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PubMed2026

The composition of the periostracum in the razor clam Sinonovacula constricta and the mantle's response to sulfide.

The razor clam Sinonovacula constricta inhabits sulfide-rich intertidal sediments and exhibits remarkable tolerance to this toxicant, yet the role of its periostracum in sulfide adaptation remains poorly understood. In this study, we investigated the composition and structure of the periostracum proteins, and the response of the mantle to sulfide stress. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed that the periostracum is approximately 10 μm thick and contains 1.43 wt% sulfur, and proteomic analysis further confirmed the presence of organic sulfur (Cys/Met-rich proteins), suggesting its involvement in sulfur deposition. Using LC-MS/MS, we identified 77 high-confidence proteins from the periostracum, which were classified into six functional categories: enzymes, framework proteins, immune-related proteins, calcium ion-related proteins, other proteins, and proteins with unknown functions. Phylogenetic analyses of representative proteins revealed bivalve-specific evolutionary patterns, with several proteins exclusively present in Bivalvia, such as Unknown protein 2 and 7, which possess signal peptides and low-complexity domains. For the sulfide exposure experiment, razor clams were subjected to three Na2S concentrations (0, 10, and 100 μM). qPCR analysis showed that, compared with the control group, Chitin-binding protein 3 and Tyrosinase were significantly upregulated in the mantle, peaking in the 100 μM group at 48 h (5677.84-fold and 157.20-fold, respectively), whereas Collagen and Cadherin 3 were generally suppressed. This study represents one of the most comprehensive proteomic profiles of the razor clam periostracum and highlights the mantle's potential role in sulfide tolerance, offering insights for sulfur-tolerant aquaculture breeding and bioremediation applications.

Animals↗

PubMed2026

Quantitative N-glycoproteomic analysis reveals glycosylation signatures of plasma immunoglobulin G in sepsis.

INTRODUCTION: Sepsis is a life-threatening condition resulting from organ dysfunction due to a dysregulated immune response to infection. Immunoglobulin G (IgG) plays a role in modulating immune responses. However, the precise IgG subclass-specific N-glycosylation profiles in patients with sepsis remain poorly characterized. METHODS: This study aimed to define the site-specific N-glycosylation signatures of plasma IgG subclasses in sepsis patients with different prognoses using quantitative glycoproteomics. By employing our established GlycoQuant strategy, we quantified the intact N-glycopeptides (IGPs) of IgG subclasses in 40 healthy controls and 40 sepsis patients with a clear prognosis. RESULTS: We identified 12 IGPs with altered abundances between patients with sepsis and healthy controls. After Benjamini-Hochberg (BH) correction of the 31 outcome-stratified IGP comparisons, IGP24 and IGP25 remained significant and met the prespecified fold-change criterion. Global BH correction across 124 IGP-clinical parameter correlations retained positive associations of IGP19, IGP22, and IGP23 with procalcitonin (PCT). In exploratory outcome-stratified ROC analyses, candidates were selected using the original unadjusted P-value and fold-change screen; five IGPs were evaluated, with IGP25 and IGP24 yielding the highest individual AUCs. Collectively, our findings underscore the potential of IgG subclass-specific glycosylation profiling as a novel translational approach for clinical applications in sepsis management. SIGNIFICANCE: Sepsis remains a leading cause of global mortality, with patient outcomes heavily dependent on timely diagnosis and accurate prognosis. The dysregulated host immune response, particularly involving immunoglobulins, is central to its pathophysiology. This study provides a significant advance in the field of clinical glycoproteomics by applying a quantitative, site-specific strategy to delineate the plasma IgG subclass N-glycosylation landscape in sepsis. We report, for the first time, a panel of subclass-specific intact IgG N-glycopeptides (IGPs) that are significantly altered in sepsis patients compared to healthy controls. The identified IGPs not only demonstrate diagnostic and prognostic potential but also show a significant correlation with procalcitonin, a key clinical severity index. These findings bridge a critical knowledge gap by moving beyond bulk IgG glycosylation analysis to subclass-resolved profiling, offering novel molecular insights into sepsis immunopathology. The identified glycosylation signatures hold substantial translational promise as a foundation for developing innovative, glycan-based biomarker panels to improve the precision management of this heterogeneous and life-threatening syndrome.

