics revealed V. berus venom enrichment in PLA2 (34.6%), SVMP (14.6%), and CRiSP (15.6%), whereas V. nikolskii venoms were more variable. Pooled V. nikolskii venoms showed SVMP abundance (35.2-41.1%), contrasting with lower levels in individual samples. Antivenom binding was stronger for V. berus but weaker and more variable across individual and pooled V. nikolskii samples. Antivenom targeted PLA2/VEGF, CRiSP (only in V. berus), and Kunitz-type proteins. In vivo neutralization assay demonstrated strong protection against V. berus but not V. nikolskii venom. These findings reveal substantial compositional and antigenic variability in V. nikolskii venoms, highlight discrepancies between pooled and individual ones, and underscore the need for region-specific and functionally validated antivenom evaluation.

Animals↗

PubMed2026

Benchmarking the OptiSpray-μPAC Workflow against a Traditional Nanospray Capillary Interface for Multiplexed Quantitative Proteomics.

Nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) underpins modern quantitative proteomics, yet the column-to-mass spectrometer interface remains an important yet often underappreciated determinant of analytical depth, sensitivity, and reproducibility. Here, we benchmark an integrated workflow comprising the newly developed OptiSpray ion source and a micropillar array column (μPAC) cartridge against a conventional Nanospray Flex Source with an Accucore resin-packed capillary column. We performed a TMTpro 18-plex experiment across nine human cell lines on a FAIMS Pro-equipped Orbitrap Exploris 480. Following basic-pH reversed-phase fractionation, 12 fractions were analyzed on both workflow configurations under matched chromatographic gradient and acquisition conditions. Across both configurations, we quantified >9000 protein groups with highly comparable quantitative reproducibility and principal component clustering. Direct comparison of protein abundance ratios across cell lines showed agreement (Pearson R2 ≈ 0.7-0.8) without systematic bias. These results were achieved without workflow-specific optimization of the OptiSpray-μPAC platform, enabling direct transfer of established acquisition methods. Despite differences in column architecture, both configurations delivered comparable proteome coverage and quantitative fidelity. These findings establish the OptiSpray-μPAC workflow as a standardized alternative to conventional capillary-based interfaces, offering simplified operation while preserving quantitative performance.

Humans↗

PubMed2026

Nutrikinetics and bioavailability of Promunel®, a standardized poplar-type propolis phenolic extract: a double-blinded, placebo-controlled, cross-over, randomized trial.

Brown poplar-type propolis has been recognized and used for centuries to help prevent upper respiratory tract infections (URTIs). However, the scarce, incomplete information in humans on the nutrikinetics and bioavailability of its phenolic constituents, combined with a lack of standardization in its phenolic content and profile pose major challenges to develop bioactive ingredients. Thus, the aim of this study was to establish the nutrikinetics and total bioavailability (NKBA) parameters of brown poplar-type propolis phenolics in humans using the Standardized Propolis Extract (SPE) Promunel®. To achieve this, a 48 h NBKA study was conducted following a double blinded, randomized, placebo-controlled, cross-over design in healthy humans (n = 10) with two doses of SPE (1X = 400 mg or 4X = 1600 mg). Phenolic compounds were detected, identified and quantified in the extract, plasma and urine through different LC-MS/UV technologies. The SPE used is a rich (304.44 ± 15.61 µmol mg-1) and diverse source of phenolic compounds (5 sub-families). A total of 63 and 85 phenolic metabolites were identified and quantified in plasma and urine, mostly in the form of glucuronides and sulfates. In plasma, phenolic metabolites reached Cmax (1.22 ± 0.20 for 1X and 4.80 ± 0.48 µM for 4X) after 1 h of SPE intake, while urinary excretion occurred mostly during the first 3 h after. The total net bioavailability of SPE phenolic compounds at 48 h was 57.16 ± 5.71% for 1X and 43.82 ± 6.77% for 4X. Generally, the data between SPE 1X and 4X were proportional, indicating that a higher dose does not substantially modulate total net bioavailability. Overall, our data shows that brown poplar-type SPE phenolic compounds are highly bioavailable in the form of cinnamic acid and flavonoid conjugates, and that these compounds are rapidly absorbed and eliminated through the urine. Our results suggest that, for a sustained presence in circulation, brown poplar-type propolis supplements should be consumed more than once a day.

Humans↗

PubMed2026

Hex-MASP for mapping the whole-tissue spatial proteome and the intrabrain distribution of monoclonal antibodies.

Whole-tissue level spatial proteomics provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the micro-scaffold assisted spatial proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized projection micro-stereolithography 3D-printing, achieving high spatial resolution, sampling robustness, and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Applied to mouse brain, hex-MASP achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the identification of many unreported regionally enriched proteins across brain structures. We further applied hex-MASP to investigate the intrabrain distribution of intracerebroventricularly dosed antibody therapeutics and related proteins, which enabled whole-tissue mapping of protein drugs revealed insights into antibody brain penetration and distribution. Hex-MASP represents a robust, scalable platform for whole-tissue spatial proteomics.

