Journal of Proteome Research最新文献

Multiplexing samples in mass-spectrometry-based proteomics has long been accomplished by isotopologues of small molecules. These chemically identical "mass tags" conjugate to peptides to encode samples with different mass offsets for parallel analysis. The current state of the art for multiplexing with nonisobaric mass tags was recently improved from 3-plex to 9-plex, but what is the largest plex size that can be reasonably achieved with current technology? A full answer to this question requires evaluating current mass spectrometry hardware, facets of which have been well investigated by others. However, it may be underappreciated that multiplexing 1000s of samples with mass tags does not actually require 1000s of isotopes or 1000s of synthesis steps to create. Nonintuitively, high-plex mass tags can require relatively few different isotopes. The focus of this exposition is to characterize the potential of the differential mass defect to create tens to over a thousand isotopologues of small molecules and how careful combinations of these small molecules can combinatorially scale the plex size to minimize the number of synthetic steps. Importantly, we show using formulas from publicly reported compounds that plex sizes in the hundreds, an order of magnitude greater than the state of the art, are achievable using molecules comparable in size to existing commercial tags and that going beyond hundreds may require larger molecules. Additionally, we show how plexing with the differential mass defect allows significantly higher-purity yields during reagent creation for a given plex size. In summary, we investigated and present here how the differential mass defect must be utilized to create high-plex mass tags with limited dopable sites.

Nonhistone acetylation is a key epigenetic mechanismin osteoarthritis(OA), but its specific role in cartilage degeneration remains unclear.Here, we integrated transcriptomics, proteomics, and acetyl-proteomicsto systematically analyze molecular changes in rat OA cartilage. Multiomicscoenrichment identified the ECM-receptor interaction pathway as thecore target, reprogrammed by increased acetylation of Fn1, Tnc, andItgb1. Using 113 machine-learning strategies, we identified 11 corediagnostic genes, with Wisp3 showing the highest efficacy validatedin independent data sets. In vitro experiments and single-cell RNA-seqconfirmed that Wisp3 downregulation disrupts ECM homeostasis. Thisstudy reveals that nonhistone acetylation drives OA by reprogrammingthe ECM-receptor interaction pathway and provides novel early diagnosticbiomarkers.

Late-onset depression (LOD) is acommon subtype of depression inthe elderly, characterized by high disability and recurrence rates,along with poorly understood biological correlates, posing a seriousthreat to the health of the aging population; moreover, the molecularcorrelates and diagnostic targets for LOD remain largely unknown.Aging and stress are recognized as two core risk factors for LOD;however, whether they interact and how such interaction is associatedwith the depressive phenotype remain unclear, so we established arat model of LOD to analyze the interactive effects of aging and stresson depressive-like behaviors, and combined this with cerebrospinalfluid proteomics to explore potential protein correlates and provideexperimental evidence for identifying candidate therapeutic targets.In terms of results, aging and stress exhibited a synergistic associationwith depressive-like behaviors in LOD rats, and enrichment was observedin extracellular matrix and sphingolipid metabolic pathways; furthermore,several proteins─including ceramide, MMP2, TIMP2, SPARC, FBN1,and collagen─were identified as candidate correlates worthyof further investigation. In conclusion, stress and aging exert interactiveeffects on behavioral phenotypes in LOD rats, with associated alterationsin extracellular matrix and sphingolipid pathways, and key moleculesincluding Cer, MMP2, Timp2, and Sparc warrant further investigation.

Developing effective therapeutic approaches for tau-associatedneurodegenerative diseases, including frontotemporal dementia andAlzheimer’s disease, remains extremely challenging due to theircomplex pathogeneses. Membrane proteins constitute a large proportionof current therapeutic and drug delivery targets because of theirkey roles in essential cellular processes. Here, we examined alterationsin membrane protein abundances in the prefrontal cortex of male andfemale mouse models of human tauopathy, JNPL3 mice, compared withsex-matched wild-type (WT) mice using quantitative proteomics. Thedata-independent acquisition (DIA) proteomics analysis revealed variousalterations in membrane proteins involved in different altered pathwaysin male JNPL3 mice (e.g., nucleotide metabolism and translation) andfemale JNPL3 mice (e.g., mitochondrial function). In addition, wefound sex-associated changes in the abundances of several proteinspreviously reported to be involved in tau pathology, such as Reelin,DARPP-32, Zyxin, Tbce, and Gng5, in JNPL3 mice. Our multiple-reactionmonitoring absolute quantitative analysis of transporter proteinsrevealed reduced abundance of the main drug efflux transporter ABCB1in male JNPL3 mice compared with WT animals, with no changes in femalegroups. All in all, we provide novel information on membrane proteomicchanges in male and female mouse models of human tauopathy, JNPL3mice, which could aid in developing treatments for tau-associatedneurodegenerative diseases and facilitate the prediction of neuropharmacokineticsin female and male patients.