Humans↗

PubMed2026

2-Mercaptoethanol/DMSO Workflow Enables Highly Reproducible Quantitative Proteomics.

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase spectral complexity in mass spectrometry and reduce quantitative accuracy. Here, we established a reproducibility-focused 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow and systematically evaluated its quantitative performance at the proteome-wide level. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6- to 1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage with the optimized 2-ME treatment. Quantitative reproducibility was also enhanced, with the peptide quantified CV ≤ 20% increasing from 61.4% with IAA treatment to 86.1% with 2-ME treatment, and protein quantified CV ≤ 20% increasing from 80.6% with IAA treatment to 93.5% with 2-ME treatment. Application of this new workflow to ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.

Animals↗

PubMed2025

Multi-Omics and Integrative Analytics in Natural Products Discovery.

Natural products (NPs) have long been an essential source of new bioactive compounds for drug discovery; however, traditional methods for screening and isolating these compounds can be slow and often yield diminishing returns. Fortunately, advanced multi-omics and computational approaches present powerful solutions to these challenges. This review highlights innovative methodologies that integrate metabolomics, genomics, transcriptomics, and proteomics with bioinformatics and analytical chemistry to accelerate NP discovery. For instance, untargeted metabolomics platforms like high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Global Natural Products Social (GNPS) molecular networking allow for comprehensive profiling of new compounds, while targeted isotope-labeling strategies enhance this process. Additionally, genome and metagenome mining tools such as antibiotics and secondary metabolite analysis shell (antiSMASH), Deep Biosynthetic Gene Cluster (DeepBGC), and Pipeline for Reconstructing Integrated Syntheses of Metabolites (PRISM) quickly identify biosynthetic gene clusters (BGCs) in both cultured and uncultured organisms, often using heterologous expression to validate products. Transcriptomic analyses, including RNA sequencing (RNA-seq), co-expression networks, and fluxomics, help clarify how pathways are regulated, while quantitative proteomics techniques like tandem mass tags/isobaric tags for relative and absolute quantitation (TMT/iTRAQ) and label-free methods, along with chemoproteomics approaches such as cellular thermal shift assay and thermal proteome profiling (TPP), uncover molecular targets and their mechanisms of action. This review also places significant emphasis on the role of artificial intelligence (AI) and machine learning (ML) in integrating multi-omics data, spanning activities from constructing gene-metabolite correlation networks to leveraging knowledge graphs and graph neural networks for data fusion and functional prediction. Finally, this review concludes by discussing the synergistic benefits of multi-omics for natural-product discovery, addressing current technical challenges, and exploring future directions toward high-throughput, intelligent data integration for next-generation NP research.

Biological Products↗

PubMed2024

A Proteogenomic Pipeline for the Analysis of Protein Biosynthesis Errors in the Human Pathogen Candida albicans.

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37 °C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C. albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an in vivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C. albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C. albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity.

Candida albicans↗

PubMed2026

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba↗

PubMed2025

Mass spectrometry-based ligand binding assays in biomedical research.

INTRODUCTION: Ligand binding assays combining immunoaffinity enrichment steps with mass spectrometry (MS) readout have gained attention as a highly specific and sensitive tool for protein quantification. These techniques typically combine enzymatic fragmentation of the sample or enriched protein with capture on the protein or peptide-level for quantification. Antibodies ensure specific target recognition, while MS offers quantitative accuracy with isotopically labeled internal standards. This dual approach supports a broad dynamic range, enabling protein measurements from picomolar to nanomolar levels. These methods have diverse applications, from quantifying signaling proteins in basic research to biomarker monitoring in clinical trials and analyzing the pharmacokinetics of therapeutic proteins. AREAS COVERED: This review delves into the diverse workflows of immunoaffinity-MS, shedding light on the innovative strategies employed, their practical applications, efficacy, and inherent limitations in the realm of protein quantification. EXPERT OPINION: Immunoaffinity-MS has transformed protein analysis, but widespread adoption is hindered by complex workflows, high instrument costs, and limited capture molecule availability. Efforts to enhance automation, standardize workflows, and advance technological innovation aim to overcome these barriers. Improvements in mass spectrometer sensitivity, advances in recombinant capture technologies, and support from public initiatives are poised to further improve the reliability and accessibility of this method.

