Capture of the Mouse Organ Membrane Proteome Specificity in Peptidisc Libraries

Journal of Proteome Research, Год журнала: 2024, Номер 23(2), С. 857 - 867

Опубликована: Янв. 17, 2024

Membrane proteins, particularly those on the cell surface, play pivotal roles in diverse physiological processes, and their dysfunction is linked to a broad spectrum of diseases. Despite being crucial biomarkers therapeutic drug targets, low abundance hydrophobic nature pose challenges isolation quantification, especially when extracted from tissues organs. To overcome these hurdles, we developed membrane-mimicking peptidisc, enabling membrane proteome water-soluble library conducive swift identification through liquid chromatography with tandem mass spectrometry. This study applies method across five mice organs, capturing between 200 450 plasma proteins each case. More than just protein identification, peptidisc used estimate relative linking cell-surface molecular functions organ biological roles, thereby contributing ongoing discourse specificity. contribution holds substantial potential for unveiling new avenues exploration downstream applications involving knowledge proteome.

Язык: Английский

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Spatiotemporally resolved mapping of extracellular proteomes via in vivo-compatible TyroID

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Март 15, 2025

Extracellular proteins play pivotal roles in both intracellular signaling and intercellular communications health disease. While recent advancements proximity labeling (PL) methods, such as peroxidase- photocatalyst-based approaches, have facilitated the resolution of extracellular proteomes, their vivo compatibility remains limited. Here, we report TyroID, an vivo-compatible PL method for unbiased mapping with high spatiotemporal resolution. TyroID employs plant- bacteria-derived tyrosinases to produce reactive o-quinone intermediates, enabling multiple residues on endogenous bioorthogonal handles, thereby allowing identification via chemical proteomics. We validate TyroID's specificity by proteomes HER2-neighboring using affibody-directed recombinant tyrosinases. Demonstrating its superiority over other enables including HER2-proximal tumor xenografts, quantifying turnover plasma hippocampal-specific live mouse brains. emerges a potent tool investigating protein localization molecular interactions within living organisms. is that maps through efficiently labels study interactions.

Язык: Английский

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Proteomic Insight Into Alzheimer's Disease Pathogenesis Pathways

PROTEOMICS, Год журнала: 2025, Номер unknown

Опубликована: Янв. 10, 2025

ABSTRACT Alzheimer's disease (AD) is a leading cause of dementia, but the pathogenesis mechanism still elusive. Advances in proteomics have uncovered key molecular mechanisms underlying AD, revealing complex network dysregulated pathways, including amyloid metabolism, tau pathology, apolipoprotein E (APOE), protein degradation, neuroinflammation, RNA splicing, metabolic dysregulation, and cognitive resilience. This review examines recent proteomic findings from AD brain tissues biological fluids, highlighting potential biomarkers therapeutic targets. By examining landscape them, we aim to deepen our understanding support developing precision medicine strategies for more effective interventions.

Язык: Английский

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Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation

Cells, Год журнала: 2025, Номер 14(2), С. 103 - 103

Опубликована: Янв. 12, 2025

Glycosylation plays a critical role in various biological processes, yet identifying specific glycosyltransferase substrates remains challenge due to the complexity of glycosylation. Here, we employ proximity labeling with biotin ligases BASU and TurboID map proximitome MGAT3, responsible for biosynthesis bisecting GlcNAc structure, HEK293T cells. This approach enriched 116 189 proteins, respectively, 17 common shared GlcNAc-bearing proteome obtained via intact glycopeptide enrichment methods. Gene ontology analysis revealed that proteins were predominantly localized exosome, endoplasmic reticulum, Golgi apparatus, consistent subcellular localization MGAT3 substrates. Notably, four novel substrates, GOLM2, CCDC134, ASPH, ERO1A, confirmed bear modification, validating utility method. Furthermore, observed modification inhibits breast cancer progression by promoting degradation α-galactosidase A (GLA). These findings demonstrate efficacy provide insights into functional impact modification.

