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

• DOI: 10.1016/j.mcpro.2023.100546
• Publication Date: 2023-04-14T09:12:00Z
• Authors: Sydney Sunna; Christine Bowen; Hollis Zeng; Sruti Rayaprolu; Prateek Kumar; Pritha Bagchi; Eric B. Dammer; Qi Guo; Duc M. Duong; Sara Bitarafan; Aditya Natu; Levi Wood; Nicholas T. Seyfried; Srikant Rangaraju
• Keywords: Profiling (computer programming)

•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. 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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