Mass Spectrometry↗

PubMed2026

Development of a High-Sensitivity Glycoproteomics Approach for Fc-Specific Quantification of IgG Core Fucosylation in Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers complex neuroinflammatory cascades that involve sustained immune activation and dysregulated antibody effector functions. Immunoglobulin G (IgG) Fc N-glycosylation, particularly core fucosylation, critically modulates immune signaling through altered Fcγ receptor (FcγR) interactions; however, its role in TBI remains unexplored. Here, we developed a high-sensitivity, mass spectrometry-based glycoproteomics method for the systematic analysis of IgG Fc core fucosylation dynamics following TBI. The approach integrates Fc-specific enzymatic truncation with GlycINATOR (EndoS2) and tryptic digestion, followed by high-resolution LC-MS/MS profiling, enabling confident identification of truncated Fc glycopeptides. Furthermore, a targeted parallel reaction monitoring (PRM) strategy allowed direct quantification of core fucosylated and afucosylated glycopeptides from 10 μg of crude serum protein, eliminating the need for IgG purification. Our results reveal time-dependent and subclass-specific remodeling of IgG Fc fucosylation postinjury, characterized by an overall reduction in fucosylated species and a relative increase in afucosylation. Collectively, this study establishes a scalable analytical platform for Fc-specific glycosylation profiling and identifies IgG core fucosylation as a candidate molecular indicator of immune dysregulation in TBI, providing new insights into post-traumatic immune regulation.

Brain Injuries, Traumatic↗

PubMed2026

Proteomic profiling of bone for the estimation of post-mortem interval and post-mortem submersion interval: a systematic review.

Accurate estimation of the Post-Mortem Interval (PMI) and Post-Mortem Submersion Interval (PMSI) remains a persistent challenge in forensic science, especially when traditional morphological and entomological methods fail due to advanced decomposition or in aquatic environments. Proteomic profiling of bone tissues has recently emerged as a promising approach, leveraging the predictable degradation patterns of bone proteins to estimate time since death more reliably. This systematic review, conducted in accordance with PRISMA guidelines, analyzed 24 peer-reviewed studies focusing on the application of proteomic techniques to bone tissue for PMI and PMSI estimation. The included studies were evaluated based on sample type, analytical techniques used, identified biomarkers, environmental conditions assessed, and the overall reliability and reproducibility of the findings. The review found that specific bone proteins, particularly collagen, osteocalcin, fetuin-A, etc. exhibited consistent degradation patterns that correlated strongly with elapsed post-mortem time. Cortical bone was identified as a more stable and informative matrix compared to trabecular bone. Mass spectrometry, especially LC-MS/MS, emerged as the predominant analytical technique due to its high sensitivity and accuracy in detecting low-abundance proteins over extended PMIs and PMSIs. However, protein degradation rates were significantly influenced by environmental variables such as temperature, humidity, soil pH, and microbial activity. This review also emphasizes the transformative role of bone proteomics in advancing forensic science while identifying key gaps that must be addressed to achieve global standardization and practical implementation in diverse forensic contexts. The integration of proteomics with other emerging technologies, such as machine learning algorithms and computational modeling, may further enhance the precision of PMI and PMSI estimation in future applications.

Postmortem Changes↗

PubMed2026

Relaxin-2: Shaping the Proteomic Landscape of Skeletal Muscle Physiology, Glucose Trafficking, and Mitochondrial Function in Rat.

