Zikomo pochezera Nature.com.Mtundu wa msakatuli womwe mukugwiritsa ntchito uli ndi chithandizo chochepa cha CSS.Kuti mudziwe zambiri, tikupangira kuti mugwiritse ntchito msakatuli wosinthidwa (kapena kuletsa Compatibility Mode mu Internet Explorer).Pakadali pano, kuti tithandizire kupitilizabe, tidzapereka tsambalo popanda masitayilo ndi JavaScript.
Njira zolembera za ma enzymatic proximity zotengera ma esters otsegulidwa kapena ma phenoxy radicals amagwiritsidwa ntchito kwambiri popanga mapu a subcellular proteins ndi protein interactors m'maselo amoyo.Komabe, ma esters omwe adatsegulidwa sachitapo kanthu, zomwe zimapangitsa kuti pakhale ma radius ambiri, ndipo ma phenoxy radicals opangidwa ndi mankhwala a peroxide amatha kusokoneza njira za redox.Apa tikunena za njira ya proximity labeling dependent photoactivation (PDPL) yopangidwa polumikiza puloteni ya miniSOG photosensitizer ndi puloteni yosangalatsa.Kuyambitsidwa ndi kuwala kwa buluu ndikuwongoleredwa ndi nthawi yowonekera, okosijeni wa singlet amapangidwa ndiyeno zotsalira za histidine zotsatiridwa ndi aniline zimatheka.Timawonetsa kukhulupirika kwake kwakukulu kudzera mu mapu a organelle-specific proteinome.Kuyerekeza mbali ndi mbali kwa PDPL ndi TurboID kukuwonetsa kufalikira kwatsatanetsatane komanso kokwanira kwa PDPL.Kenaka, tinagwiritsa ntchito PDPL ku coactivator yokhudzana ndi matenda BRD4 ndi E3 Parkin ligase ndipo tinapeza ogwirizanitsa omwe poyamba sankadziwika.Pogwiritsa ntchito kuwonetsetsa mopitirira muyeso, magawo awiri osadziwika, Ssu72 ndi SNW1, adadziwika kwa Parkin, yemwe kuwonongeka kwake kumayendetsedwa ndi njira ya ubiquitination-proteasome.
Kudziwika bwino kwa ma netiweki a protein kumapangitsa kuti pakhale njira zambiri zoyambira ma cell.Choncho, mapu olondola kwambiri a spatiotemporal okhudzana ndi mapuloteni adzapereka maziko a mamolekyu kuti adziwe njira zamoyo, matenda a matenda, ndi kusokoneza kuyanjana kumeneku pofuna kuchiza.Kuti izi zitheke, njira zomwe zimatha kuzindikira kuyanjana kwakanthawi m'maselo amoyo kapena minofu ndizofunika kwambiri.Affinity Purification Mass Spectrometry (AP-MS) yakhala ikugwiritsidwa ntchito kuzindikiritsa anthu omwe amamanga ma protein of interest (POIs).Ndi chitukuko cha kuchuluka kwa ma proteomics, Bioplex3.0 idapangidwa, nkhokwe yayikulu kwambiri yama protein network yotengera AP-MS.Ngakhale AP-MS ndi yamphamvu kwambiri, ma cell lysis ndi dilution masitepe pamayendetsedwe a ntchito amakondera pazolumikizana zofooka komanso zosakhalitsa ndikuyambitsa zinthu zakale za post-lysis monga ma spurious interaction awiriawiri omwe alibe compartmentalization isanachitike lysis.