Язык: Английский

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Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Окт. 26, 2023

Abstract Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS have been identified. Here, we use antibody-directed proximity biotinylation to define the close adhesion molecule Neurofascin. To determine distributions of identified proteins, CRISPR-mediated genome editing for insertion epitope tags endogenous proteins. We identify Contactin-1 (Cntn1) as an protein. Cntn1 is enriched at through interactions with Neurofascin NrCAM. further show that contributes assembly extracellular matrix, regulates innervation by inhibitory basket cells cerebellum chandelier cortex.

Язык: Английский

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Creative approaches using proximity labeling to gain new biological insights

Trends in Biochemical Sciences, Год журнала: 2023, Номер 49(3), С. 224 - 235

Опубликована: Дек. 30, 2023

At its most fundamental level, life is a collection of synchronized cellular processes driven by interactions among biomolecules. Proximity labeling has emerged as powerful technique to capture these in native settings, revealing previously unexplored elements biology. This review highlights recent developments proximity labeling, focusing on methods that push the technologies beyond classic bait-prey paradigm, such RNA–protein interactions, ligand/small-molecule–protein cell surface protein and subcellular trafficking. The advancement address different biological problems will accelerate our understanding complex systems make up life.

Язык: Английский

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Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines

Molecular & Cellular Proteomics, Год журнала: 2023, Номер 22(6), С. 100546 - 100546