Relaxin-2 is a hormone with robust beneficial effects on the heart and blood vessels and potential as a therapy for cardiovascular (CV) disease. Considering the interorgan communication between skeletal muscle and heart, and the relation between muscle quality/composition and CV events, we hypothesize that relaxin-2 may regulate skeletal muscle physiology and metabolism. We aim to evaluate the impact of relaxin-2 on the proteome of skeletal muscle from healthy Sprague-Dawley rats. Animals were treated with 0.4 mg/kg/day of serelaxin (recombinant form of human relaxin-2) or vehicle (PBS) for 2 weeks employing subcutaneous osmotic minipumps. Skeletal muscle protein identification and quantification were performed by LC-MS/MS using a Data-Independent Acquisition (DIA)-Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH) method. SWATH/MS quantitative analysis identified that relaxin-2 significantly decreased 95 proteins and significantly increased 32 proteins in rat skeletal muscle when compared to control rats. From these, 34 proteins were associated with muscle function, myogenesis, muscle differentiation and/or regeneration, 20 are mitochondrial proteins (six from the complexes of the electron transport chain), and 10 proteins participate in glucose metabolism. Qualitative data-dependent workflow analysis identified 35 proteins exclusive to the skeletal muscle of the relaxin-2-treated group: eight proteins related to processes of skeletal muscle function (size, ion homeostasis or organization of caveolae structures and cytoskeleton) and myogenesis, and two proteins involved in muscle differentiation. Our work highlighted for the first time the role of relaxin-2 in crucial processes of muscle physiology and energetic metabolism, which could influence several processes involved in myopathy and CV.

Animals↗

PubMed2026

Subcellular Proteomic Analyses Reveal REEP5 Knockdown in the Mouse Heart Disrupts Mitochondrial Networks.

Receptor Expression-Enhancing Protein 5 (REEP5) is a cardiac-enriched, membrane-shaping protein localized to the sarco(endo)plasmic reticulum (SR/ER), where it supports membrane network architecture and cardiomyocyte function. While REEP5 has been implicated in calcium handling and contractility, its role in regulating inter-organelle communication and mitochondrial homeostasis remains less well-understood. In this study, we used recombinant adeno-associated virus serotype 9-mediated shRNA knockdown of Reep5 in mouse hearts, combined with subcellular fractionation and data-independent acquisition mass spectrometry, to define proteomic remodeling across microsomal (SR/ER), mitochondrial, and cytosolic compartments. Loss of REEP5 altered the composition of SR/ER membrane-shaping proteins, including upregulation of RTN4, ATL3, and CKAP4, suggesting a partial compensatory response. Microsomal, mitochondrial and cytosolic proteomes exhibited broad reorganization, with enrichment of proteins involved in redox adaptation and proteostasis, alongside depletion of mitochondrial import machinery and antioxidant enzymes. Imaging of isolated cardiomyocytes confirmed fragmented mitochondrial networks and increased reactive oxygen species, consistent with proteomic signatures of disrupted mitochondrial dynamics and oxidative stress. Gene ontology enrichment across all fractions highlighted widespread dysregulation in organelle-specific processes, including translation, protein localization, and metabolic remodeling. Notably, several altered pathways converged on mitochondria-associated membranes, suggesting that REEP5 may support SR/ER-mitochondria tethering and functional crosstalk. These findings position REEP5 as a key regulator of organelle homeostasis in the heart and underscore how its loss disrupts mitochondrial integrity and inter-organelle communication across cellular compartments.

Animals↗

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↗

PubMed2026

Proteomic profiling of the aqueous extract from the antennal gland of the Pacific white shrimp, Litopenaeus vannamei.