Pofuna kuthana ndi izi, ma amino acid omwe si achilengedwe (UAA) okhala ndi magulu ophatikizika komanso nsanja zolembera za enzymatic (PL) (mwachitsanzo APEX ndi BioID)5 apangidwa.Ngakhale kuti njira ya UAA yakhala ikugwiritsidwa ntchito bwino muzochitika zambiri ndipo imapereka chidziwitso pa zomatira mwachindunji za mapuloteni, kukhathamiritsa kwa malo oyikapo UAA kumafunikabe.Chofunika kwambiri, ndi njira yolembera stoichiometric yomwe ilibe kusintha kothandizira pakulemba zochitika.Mosiyana, njira za enzymatic PL, monga njira ya BioID, zimaphatikizira biotin ligase ku POI7, yomwe pambuyo pake imayambitsa biotin kupanga biotinyl-AMP ester yapakatikati.The enzyme motero imathandizira ndikutulutsa "mtambo" wa biotin womwe umalemba zotsalira za lysine.Komabe, BioID imafuna maola opitilira 12 kuti ipeze chizindikiro chokwanira cholembedwa, chomwe chimalepheretsa kugwiritsidwa ntchito kwake ndikusintha kwakanthawi.Pogwiritsa ntchito chisinthiko cholunjika chotengera chiwonetsero cha yisiti, TurboID idapangidwa kutengera BioID kuti ikhale yogwira ntchito bwino, kulola kulemba bwino ndi biotin mkati mwa mphindi 10, kulola kuti njira zosinthira ziziwerengedwa.Chifukwa TurboID ndi yogwira ntchito kwambiri komanso ma biotin amtundu wa endogenous ndi okwanira kuti alembe zilembo zapang'onopang'ono, kulemba zilembo zakumbuyo kumakhala vuto lomwe lingakhale vuto ngati kuwonjezeredwa kwanthawi yayitali kumafunika powonjezerapo ma biotin akunja.Kuonjezera apo, ma esters otsegulidwa sachita bwino (t1/2 ~ 5 min), zomwe zingapangitse kuti pakhale malo ozungulira kwambiri, makamaka pambuyo podzaza mapuloteni oyandikana nawo ndi biotin 5. Mwa njira ina, kuphatikizika kwa majini a injiniya ascorbate peroxidase (ie biotin- phenol radicals ndipo amalola kulembedwa kwa mapuloteni mkati mwa mphindi imodzi9, 10. APEX imagwiritsidwa ntchito kwambiri pozindikira ma proteomes a subcellular, membrane protein complexes, ndi cytosolic signing protein complexes11, 12. njira zama cellular.
Choncho, njira yatsopano yomwe ingathe kupanga mitundu yowonjezereka yodziwika bwino yotchedwa-radius kupondereza mitundu yokhala ndi malo olondola kwambiri komanso osakhalitsa popanda kusokoneza kwambiri njira zama cell idzakhala yofunikira kuwonjezera pa njira zomwe zilipo kale. Pakati pa zamoyo zowonongeka, mpweya wa singlet udadzutsa chidwi chathu chifukwa cha moyo wake waufupi komanso utali wocheperako (t1/2 <0.6 µs m'maselo)13. Pakati pa zamoyo zowonongeka, mpweya wa singlet udadzutsa chidwi chathu chifukwa cha moyo wake waufupi komanso utali wocheperako (t1/2 <0.6 µs m'maselo)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии 3,1/3 (t. Pakati pa mitundu yogwira ntchito, okosijeni wa singlet adakopa chidwi chathu chifukwa cha moyo wake waufupi komanso ma radius ochepa (t1/2 <0.6 µs m'maselo)13.在活性物质中，单线态氧因其寿命短和扩散半径有限（细胞中t1/2 <0.6 µs）而引起了我们的 1/2 <0.6 µs）而引起了我們的注意13 Среди активных форм наше внимание привлекает синглетный кислород из-за его короткого времени жизни и ограниченного радиуса дифф6/зим2 (канглетный кислород). Pakati pa mitundu yogwira ntchito, okosijeni wa singlet amakopa chidwi chathu chifukwa cha moyo wake waufupi komanso ma radius ochepa (t1/2 <0.6 μs m'maselo).Mpweya umodzi wa okosijeni wanenedwa kuti umatulutsa methionine, tyrosine, histidine ndi tryptophan mwachisawawa, ndikupangitsa kuti 14,15 ikhale yogwirizana ndi amine kapena thiol based probes16,17.Ngakhale okosijeni wa singlet wakhala akugwiritsidwa ntchito kuyika ma subcellular compartment RNA, njira zobwezeretsanso zolembera zoyandikira za POI sizinadziwikebe.Apa, tikuwonetsa nsanja yotchedwa photoactivation-dependent proximity labeling (PDPL), komwe timagwiritsa ntchito kuwala kwa buluu kuunikira ma POI ophatikizidwa ndi miniSOG photosensitizer ndikuyambitsa m'badwo wa okosijeni wa singlet kuti oxidize zotsalira zotsalira, zotsatiridwa ndi zosinthidwa zomwe zili ndi amine kuti zilowetse ma probe amakhemikolo. maselo amoyo apakati..Tidayesa gulu lazofufuza zamankhwala kuti tiwonjezere kutsimikizika kwa ma tag ndikuzindikira malo osinthidwa pogwiritsa ntchito mayendedwe otseguka a proteinomics.Kuyerekeza mbali ndi mbali kwa PDPL ndi TurboID kukuwonetsa kufalikira kwatsatanetsatane komanso kokwanira kwa PDPL.Tidagwiritsa ntchito njira iyi pazizindikiro zapadera za subcellular proteome ndi chizindikiritso chonse cha proteome cha omwe amamangiriza omwe amamangirira mapuloteni okhudzana ndi khansa a BRD4 ndi E3 ligase Parkin yokhudzana ndi matenda a Parkinson, zomwe zidatsimikizira kuti mapuloteni odziwika komanso osadziwika kuyanjana..Kuthekera kwa PDPL kuzindikira magawo a E3 m'mapuloteni akulu akulu kumayimira nthawi yomwe kuzindikira zomangira zosalunjika kumafunikira.Magawo awiri osadziwika a parkin omwe amayanjanitsidwa ndi ubiquitination-proteasome atsimikiziridwa mu situ.