Immune dysfunction in hematological malignancies arisesfrom bothdisease-related alterations and therapeutic interventions, yet comprehensiveapproaches for characterizing the immune status remain limited. Weevaluated an integrated immune profiling strategy combining antigen-specificserology and high-dimensional B-cell phenotyping. Thirty-eight patientswith B-cell hematological malignancies receiving ibrutinib-, lenalidomide-,and rituximab-based therapies, or no treatment, were analyzed. Antigen-specifichumoral responses were assessed by using a customized protein microarraycontaining 37 microbial antigens, enabling parallel IgG and IgM profiling.B-cell subpopulations were characterized by spectral flow cytometry.Multiomics factor analysis was applied to integrate serological andcellular data sets and identify major sources of immune variability.Distinct serological and cellular immune profiles were observed acrosstherapeutic groups. Patients receiving ibrutinib displayed lower IgGlevels against several microbial antigens and alterations in multipleB-cell compartments, whereas patients receiving lenalidomide-basedtherapy showed comparatively preserved B-cell populations and broaderantigen-specific IgG reactivity. Rituximab-containing regimens wereassociated with marked reductions in several B-cell subsets. Integratedanalysis identified cellular immune features as the major contributorsto interpatient heterogeneity, which suggests the presence of treatment-associatedimmune patterns within the studied cohort. Therefore, integrated serologicaland cellular profiling provides a scalable framework for characterizingan immune fingerprint with potential prognostic value in patientswith B-cell hematological malignancies. These findings support furtherinvestigation of immune monitoring approaches in larger longitudinalcohorts.

Breastcancer cases are rising annually across the globe, withtreatment options limited and slim survival chances at the end stageof cancer; the need for an early diagnosis is crucial. The presentstudy aims to explore a noninvasive method for breast cancer detectionby analyzing urine collected from patients. In this preliminary study,LC-MS-based profiling has been employed on the proteins in urine samplesfrom Gujarat-based breast cancer patients to find potential diagnosticbiomarkers. Differential analysis using limma with Benjamini–Hochbergcorrection revealed 57 proteins that were upregulated in breast cancer,while 61 proteins were significantly downregulated (p-value ≤0.05, FC ≥ 2). ROC curve analysis yielded AUC values above0.7 for candidate proteins, and functional enrichment analysis showedinvolvement of PI3K/AKT signaling, phosphoinositide metabolism, andRNA processing pathways. Based on FDR-controlled differential abundance,individual ROC performance, and biological relevance, TMC5, SFPQ,SLC27A5, PLCH2, SPG11, and INPP4B were prioritized as exploratorycandidate proteins. These proteins demonstrated potential as noninvasiveurinary biomarkers for detection of breast cancer, requiring targetedvalidation in an independent, clinically characterized cohort beforeinvestigating their diagnostic utility.

Prey rely on chemicalcues to detect predators, especially in coastalecosystems where danger is concealed by turbid waters. Juvenile oysters(Crassostrea virginica) respond toblue crab (Callinectus sapidus) chemicalcues by growing stronger shells, while mud crabs (Panopeus herbstii) reduce foraging to avoid detection. However,the chemical composition of marine fear cues is largely unknown, partlydue to methodological challenges. In this study, metabolomics wasused to pinpoint predator metabolites that correlated with inducedjuvenile oyster defenses when exposed to the urine of blue crabs fedeither oysters or mud crabs and to determine the chemical compositionof these fear cues. Genetic algorithms applied to urine mass spectracoupled with bioassay data confirmed that metabolites from blue craburine are responsible for fear responses by oysters. Sixteen compounds(of over 700) were determined to be potentially important cues usedby juvenile oysters to detect blue crabs, with 3-(1-methyl-1H-imidazol-4-yl) propanoic acid identified as the mostimportant contributor to this effect. These results indicate thatoysters rely on certain metabolites that are ubiquitous in a varietyof chemical blends to evaluate predation risk, and the response ofjuvenile oysters is not dependent on a singular metabolite but onnumerous metabolites working in conjunction.