The antennal gland (AnG) of decapod crustaceans has been proposed as a potential source of bioactive molecules involved in chemical communication; however, its protein composition remains largely unexplored. Here, we present the first reference proteomic map of the aqueous extract from the antennal gland of the Pacific white shrimp Litopenaeus vannamei. Protein extracts from immature and mature females were analyzed using an integrated workflow combining one-dimensional SDS-PAGE, reverse-phase high-performance liquid chromatography (RP-HPLC), and nanoLC-tandem mass spectrometry. Electrophoretic and chromatographic analyses revealed a high degree of qualitative similarity between reproductive stages. SDS-PAGE resolved six major protein bands (∼227, 166, 77, 42, 35, and 17 kDa), most comprising multiple co-migrating proteins as revealed by LC-MS/MS. Hemocyanin was identified as the predominant protein and was detected across several electrophoretic bands. Additional proteins were associated with innate immunity, including β-1,3-glucan-binding protein and coagulable hemolymph protein; reproductive processes, including vitellogenin, spermatogonial stem-cell renewal factor, farnesoic acid O-methyltransferase, estrogen sulfotransferase, and prostaglandin reductase 1; as well as energy metabolism, protein homeostasis, cytoskeletal organization, and intracellular trafficking. Because several identified proteins are widely distributed or known hemolymph components, their detection cannot be assumed to reflect AnG-specific expression or function. Collectively, these findings establish a molecular reference for the L. vannamei AnG and reveal protein components associated with multiple physiological processes. This dataset provides a proteomic framework for future comparative and functional studies aimed at elucidating antennal gland physiology and experimentally evaluating the potential involvement of proteinaceous or peptide-based molecules in chemical communication in decapod crustaceans.

Animals↗

PubMed2024

Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA.

Ubiquitin-specific protease 7 (USP7) is implicated in many cancers including colorectal cancer in which it regulates cellular pathways such as Wnt signaling and the P53-MDM2 pathway. With the discovery of small-molecule inhibitors, USP7 has also become a promising target for cancer therapy and therefore systematically identifying USP7 deubiquitinase interaction partners and substrates has become an important goal. In this study, we selected a colorectal cancer cell model that is highly dependent on USP7 and in which USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, and cell-cell adhesion. We then used inducible knockdown of USP7 followed by LC-MS/MS to quantify USP7-dependent proteins. We identified the Ajuba LIM domain protein as an interacting partner of USP7 through co-IP, its substantially reduced protein levels in response to USP7 knockdown, and its sensitivity to the specific USP7 inhibitor FT671. The Ajuba protein has been shown to have oncogenic functions in colorectal and other tumors, including regulation of cell-cell adhesion. We show that both knockdown of USP7 or Ajuba results in a substantial reduction of cell-cell adhesion, with concomitant effects on other proteins associated with adherens junctions. Our findings underlie the role of USP7 in colorectal cancer through its protein interaction networks and show that the Ajuba protein is a component of USP7 protein networks present in colorectal cancer.

Ubiquitin-Specific Peptidase 7↗

PubMed2001

Mass spectrometry compatibility of two-dimensional gel protein stains.

As proteomic technology evolves, protein staining sensitivity is constantly being improved, enabling researchers to better visualize the proteome of their system. The current challenge is to balance the limits of detection of protein visualization with those of the mass spectrometric methods. In this report, mass spectra generated from human serum or rat liver proteins stained with either colloidal Coomassie blue, Daiichi silver, SYPRO Orange, SYPRO Red, SYPRO Ruby, or SYPRO Tangerine are compared. It has been concluded that the newest generation of fluorescent protein stains, compared with traditional staining methods, are more compatible to matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The number of database matches obtained using each mass spectrometry method and the percent sequence coverage obtained from trypsin digested proteins stained using these six methods is provided.

Amino Acid Sequence↗

PubMed2000

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.

Animals↗

PubMed2026

Schisantherin B mitigates cisplatin-induced ototoxicity by modulating the CNPY2-PERK/CHOP signaling axis.