Опубликована: Апрель 14, 2023

•Cytosolic TurboID biotinylates >50% of the proteome in microglia and neuronal cells.•TurboID-NES has minimal impacts on cellular proteomic composition function.•About 1340 proteins labeled by differentiate from neurons vitro.•TurboID profiling captures microglial activation lipopolysaccharide. Different brain cell types play distinct roles development disease. Molecular characterization cell-specific mechanisms using type–specific approaches at protein (proteomic) level can provide biological therapeutic insights. To overcome barriers conventional isolation-based methods for proteomics, vivo labeling with proximity-dependent biotinylation cytosolic biotin ligase TurboID, coupled mass spectrometry (MS) proteins, emerged as a powerful strategy proteomics native state cells without need isolation. complement proximity approaches, vitro studies are needed to ensure that proteomes approach representative whole-cell capture responses stimuli disruption processes. address this, we generated murine neuroblastoma (N2A) (BV2) lines stably expressing biotinylate downstream purification analysis MS. TurboID-mediated captured 59% BV2 65% N2A under homeostatic conditions. endolysosome, translation, vesicle, signaling synaptic, neuron projection, microtubule neurons. expression minimally impacted did not affect lipopolysaccharide-mediated cytokine production or resting respiration cells. MS biotin-labeled impact lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory (Il1a, Irg1, Oasl1) decreased anti-inflammatory (Arg1 Mgl2). The is complex organ possessing heterogeneous populations neurons, glia, vascular orchestration interactions within (cell autonomous) between (noncell support higher processes critical development, aging, neurodegeneration. Protein-level analyses expand upon other systems-level analyses, genomics transcriptomics. Specifically, profile total abundances, identify post-translational modifications, resolve protein-level changes occurring subcellular compartments. A central challenge neuroproteomics difficulty obtaining tissue. Traditional isolating fluorescence-activated sorting magnetic-activated require fresh tissue, harsh laborious processing itself poses challenges (1Hulett H.R. Bonner W.A. Barrett J. Herzenberg L.A. Cell sorting: automated separation mammalian function intracellular fluorescence.Science. 1969; 166: 747-749Crossref PubMed Scopus (2) Google Scholar, 2Schmitz B. Radbruch A. Kümmel T. Wickenhauser C. Korb H. Hansmann M.L. et al.Magnetic activated (MACS) — new immunomagnetic method megakaryocytic isolation: comparison different techniques.Eur. Haematol. 1994; 52: 267-275Crossref (112) Scholar). majority adult do survive isolation process, sampling bias healthier non-neuronal able withstand process limits disease states. In addition, contamination derived nontarget persists. maintaining integrity motivated field innovate novel applying systems. One achieve uses BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) which mutated methionyl-tRNA synthetase incorporates azidonorleucine, methionine analog, into newly synthesized peptides (3Tanrikulu I.C. Schmitt E. Mechulam Y. Goddard III, Tirrell D.A. Discovery Escherichia coli mutants efficient azidonorleucine vivo.Proc. Natl. Acad. Sci. U. S. 2009; 106: 15285-15290Crossref (98) 4Link A.J. Vink M.K.S. Agard N.J. Prescher J.A. Bertozzi C.R. aminoacyl-tRNA activity through cell-surface display noncanonical amino acids.Proc. 2006; 103: 10180-10185Crossref (151) 5Dieterich D.C. Link Graumann Schuman E.M. Selective identification bioorthogonal tagging (BONCAT).Proc. 9482-9487Crossref (587) Subsequently, lysates homogenates undergo click chemistry biotin-alkyne azidonorleucine-containing peptides. By driving promoter enriching biotinylated streptavidin affinity (AP), BONCAT purify translated (6Kunkle B.W. Grenier-Boley Sims R. Bis J.C. Damotte V. Naj A.C. al.Genetic meta-analysis diagnosed Alzheimer's identifies risk loci implicates Aβ, tau, immunity lipid processing.Nat. Genet. 2019; 51: 414-430Crossref (1281) 7Alvarez-Castelao Schanzenbächer C.T. Hanus Glock Tom Dieck Dörrbaum A.R. al.Cell-type-specific metabolic nascent vivo.Nat. Biotechnol. 2017; 35: 1196-1201Crossref (108) advantage this lies its ability label low-abundant proteins. limitation may be low depth biases toward high turnover. thus far been applied characterize excitatory inhibitory mice rats both vivo, ex contexts 8Hodas J.J. Nehring Höche N. Sweredoski M.J. Pielot Hess al.Dopaminergic modulation hippocampal neuropil identified (BONCAT).Proteomics. 