Salmonella Pullorum (SP) infection hasbeen proposedas an experimental model of inflammation in late-stage chicken embryos(CEs). We refined this model by inoculating 9.5 day embryos via allantoiswith a low SP dose and evaluating outcomes at 24 and 48 h postinfection.This approach aims to provide a framework for systematically exploringearly molecular events triggered by infection and inflammation whileusing the CE as an ethically acceptable and biologically relevantmodel of systemic inflammation. Clinical parameters (mortality andweight), serum proteomics, and untargeted metabolomics were assessed.Proteomics was performed by LC–MS/MS, followed by PANTHER andKEGG functional enrichment; metabolomics was performed to assess serumalterations. Mortality increased at 48 h postinfection (30%), andsurviving embryos showed a trend toward reduced weight. Proteomicprofiling revealed an increase in overall protein concentrations inthe infected group compared to the control, despite high zero inflation,with α-2-macroglobulin significantly upregulated at 48 h, indicatingan endogenous immune response. For pathway-level enrichment, Bayesiananalysis suggested subtle effects on proteome dynamics. Metabolomicsidentified time-dependent changes, including increased levels of α-tocopherol-relatedand phenylalanine-derived metabolites and decreased levels of oxidizedfatty acid derivatives at 48 h. These findings support the CE modelbefore 12 days as an ethically refined and sensitive system for studyingacute inflammatory responses.

Etomidate has been linked to addictive behaviors andneurotoxicand hepatotoxic effects. We established a mouse exposure model acrossgraded etomidate doses and assessed reward using conditioned placepreference (CPP). Proteomic and metabolomic profiling of cerebraltissue and right hepatic lobe tissue was performed using data-independentacquisition (DIA) liquid chromatography–tandem mass spectrometryand quadrupole time-of-flight mass spectrometry, respectively; integratedmulti-omics candidates were verified by parallel reaction monitoring(PRM). Both low- and high-dose etomidate significantly increased CPPscores, indicating robust rewarding properties. Neuronal and hepaticlesions increased with dose, with the prefrontal cortex and ventraltegmental area showing the greatest brain vulnerability. Integratedproteomic and metabolomic analyses indicated shared biotransformation-and stress-response features across brain and liver, with brain changesmainly involving xenobiotic metabolism, oxidative stress-related detoxification,and synaptic signaling and cAMP–RAS signaling, and liver changesmainly involving cytochrome P450-mediated biotransformation, lipidand fatty-acid metabolism, and lethal-stage catabolic remodeling.Metabolomics showed dose-stratified pathway disruption: low-dose exposureaffected aminoacyl-tRNA and amino acid biosynthesis, high-dose exposureperturbed amino acid and carbon metabolism, and lethal doses alteredpurine metabolism and glutamatergic pathways. The main innovationof this study lies in establishing a dose-stratified mouse model thatintegrates CPP behavior, brain and liver pathological injury, andcross-organ proteomic–metabolomic profiling, thereby extendingexisting evidence of etomidate-related reward and toxicity and delineatinga combined molecular profile of etomidate-induced reward-related behaviorand organ toxicity.

Histone deacetylase-containingcomplexes play centralroles inchromatin regulation, yet the structural organization of their intrinsicallydisordered regions (IDRs) remains poorly understood. Here, we identifythe previously uncharacterized protein C16orf87, which we renamedMHAP1, as a binding partner of HDAC2 and MIER1. Because all threeproteins contain extensive IDRs that remain difficult to study usingsingle structural analysis methods, we employed an integrative structuralapproach combining cross-linking mass spectrometry (XL-MS) with integrativestructural modeling to define the architecture of the complex. Theresulting integrative structural model reveals that the previouslyuncharacterized C-terminal IDR of HDAC2 acts as a key interactionhub, engaging both the ELM2 domain of MIER1 and the N- and C-terminalregions of MHAP1. These findings provide the first structural frameworkfor the interactions among HDAC2:MIER1:MHAP1 and demonstrate how intrinsicallydisordered regions can mediate assembly of a chromatin-associateddeacetylase complex. Finally, this research further demonstrates theability of XL-MS to facilitate integrative structural modeling ofprotein complexes. Mass spectrometry data may be accessed throughProteomeXchange via PXD082280 and the MassIVE repository MSV000102752.