Irreversible cisplatin-induced hearing loss (CIHL) is a refractory chemotherapy-related adverse effect with limited clinical treatments. Schisantherin B (STB), a lignan isolated from Schisandra chinensis, is widely recognized for its neuroprotective properties, while its role in auditory injury remains unclear. Herein, we found that STB alleviated cisplatin-induced ototoxicity in House Ear Institute Organ of Corti 1 (HEI-OC1) cells and guinea pig models, protecting cochlear hair cells, synaptic ribbons and spiral ganglion neurons, and partially restoring auditory brainstem response (ABR) thresholds. Furthermore, combined drug affinity responsive target stability (DARTS) assay, the cellular thermal shift assay (CETSA), and the surface plasmon resonance (SPR) assay, we confirmed STB directly binds to the canopy FGF signaling regulator 2 (CNPY2), a key initiator of endoplasmic reticulum (ER) stress. Notably, consistent dual in vitro and in vivo validation confirmed that STB exerts no regulatory effect on CNPY2 protein abundance, yet suppressed the downstream Protein kinase R-like endoplasmic reticulum kinase / C/EBP homologous protein (PERK/CHOP) signaling cascade and ER stress-mediated apoptosis. Moreover, molecular docking and co-immunoprecipitation (co-IP) validated the physical binding of STB to CNPY2 and the endogenous interaction between CNPY2 and PERK. Additionally, CNPY2 overexpression and shRNA knockdown further verified this functional relationship. Integrated proteomic and transcriptomic analyses showed STB partially reversed cisplatin-triggered inflammation and excessive ER stress. Collectively, our results suggest STB may serve as a potential otoprotective agent. The CNPY2-PERK/CHOP axis is closely linked to cisplatin-induced cochlear damage and offers a feasible target for intervention against CIHL. Abbreviations: CIHL, cisplatin-induced hearing loss; STB, Schisantherin B; HEI-OC1, house ear institute organ of corti 1; ABR, auditory brainstem response; DARTS, drug affinity responsive target stability; CETSA, cellular thermal shift assay; SPR, surface plasmon resonance; CNPY2, canopy FGF signaling regulator 2; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; co-IP, co-immunoprecipitation; STA, Schisantherin A; STC, Schisantherin C; dB SPL, decibels sound pressure level; EDTA, ethylenediaminetetraacetic acid; dB SPL, decibels sound pressure level; SGN, spiral ganglion neuron; IHCs, inner hair cells; OHCs, outer hair cells; CCK-8, Cell Counting Kit-8; OD, optical density; ODb, blank sample, ODc, control sample; NC, negative control; PVDF, polyvinylidene difluoride; RT, room temperature; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; DMSO, dimethyl sulfoxide; KDs, equilibrium dissociation constants; SP, standard precision; SEM, standard error of the mean; HSD, honestly significant difference; Ctrl, control group; CV, cell viability; Kd, dissociation rate constant; Ka, association rate constant; STS, sodium thiosulfate; UPR, unfolded protein response; BLB, blood-labyrinth barrier.

Apoptosis↗

PubMed2026

Protein profiling and GC-MS product analysis provide insights into lignite solubilization and bioconversion by Lysinibacillus sphaericus strain SH19.

Lignite biosolubilization offers a mild route for valorizing low-rank coal, although the microbial processes that accompany solubilization remain incompletely defined. Here, an endogenous isolate designated Lysinibacillus sphaericus strain SH19 was evaluated using nitric-acid-pretreated Shengli lignite. Under the selected working conditions (4 M nitric-acid pretreatment, initial pH 8, 40°C, and 16 days), the apparent solubilization rate reached 66.81%. Changes in A450, residual solid mass, culture pH, and extracellular protein concentration showed that chemical pretreatment and bacterial culture were both associated with the release of soluble lignite-derived material. SDS-PAGE and two-dimensional electrophoresis revealed treatment-associated differences in extracellular and intracellular protein patterns. LC-MS/MS analysis of excised protein spots yielded 85 candidate protein assignments; the revised supplementary table reports PEAKS scores, sequence coverage, peak area, and unique-peptide counts and highlights the limited support for several entries. GC-MS analysis produced 33 tentative library assignments in the solubilized fraction, but siloxane- and silyl-related signals were treated as possible analytical background, and no pathway was inferred from these assignments alone. Together, the data identify strain SH19 as a promising lignite-biosolubilizing isolate and provide candidate proteins and product signals for future validation. The proposed process model remains exploratory because direct enzyme assays, inhibitor experiments, carbon-balance measurements, transcriptomic or genetic validation, complete GC-MS blank subtraction, and authentic-standard confirmation were not available.

Bacillaceae↗