Photodynamic therapy (PDT)19 ndi chromophore-assisted laser inactivation (CALI)20, momwe kuwala kwa kuwala kokhala ndi photosensitizer kumatulutsa mpweya wa singlet, kumatha kulepheretsa mapuloteni omwe amawatsata kapena kuchititsa kufa kwa maselo.Popeza mpweya wa singlet ndi chinthu chomwe chimagwira ntchito kwambiri chokhala ndi mtunda wongoyerekeza wa pafupifupi 70 nm, okosijeni wocheperako mozungulira photosensitizer amatha kuwongoleredwa.Kutengera lingaliro ili, tidaganiza zogwiritsa ntchito mpweya wa singlet kuti tikwaniritse zilembo zama protein complexes m'maselo amoyo.Tapanga njira ya PDPL chemoproteomic kuti tikwaniritse ntchito zinayi: (1) kulimbikitsa mbadwo wa oxygen yogwira ntchito mofanana ndi njira ya PL enzymatic;(2) perekani zolemba zokhazikika pakanthawi koyambitsa kuwala;(3) posintha (4) Pewani kugwiritsa ntchito ma cofactors amkati (monga biotin) kuti muchepetse maziko, kapena gwiritsani ntchito ma reagents osokoneza bongo (monga peroxides) kuti muchepetse kukhudzana ndi kupsinjika kwa chilengedwe.
Ma Photosensitizer atha kugawidwa m'magulu awiri kuphatikiza ma cell ang'onoang'ono olemera a fluorophores (monga rose bengal, methylene blue)22 ndi mapuloteni ang'onoang'ono okhala ndi ma genetic (monga miniSOG, KillerRed)23.Kuti tikwaniritse mapangidwe okhazikika, tinapanga nsanja ya PDPL ya m'badwo woyamba powonjezera mapuloteni a photosensitizer (PS) ku POI24,25 (Chithunzi 1a).Ikayatsidwa ndi kuwala kwa buluu, singlet oxygen imatulutsa ma proximal nucleophilic amino acid zotsalira, zomwe zimapangitsa kuti pakhale polarity yomwe ili ndi electrophilic ndipo imatha kuchitanso ndi amine probe nucleophiles16,17.Pulogalamuyi idapangidwa ndi chogwirira cha alkyne kuti ilole chemistry ndikutsitsa kuti iwonetse mawonekedwe a LC/MS/MS.