Animals↗

PubMed2026

Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Blood Platelets↗

PubMed2026

An Integrated Proteomics and Genomics Approach to Identify Essential Protein Kinases During Human Trophoblast Development.

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrete human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile the proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1, and TNIK, that are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKCζ are essential for STB development, whereas PTK2B, SRC, TRIO, and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

Humans↗

PubMed2026

Transcriptomic and proteomic signatures underlying nymphal adaptation and foam production in the forage pest Mahanarva spectabilis.

The spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) is an important pest of forage grasses in South America, where its nymphs cause pasture damage by feeding on xylem sap and producing a characteristic foam that protects them against environmental stressors. To investigate the molecular basis of this adaptation, we integrated RNA-seq analysis of nymphs with LC-MS/MS proteomics of the Batelli gland, the primary source of foam secretion. De novo assembly of 100,666 unigenes revealed broad functional diversity, with strong representation of detoxification enzymes (CYP450s, GSTs, UGTs, carboxylesterases), transporters and ion pumps, cuticle proteins, and stress- and immunity-related genes. Nearly 16% of loci exhibited alternative splicing, particularly within detoxification, chemosensory and osmoregulatory gene families, highlighting evidence of transcriptomic variability. Signal peptide and secreted protein predictions identified 168 high-confidence candidate secreted proteins, including detoxification enzymes, proteases, structural proteins and immune-related factors, several of which are consistent with antimicrobial and surfactant-related functions. Proteomic profiling of the Batelli gland confirmed 500 proteins, enriched in chaperones, metabolic enzymes, detoxification pathways and osmoregulatory components, with the most abundant proteins corresponding to Hsp70 chaperones, ATP synthases, cuticle proteins and carbonic anhydrases. Together, these results provide an integrative transcriptomic and proteomic overview for M. spectabilis nymphs, highlighting genes and proteins associated with xylem feeding, foam production and responses potentially related to environmental stress tolerance. This comprehensive dataset not only advances the understanding of spittlebug biology but also identifies candidate molecular targets that may inform innovative strategies for controlling nymphal stages and mitigating spittlebug damage in forage systems.

Animals↗

PubMed2026

Comparative Genome-Wide Association Studies of Metabolites and Grain-Related Traits in Common Wheat.

The metabolome is highly diverse and the closest layer to phenotype; therefore, it is commonly regarded as a bridge between the genome and phenome in plants. Here, we performed large-scale metabolome analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 33 grain-related traits in a diverse panel of natural accessions and a recombinant inbred line (RIL) population. We identified a new network of 2286 associations between 947 metabolites and 33 grain-related traits. Systematic integration of metabolic genome-wide association study (mGWAS) and metabolic quantitative trait locus (mQTL) analyses identified 33 566 significant single-nucleotide polymorphisms (SNPs) and 3128 mQTL. Thirteen annotated metabolites co-localized within a physical interval on 7A. Integration of metabolite-based and phenotype-based GWAS and QTL revealed an overlapped region for gibberellin A4 (GA4) content and grain roundness on 4A. Phenotyping of an ethyl methanesulfonate (EMS)-induced mutant confirmed the role of TaSDR in regulating GA4 content and grain morphology. These findings provide novel insights into the metabolic pathways influencing key grain-related traits and advance our understanding of the complex molecular mechanisms regulating grain metabolites and phenotypes in wheat. The identified metabolic markers and candidate genes provide valuable targets for molecular breeding programs aimed at improving wheat yield and quality.

QTL↗

PubMed2026

Synaptic Proteome Divergence in the Prefrontal Cortex of Tame and Aggressive Red Foxes (Vulpes vulpes).

The biological mechanisms behind aggressive and affiliative behaviors are difficult to pinpoint. In the Farm-Fox Experiment, conventional foxes were selectively bred since 1959 in two different directions, one for tame and another for aggressive response to humans. The distinct differences in social behavior of tame, aggressive, and conventional populations are genetically based and the three populations live in conditions that control for factors that could impact social reactions, such as environment and social experiences. Genomic and transcriptomic studies of genetic differences among the fox populations have highlighted genes involved in synaptic processes in the prefrontal cortex. To investigate how the synaptic mechanisms differ between the three fox populations, synaptosomes were isolated from prefrontal and premotor cortex extracts of sixteen female foxes. Tandem mass tags with liquid chromatography tandem mass spectrometry (LC-MS) were used to identify and quantify the relative abundance of the proteins. The results were sorted into protein groups and compared between populations using a limma analysis to determine proteins with differential expression (DE). In the tame versus aggressive comparison, 174 protein groups were found to be DE, while only five were found in the conventional versus aggressive comparison. Most DE protein groups had lower fold expression in the aggressive population compared to tame and aggressive populations. ADGRB2 was found to be the most DE protein group, with 11-fold higher expression in aggressive foxes than in tame foxes. ADGRB2 was previously shown to affect depression-like behavior in mice and is involved in the vascular endothelial growth factor signaling pathway, that is known to influence neurogenesis. Enrichment analyses on the DE protein groups found gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in the tame versus aggressive comparison, including multiple, highly enriched terms involving ribosome and translation. Local translation at synapses plays an important role in synaptic plasticity and, as a result, can profoundly influence behavior. This study highlighted potential mechanisms that could underly the behavioral differences between tame and aggressive foxes.