2012; 12: 2464-2476Crossref (53) 9Di Paolo Farias Garat Macklin Ignatchenko Kislinger al.Rat sciatic nerve axoplasm enriched ribosomal during regeneration processes.J. Proteome Res. 2021; 20: 2506-2520Crossref (7) date, extension glial yet published. contrast labels only proximity-labeling techniques rely ligases, nearby interactors. BioID promiscuous engineered site-specific ligase, BirA, endogenously produced (10Choi-Rhee Schulman Cronan J.E. Promiscuous ligase.Protein 2004; 13: 3043-3050Crossref (174) Because nontoxic, technology opened up opportunities applications, though reaction kinetics takes place over 18 24 h (11Uezu Kanak D.J. Bradshaw T.W. Soderblom E.J. Catavero C.M. Burette al.Identification an elaborate mediating postsynaptic inhibition.Science. 2016; 353: 1123-1129Crossref (184) Alice Ting's group used yeast surface display–mediated directed evolution improve introducing 15 mutations catalytic domain relative wildtype BirA (12Branon T.C. Bosch Sanchez A.D. Udeshi N.D. Svinkina Carr S.A. al.Efficient living organisms TurboID.Nat. 2018; 36: 880-887Crossref (672) This termed robustly promiscuously animal models toxicity, little 10 min culture systems 13Cho K.F. Branon Myers Ting A.Y. Proximity split-TurboID.Nat. Protoc. 2020; 15: 3971-3999Crossref (102) Versatile fused interest map interactomes, targeted compartment interest, exported out nucleus (14Szczesniak L.M. Bonzerato C.G. Wojcikiewicz R.J.H. Identification Bok interactome labeling.Front. Dev. Biol. 9689951Crossref (6) 15Xiong Z. Lo H.P. McMahon K.A. Parton R.G. Hall T.E. dependent labelling zebrafish profiling.Bio 11: e4178Crossref 16Artan M. Barratt Flynn S.M. Begum F. Skehel Nicolas al.Interactome Caenorhabditis elegans synapses TurboID-based labeling.J. Chem. 297101094Abstract Full Text PDF 17Mair Xu S.L. Bergmann complexes cell-type-specific organellar Arabidopsis enabled TurboID.Elife. 8https://doi.org/10.7554/eLife.47864Crossref (104) 18Takano Soderling S.H. Tripartite synaptomics: vivo.Neurosci. 173: 14-21Crossref (8) 19Li Frankenfield A.M. Houston Sekine Hao L. Thiol-cleavable chemical enzymatic application mitochondrial proteomics.J. Am. Soc. Mass Spectrom. 32: 2358-2365Crossref (11) biotin-label proximal intermembrane contact sites, split-TurboID was recently developed two inactive fragments reconstitute presence rapamycin (13Cho 20Cho Rajeev Thoudam al.Split-TurboID enables contact-dependent cells.Proc. 11712143Crossref (117) When driven promoter, cellularly their These advancements, enable robust, targeted, nontoxic proteomes, have yielded promising applications neuroproteomics. Recently, perisynaptic-cleft discovery astrocyte (18Takano 21Takano Wallace J.T. Baldwin K.T. Purkey Uezu Courtland J.L. al.Chemico-genetic astrocytic control inhibition vivo.Nature. 588: 296-302Crossref (78) transgenic mouse model conditional nuclear export sequence (TurboID-NES) also region-specific signatures CamKIIa Aldh1l1 astrocytes (21Takano 22Rayaprolu Bitarafan Santiago J.V. Betarbet Sunna Cheng al.Cell type-specific resolves regional differences brain.Nat. Commun. 2022; 2927Crossref (12) recent advances position (CIBOP) pathological contexts. anticipation TurboID-NES it important establish effects global molecular It breadth TurboID-NES, determine how reflective these untransduced (WC) any inherent approach. order use relevant identity, test if types, such microglia. Finally, ascertain whether induced inflammatory efficiently reliably data interpreting results aim vivo. answer questions controlled experimental conditions, immortalized express V5-TurboID-NES excluding nucleus. We examined extent coverage cells, (LPS)-stimulated label-free quantitation (LFQ) found preserves states challenge. complete table antibodies reagents provided (Tables 1 2).Table 1Antibodies corresponding dilutionsAntibodyManufacturerCatalog numberDilutionRabbit anti-V5Abcamab2065661:500Rat anti-α-tubulinMilliporeSigmaMAB18641:1000Goat anti-β-actinSanta Cruz Biotechnologysc-16151:1000Rabbit anti-histone H3Abcamab17911:5000Donkey anti-rabbit 800InvitrogenA113741:10,000Donkey anti-goat 680LI-CORA210841:10,000Donkey 800LI-CORA113701:10,000Goat anti-rat 800LI-COR926-322191:10,000Streptavidin DyLight 594ThermoFisher218421:1000Streptavidin, Alexa-Fluor 680 conjugateInvitrogenS323581:10,000DAPIRoche102362760011 μg/ml Open tab Table 2Reagents manufacturer catalog numbersReagentManufacturerCatalog numberStartingBlockT20ThermoFisher37543HALT protease & phosphatase inhibitor cocktailThermoFisher78446Dulbecco's modified Eagle's mediumGibco11965-092Penicillin–streptomycinGibco15140-122Fetal bovine serum (FBS)Gibco26140-7079BiotinSigma–AldrichB4639-100mgLPSSigma–AldrichL4391-1mgPuromycinSigma–AldrichP96204% Paraformaldehyde PBSThermo ScientificJ19943-K20.05% Trypsin–EDTAGibco253000054Reagent AThermoFisher23222Reagent BThermoFisher23224Bovine albumin standardsThermoFisher23208LB mediumSigma–Aldrich28713-500G-FSeahorse Flux PackAgilent102601-100Seahorse XF MediaAgilent103575-100Sodium pyruvateSigma–AldrichS8636-100mLl-glutamineSigma–AldrichG7513-100mLGlucoseSigma–AldrichG8769-100mLOligomycinSigma–Aldrich75351-5mgCarbonyl cyanide-4 (trifluoromethoxy) phenylhydrazoneSigma–AldrichC2920-10mgRotenoneSigma–AldrichR8875-1GAntimycin-ASigma–AldrichA8674-25mg were cultured filtered Dulbecco's medium supplemented glucose l-glutamine containing 1% penicillin–streptomycin 10% fetal serum. All media vacuum-filtered 0.2 μm filters. incubated 37 °C 5% CO2 until reaching 80% confluency. splitting regimen took twice weekly, plating one million onto 100 mm plate final volume ml media. preparation experiments, reached 95% confluency 150 plates. Transduced kept 2 puromycin being plated MS, wherein they forwent treatment. V5-TurboID-NES_pCDNA3 plasmid gift (Addgene #107169). Plasmids transformed competent strain DH5α according protocols. Briefly, DH5 α thawed ice before aliquoting 50 μl 1.5 tubes. Constructs diluted 1:1000 autoclaved Milli-Q water. LB prepared diluting 20 g broth, Vegitone 1000 H2O. aliquots DH5α-competent 5 constructs mixed turning tubes upside down. plasmids 30 ice. Following incubation, samples underwent heat shock 42 s placed min. About 500 prewarmed added each sample rotor shake 225 rpm h. Plasmid DNAs purified QIAfilter kits (Midi pre kit; Qiagen; no.: 12243) following manufacturer's protocol. Restriction sites (underlined) introduced via PCR primers (V5.bstb.S; 5′-gcgcctactctagagctagcgaattcgaagccaccatgggcaagcccatccccaa-3′) (nes.Bam.A; 5′-agaaggcacagtcggcggccgcggatccttagtccagggtcaggcgctccagggg-3′). subcloned pCDH-EF1-MCS-BGH-PGK-GFP-T2A-Puro (CD550A-1) sequenced. transduced puromycin-resistant lentivirus construct NES (V5-TurboID-NES) GFP connected T2A linker. Emory University's Viral Vector Core. Given titer × 109 I.U./ml, experimented multiplicities infection (MOIs) 10. triplicates, three wells type received either MOI no virus (untransduced control). 48 transduction, half replaced media, split 1:3 72 transduction. Puromycin selection began 96 after concentration μg/ml. For week, every day puromycin-containing remove nonadhering After weekly maintained puromycin. validated screening procedure assessing percentage GFP-positive fluorescent microscope flow cytometry. positive 3 days addition selection. >90% positive, cm dishes Cells receiving sooner, observe difference transduction efficacy MOIs seeded plating, biotin-supplemented (200 μM) LPS (1 μg/ml) supplementation (n = 6/group). replacement, taken off, centrifuged 800 room temperature (RT) debris, preserved tube. dissociated 2.5 0.05% trypsin–EDTA. trypsin dissociation, rinsed collected manually pipetting 7.5 transferred Supernatants pellets flash-frozen dry Dissociated supernatants RT. tube washed ice-cold 1× PBS. resuspended PBS LoBind Eppendorf (Eppendorf; 022431102). RT harvested urea lysis buffer (8 M urea, mM Tris, NaH2PO4, pH 8.5) HALT cocktail EDTA. then sonicated 30% amplitude thrice on–off pulses disrupt nucleic acids membrane. 4 12,700 rpm. concentrations determined bicinchoninic assay standards. Of six replicates per group, four reserved quality-control performed prior studies. well, μg resolved 12% polyacrylamide gel iBlot Transfer Stack nitrocellulose membrane BOLT transfer system. membranes StartingBlockT20 rabbit anti-V5 primary antibody overnight rapid washes Tris-buffered saline Tween-20 (TBST) followed 10-min TBST. Membranes TBS TBS. secondary visualize donkey V5-tagged TurboID-NES. again previously described undergoing imaging Odyssey Infrared Imaging System (LI-COR Biosciences). Immunofluorescent (IF) staining published modifications (22Rayaprolu ∼50,000 HCl–ethanol-treated 25 coverslips 6-well dish. supplemental duration maintenance. 50% confluency, warm sterile fixed 4% paraformaldehyde min, gently orbitally rotating (IKA; KS 260). Fixed permeabilized blocked simultaneously solution normal horse 0.25% TBST orbital rotator. (1:500 dilution) °C. incubation 4′,6-diamidino-2-phenylindole (DAPI) staining. IF imagings 60× oil-immersion objective Keyence BZ-X810. Colocalization slight previous (23Tamura Uemoto Tabata Augmented liver targeting exosomes modification cationized pullulan.Acta Biomater. 57: 274-284Crossref (105) 24Yarandi S.S. Robinson Vakili Donadoni Burdo T.H. Sariyer I.K. Characterization Nef regions SIV-infected macaques.PLoS One. 15e0241667Crossref subtracted target area (μm2) DAPI-signal overlap V5 signal (nuclear-localizing respectively) BZ-X810 Analyzer hybrid-cell-count colocalization software. Only DAPI Streptavidin analyzed analysis. Significance p values assessed two-tailed Mann–Whitney test. counts (number counted because overlapping signal) V5–DAPI 1876 1945 DyLight–DAPI naïve 2289 1638 triplicates plates grown 70% Cellular monolayers PBS, aspirated. scraped scraper 2.97 100× HALT, immediately use. slurries 1000g removed, pellet once cold obtain WC fraction, slurry 0.5 remaining 900 hypotonic (10 Hepes, 7.9, KCl, 0.1 EDTA, DTT, glycerol, PMSF, aprotinin, leupeptin, 0.1% NP-40, HALT) pellets. 15,600g cytoplasmic fractions. nuclei salt (20 0.4 NaCl, EGTA, fractions puls