Chithunzi chojambula cholemba ma protein complexes omwe ali pakati pa miniSOG.Akayatsidwa ndi kuwala kwa buluu, ma cell owonetsa miniSOG-POI amatulutsa mpweya wa singlet, womwe umasintha mapuloteni olumikizana koma osamanga.Zopangira zapakatikati za photooxidation zimalandidwa ndi zolemba zotumizirana ndi amine chemical probe kuti apange covalent adducts.Gulu la alkynyl pa kafukufuku wa chemistry limalola kudina kwa chemistry kuti kulemeretse ndi kukokera pansi ndikutsatiridwa ndi kuchuluka kwa LC-MS/MS.b Kapangidwe kakemidwe ka amine probes 1-4.c Kusanthula kwa gel oyimira fulorosenti ya mitochondrial localized miniSOG-mediated proteomic markers pogwiritsa ntchito ma probe 1-4 ndi kuchuluka kwachibale kutengera gel densitometry.Chiyerekezo cha ma sign-to-background a ma probes amankhwala adawunikidwa pogwiritsa ntchito kuyesa koyipa kupatula kuwala kwa buluu kapena kugwiritsa ntchito ma cell a HEK293T opanda mawu a miniSOG.n = 2 zitsanzo zodziyimira pawokha.Dontho lililonse likuyimira chithunzithunzi chachilengedwe.d Kuzindikira koyimilira ndi kuchuluka kwa PDPL pogwiritsa ntchito probe 3 pakupezeka kapena kusakhalapo kwa zigawo za PDPL monga c.n = 3 zitsanzo zodziyimira pawokha.Dontho lililonse likuyimira chithunzithunzi chachilengedwe.Mizere yapakati ndi ndevu zimayimira kutanthauzira ndi ± kupatuka kokhazikika.CBB: Coomassie Brilliant Blue.e Confocal imaging ya singlet oxygen yokhala ndi banga lofiira kwambiri la Si-DMA.Scale bar: 10 µm.Kujambula kwa gel ndi kuyesa kosakanikirana kunabwerezedwa paokha osachepera kawiri ndi zotsatira zofanana.
Poyamba tinayesa luso la okhwima photosensitizers miniSOG26 ndi KillerRed23, stably anasonyeza HEK293T, kuti mkhalapakati propargylamine kulemba proteome monga kafukufuku mankhwala (Supplementary Mkuyu. 1a).Kusanthula kwa gel fluorescence kunawonetsa kuti zolemba zonse za proteome zidakwaniritsidwa pogwiritsa ntchito miniSOG ndi kuwala kwa buluu, pomwe palibe cholembera chomwe chidawonedwa ndi KillerRed.Kuti tipititse patsogolo chiŵerengero cha chizindikiro ndi maziko, tinayesa ma probe a mankhwala okhala ndi aniline (1 ndi 3), propylamine (2), kapena benzylamine (4).Tinawona kuti maselo a HEK293T okha anali ndi chizindikiro chapamwamba kwambiri poyerekeza ndi kuwala kwa buluu, mwinamwake chifukwa cha endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27. Ma probe a mankhwala opangidwa ndi aniline 1 ndi 3 adapereka kutsimikizika kwabwinoko, pomwe HEK293T ikuwonetseratu miniSOG mu mitochondria kuwonetsa>> 8-kuwonjezeka kwa siginecha ya probe 3, pomwe probe 2 yomwe imagwiritsidwa ntchito munjira yolembera ya RNA CAP-seq imangowonetsa ~2.5- pindani chizindikiro kuwonjezeka, mwina chifukwa cha zosiyana reactivity zokonda pakati RNA ndi mapuloteni (Fig. 1b, c). Ma probe a mankhwala opangidwa ndi aniline 1 ndi 3 adapereka kutsimikizika kwabwinoko, pomwe HEK293T ikuwonetseratu miniSOG mu mitochondria kuwonetsa>> 