Journal Article↗

PubMed2026

Acinetobacter guillouiae, a lipolytic strain isolated from sludge capable of partially depolymerising polyethylene terephthalate: genomic, proteomic, and biochemical insights.

Acinetobacter guillouiae I-MWF was isolated by incubating amorphous polyethylene terephthalate (PET) film in sludge samples. The strain partially depolymerised PET powder with 11.3% crystallinity, as confirmed by FT-IR, HPLC-UV, and LC-MS analyses. Extracellular enzymes released terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). Genomic analysis identified 18 putative extracellular hydrolases, including lipases and esterases, each with a conserved catalytic triad. Proteomic profiling revealed expression of two triacylglycerol lipases and two additional lipase-family proteins when the strain was cultivated with PET or a PET-Tween 80 mixture. These enzymes were cloned in Escherichia coli, but most formed insoluble, inactive inclusion bodies, and one was not expressed. Molecular modelling highlighted structural features likely to influence their catalytic interaction with PET. Although the strain partially depolymerised PET powder, it was unable to grow on PET, TPA, or ethylene glycol, indicating that PET depolymerisation occurs as a side activity rather than supporting growth. Instead, A. guillouiae displayed strong lipolytic activity and a clear preference for lipid-based substrates, achieving its highest growth with Tween 80. A lipid transporter was also expressed under these conditions, suggesting adaptation to hydrocarbon-rich environments. These findings indicate that A. guillouiae I-MWF can mediate partial PET depolymerisation without assimilating the resulting monomers, while preferentially growing on lipid-like substrates.

Acinetobacter↗

PubMed2026

Improved comprehensive profiling of fecal bile acids through chemical derivatization combined with HPLC-MS/MS analysis.

Bile acids (BAs) facilitate the digestion and absorption of fats and influence lipid and glucose homeostasis, making them potential therapeutic targets for obesity and related metabolic disorders. The liver and intestinal microbiota modify BAs structurally, generating diverse chemical forms and isomers. Comprehensive profiling of the BA pool is critical for understanding their key biological functions and as a therapeutic approach for related diseases. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is usually chosen as the preferred method for BA detection due to the complex chemical structures, the wide range of actual concentrations and the complexity of fecal sample matrices. However, free BAs are difficult to ionize, resulting in low detection signals and a lack of characteristic structural fragments to assist in structural identification. In this method, the labeling reagent (2-aminoethyl) trimethylammonium (AETMA) is employed to label the carboxyl group of BAs. Compared with underivatized BAs, the detection sensitivity of unconjugated BAs was enhanced by 25-180 fold, while that of conjugated BAs increased by 6-160 fold. It also generates unique fragment ions and enhances MS response, facilitating the discovery of potential BAs. Methodological parameters were validated using 38 BAs as representatives. Through methodological validation, it was verified that the precision, recovery, matrix effect and stability parameters of the method met acceptable criteria. We also identified 61 confirmed BAs and 55 additional candidate BAs in human pooled fecal samples. It has been successfully applied to fecal BA analysis in obese populations, providing valuable insights into potential therapeutic strategies for obesity.

Tandem Mass Spectrometry↗

PubMed2026

On-filter fractionation by empFASP improves identification of membrane peptides in proteomic experiments.

Membrane proteins remain among the most analytically challenging targets in bottom-up proteomics due to their limited solubility and low abundance of protease-accessible sites within transmembrane domains. In addition, hydrophobic peptides are frequently lost during detergent removal and the on-filter processing steps. Here, we present empFASP, a straightforward on-filter-fractionation-based modification of the enhanced filter-aided sample preparation (eFASP) workflow that enhances recovery of membrane-embedded peptides otherwise lost during digestion and cleanup. The method combines controlled on-filter inversion with sequential ethyl acetate extraction at defined pH values, enabling recovery of peptide material retained on the filter and redistributed into detergent micelles. Compared with SP3 and SP4 in HEK293T lysates, empFASP increased unique hydrophobic peptide identifications by up to 48% and increased the proportion of detected transmembrane peptides. Application to mouse mitochondrial membranes and phosphatidylethanolamine-deficient and PE-containing Escherichia coli membranes showed that the additional fractions of empFASP contribute complementary recovery of hydrophobic and membrane-associated peptides, with the strongest gains observed at the peptide level. Because empFASP requires no specialized reagents or instrumentation, it can be readily implemented in standard proteomics workflows to improve coverage of membrane-embedded regions. SIGNIFICANCE: The empFASP (enhanced membrane peptide) workflow offers a practical solution to one of the persistent limitations in membrane proteomics-the underrepresentation of hydrophobic and transmembrane peptides in standard digests. By integrating simple pH-controlled extractions into an on-filter format, empFASP recovers peptides otherwise lost through adsorption or detergent micelle retention, substantially improving coverage of the membrane proteome. This method expands the analytical reach of bottom-up proteomics without requiring specialized instrumentation, making it immediately applicable for studies of membrane topology, protein-lipid interactions, and the structural consequences of altered membrane composition.

Proteomics↗