Язык: Английский

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Photoproximity labeling of endogenous receptors in the live mouse brain in minutes

Nature Chemical Biology, Год журнала: 2024, Номер unknown

Опубликована: Авг. 1, 2024

Язык: Английский

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Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics

JACS Au, Год журнала: 2023, Номер 3(12), С. 3506 - 3523

Опубликована: Дек. 13, 2023

The plasma membrane proteome is a rich resource of functionally important and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, low overall abundance, the cell surface remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here, we paired glycoprotein capture with to establish two-stage enrichment method that enables chemoproteomic profiling Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total cysteines 1,046 proteins, 1,907 residues not previously captured bulk analysis. By pairing Cys-Surf an isotopic readout, uncovered 821 ligandable cysteines, known novel sites. also robustly delineates redox-sensitive prone activation-dependent changes oxidation state sensitive addition exogenous reductants. Exemplifying capacity delineate identified redox low-density lipoprotein receptor (LDLR) impacts both localization uptake (LDL) particles. Taken together, platform, distinguished its paradigm, represents tailored approach functional therapeutic potential cysteinome.

Язык: Английский

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Proteomics-based synapse characterization: From proteins to circuits

Current Opinion in Neurobiology, Год журнала: 2023, Номер 79, С. 102690 - 102690

Опубликована: Фев. 17, 2023

The highly heterogeneous nature of neuronal cell types and their connections presents a major challenge to the characterization neural circuits at protein level. New approaches now enable an increasingly sophisticated dissection type- cellular compartment-specific proteomes, as well profiling composition specific synaptic connections. Here, we provide overview these discuss how they hold considerable promise toward unravelling molecular mechanisms circuit formation function. Finally, outlook technological developments that may bring proteomes single-synapse level within reach.

Язык: Английский

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