8-kuwonjezeka kwa siginecha ya probe 3, pomwe probe 2 yomwe imagwiritsidwa ntchito munjira yolembera ya RNA CAP-seq imangowonetsa ~2.5- pindani chizindikiro kuwonjezeka, mwina chifukwa cha zosiyana reactivity zokonda pakati RNA ndi mapuloteni (Fig. 1b, c).Ma probe a mankhwala opangidwa ndi aniline 1 ndi 3 adawonetsa kutsimikizika kwabwinoko: HEK293T, yomwe imafotokoza mokhazikika miniSOG mu mitochondria, ikuwonetsa kuchulukitsa kwa 8 kwa chizindikiro cha probe 3, pomwe probe 2, yogwiritsidwa ntchito munjira yolembera ya CAP-seq RNA, kokha imasonyeza ~ kuwonjezeka kwa chizindikiro cha 2.5, mwinamwake chifukwa cha zosiyana reactivity zokonda pakati pa RNA ndi mapuloteni (Mkuyu 1b, c).基于 的 探针 1 和 3 和 3 具有 更 好 的 增加 的 的 表达 表达 表达 的 的 的 2 的 的仅2.5- 倍信号增加，可能是由于RNA 和蛋白质之间的不同反应偏好（图1b，c）.基于 的 探针 1 和 3 和 3 的 更 的 特异性 增加 的 的 的 的 v - 倍信号增加，可能是由于RNAMa probe a mankhwala opangidwa ndi aniline 1 ndi 3 anali odziwika bwino, HEK293T adawonetsa miniSOG mokhazikika mu mitochondria, ndipo kafukufuku 3 anali ndi kuchulukitsa kwa siginecha ka 8, pomwe probe 2 ya njira yolembera ya CAP-seq RNA idangowonjezera ~ 2.5 -kuwonjezeka.mu chizindikiro, mwina chifukwa osiyana anachita zokonda pakati RNA ndi mapuloteni (mkuyu. 1b, c).Kuonjezera apo, kafukufuku wa 3 isomers ndi hydrazine probes (probes 5, 6, 7) adayesedwa, kutsimikizira kukhathamiritsa kwa probe 3 (Supplementary Fig. 1b, c).Mofananamo, kusanthula kwa gel osakaniza fluorescence kunavumbulutsa magawo ena oyezetsa bwino: kuwala kwa kuwala (460 nm), mankhwala a probe concentration (1 mM), ndi nthawi yowunikira (20 min) (Supplementary Fig. 2a-c).Kusiya chigawo chilichonse kapena sitepe ya PDPL protocol inachititsa kuti zizindikiro zisinthe kumbuyo (mkuyu 1d).Makamaka, kulembedwa kwa mapuloteni kunachepetsedwa kwambiri pamaso pa sodium azide kapena trolox, yomwe imadziwika kuti imayimitsa mpweya wa singlet.Kukhalapo kwa D2O, komwe kumadziwika kuti kukhazikitsira mpweya wa singlet, kumawonjezera chizindikiro cholembera.Kuti afufuze zakuthandizira kwa mitundu ina ya okosijeni yokhazikika pakulemba zilembo, mannitol ndi vitamini C adawonjezeredwa kuti akhazikitse hydroxyl ndi superoxide radical scavengers, motsatana, 18, 29, koma sanapezeke kuti achepetse zilembo.Kuwonjezera kwa H2O2, koma osati kuunikira, sikunapangitse kulemba (Supplementary Fig. 3a).Fluorescence singlet oxygen imaging yokhala ndi ma probe a Si-DMA inatsimikizira kukhalapo kwa oxygen ya singlet mu waya wa HEK293T-miniSOG, koma osati mu waya woyambirira wa HEK293T.Kuonjezera apo, mitoSOX Yofiira sinathe kuzindikira kupanga kwa superoxide pambuyo pa kuunikira (Mkuyu 1e ndi Supplementary Fig. 3b) 30. Deta iyi imasonyeza kuti singlet oxygen ndiyo mtundu waukulu wa okosijeni womwe umayambitsa kulembedwa kwa proteomic.Cytotoxicity ya PDPL inayesedwa kuphatikizapo kuwala kwa buluu ndi kufufuza kwa mankhwala, ndipo palibe cytotoxicity yaikulu yomwe inawonedwa (Supplementary Fig. 4a).
Kuti tiphunzire makina olembera ndikupangitsa kuti proteomic izindikirike zama protein complexes pogwiritsa ntchito LC-MS/MS, choyamba tiyenera kudziwa kuti ndi ma amino acid ati omwe amasinthidwa komanso kuchuluka kwa ma probe labels.Methionine, histidine, tryptophan ndi tyrosine zanenedwa kuti zasinthidwa ndi singlet oxygen14,15.Timaphatikiza mayendedwe a TOP-ABPP31 ndi kusaka kotseguka kopanda tsankho komwe kumaperekedwa ndi FragPipe computing platform potengera MSFragger32.Pambuyo pa kusintha kwa mpweya wa singlet ndi kulemba zolemba za mankhwala, dinani chemistry idapangidwa pogwiritsa ntchito chizindikiro chochepetsera cha biotin chokhala ndi cholumikizira cholumikizira, chotsatiridwa ndi kutambasula kwa neutravidin ndi trypsin digestion.Peptide yosinthidwa, yomwe imamangiriridwabe ku utomoni, inali photocleaved kwa LC-MS/MS kusanthula (Chithunzi 2a ndi Supplementary Data 1).Zosintha zambiri zidachitika mu proteome yokhala ndi mapu opitilira 50 a peptide (PSM) omwe adalembedwa (mkuyu 2b).Chodabwitsa, tidangowona kusinthidwa kwa histidine, mwina chifukwa cha kuchuluka kwa oxidized histidine kupita ku probes ya aniline kuposa ma amino acid ena.Malinga ndi njira yofalitsidwa ya histidine oxidation ndi singlet oxygen, 21,33 dongosolo la delta-mass la +229 Da likufanana ndi kulowetsedwa kwa probe 3 ndi 2-oxo-histidine pambuyo pa ma oxidation awiri, pamene +247 Da ndi mankhwala a hydrolysis. wa +229 Da (Wowonjezera Fig. 5).The kuunika kwa sipekitiramu MS2 anasonyeza kudalirika mkulu chizindikiritso cha ambiri y ndi b ayoni, kuphatikizapo chizindikiritso cha kusinthidwa fragment ayoni (y ndi b) (mkuyu. 2c).Kusanthula kwazomwe zimachitika m'dera la PDPL-modified histidines kunawonetsa zokonda zapakatikati zotsalira za hydrophobic pa malo a ± 1 (Supplementary Fig. 4b).Pa avareji, 1.4 histidines anazindikirika pa mapuloteni, ndi malo zolemberazi anatsimikiza ndi zosungunulira Kufikika pamwamba m'dera (SASA) ndi wachibale zosungunulira kupezeka (RSA) kusanthula (Supplementary mkuyu. 4c, d).
Kuyenda kopanda tsankho pophunzira kusankha kotsalira pogwiritsa ntchito nsanja ya FragPipe yoyendetsedwa ndi MSFragger.Zolumikizira zosinthika zimagwiritsidwa ntchito mu Click chemistry kulola kujambulidwa kwa ma peptide osinthidwa kuchokera ku streptavidin resin.Kusaka kotseguka kunayambika kuti muzindikire zosintha zambiri, komanso zotsalira zoyenera.b Perekani kuchuluka kwa zosinthidwa zomwe zikuchitika mu proteome yonse.Mapu a Peptide a PSM.c MS2 spectral annotation of histidine sites modified with probe 3. Monga chitsanzo choyimira, covalent reaction with probe 3 anawonjezera +229.0938 Da ku modified amino acid.d Kuyesa kosinthika komwe kumagwiritsidwa ntchito poyesa zolembera za PDPL.PRDX3 (H155A, H225A) ndi PRDX1 (H10A, H81A, H169A) adasamutsidwa ndi ma plasmids amtchire kuti azindikire zotsutsana ndi mbendera.e Peptide yopangidwa idachitidwa ndi miniSOG yoyeretsedwa pamaso pa kafukufuku 3 ndi zinthu zofananira ndi Δm +247 ndi +229 zidadziwika mu mawonekedwe a LC-MS.f In vitro protein-to-protein interactions opangidwa ndi miniSOG-6xHis-tag ndi anti-6xHis antibody.Antibiotin (streptavidin-HRP) ndi anti-mouse Western blot kusanthula kwa miniSOG-6xHis/anti-6xMa anti-6xMa antibody ake olembedwa ndi probe 3, malingana ndi nthawi yowunikira.Zolemba zamapuloteni amtundu uliwonse zimawonetsedwa mu kulemera kwa maselo: LC antibody light chain, HC antibody heavy chain.Zoyesererazi zidabwerezedwa mosayembekezeka kawiri ndi zotsatira zofanana.
Pakutsimikizira kwachilengedwe kwa malo olemberapo, PRDX3 ndi PRDX1 zozindikiridwa ndi ma spectrometry ambiri zidasinthidwa kuchokera ku histidine kupita ku alanine ndikuyerekeza ndi mtundu wakutchire pakuyesa kutengera kachilomboka.Zotsatira za PDPL zinasonyeza kuti kusinthaku kunachepetsa kwambiri kulemba (mkuyu 2d).Pakadali pano, zotsatizana za peptide zomwe zidadziwika pakufufuza kotseguka zidapangidwa ndikuchitidwa mu vitro ndi miniSOG yoyeretsedwa pamaso pa probe 3 ndi kuwala kwa buluu, zotulutsa zotulutsa ndi kusintha kwakukulu kwa +247 ndi +229 Da zikapezeka ndi LC-MS (Mkuyu. .2e).).Kuti tiwone ngati ma proximal proteins amatha kulembedwa mu vitro poyankha miniSOG kujambula, tidapanga njira yoyeserera yoyandikira polumikizana pakati pa mapuloteni a miniSOG-6xHis ndi anti-His monoclonal antibody in vitro (Chithunzi 2f).Pakuyesa uku, tinkayembekezera kulembedwa kwa ma antibody olemetsa komanso opepuka ndi miniSOG.Ndipotu, anti-mouse (kuzindikira maunyolo olemera ndi opepuka a anti-6xHis-labeled antibody) ndi streptavidin Western blots anasonyeza biotinylation yamphamvu ya unyolo wolemera ndi wopepuka.Makamaka, tawona miniSOG autobiotinylation chifukwa cha 6xHis tag ndi maulalo pakati pa unyolo wopepuka ndi wolemetsa, womwe ungakhale wokhudzana ndi kusiyana komwe kwafotokozedwa kale pakati pa lysine ndi 2-oxo-histidine kuyankha kwapakatikati.Pomaliza, timaganiza kuti PDPL imasintha histidine modalira moyandikana.
Cholinga chathu chotsatira chinali kuwonetsa ma subcellular proteome kuyesa kutsimikizika kwa zilembo za in situ.Choncho, ife stably anasonyeza miniSOG mu phata, mitochondrial matrix, kapena kunja ER nembanemba wa HEK293T maselo (Mkuyu 3a).Kusanthula kwa gel fluorescence kunavumbulutsa magulu ambiri olembedwa m'malo atatu ang'onoang'ono komanso mitundu yosiyanasiyana ya zilembo (mkuyu 3b).Fluorescence imaging kusanthula anasonyeza mkulu specificity wa PDPL (mkuyu 3c).Mayendedwe a PDPL adatsatiridwa ndikudina kochita ndi utoto wa rhodamine kuti afotokozere ma proteomes ang'onoang'ono pogwiritsa ntchito ma microscopy a fluorescence, ndipo ma sign a PDPL adalumikizidwa ndi DAPI, trackers mitochondrial, kapena ER trackers, kutsimikizira kukhulupirika kwakukulu kwa PDPL.Kwa malo atatu a organelle, kufananitsa mbali ndi mbali kwa PDPL ndi TurboID pogwiritsa ntchito avidin chakumadzulo blot kunasonyeza kuti PDPL inalembedwa makamaka poyerekeza ndi machitidwe awo.Pansi pa zikhalidwe za PDPL, magulu olembedwa ambiri adawonekera, akuwonetsa mapuloteni olembedwa ndi PDPL (Supplementary Fig. 6a-d).
chiwonetsero cha Schematic cha miniSOG-mediated organelle-specific proteome labeling.miniSOG imayang'ana matrix a mitochondrial kudzera kuphatikizidwira ku N-terminal 23 amino acid yaumunthu COX4 (mito-miniSOG), nyukiliya kudzera kusakanikirana kupita ku H2B (nucleus-miniSOG), ndi Sec61β kudzera pa cytoplasmic mbali ya ER membrane (ER-miniSOG ).Zizindikiro zimaphatikizapo kujambula kwa gel, kujambula kwa confocal, ndi mass spectrometry.b Zithunzi za gelisi zoyimira za mbiri ya PDPL ya organelle.CBB Coomassie Brilliant Blue.c Zithunzi zoyimilira za ma cell a HEK293T akuwonetsa mokhazikika miniSOG yokhala ndi ma cell osiyanasiyana omwe amadziwika ndi antibody otchedwa V5 (wofiira).Zolemba zazing'ono zimagwiritsidwa ntchito pa mitochondria ndi ER (zobiriwira).Mayendedwe a PDPL amaphatikizanso kuzindikira kwa miniSOG (yellow) otchedwa subcellular proteins pogwiritsa ntchito Cy3-azide click chemistry.Scale bar: 10 µm.d Mapangidwe a Volcanic a PDPL-tagged proteomes mu organelles osiyanasiyana otsimikiziridwa ndi kuchuluka kosalembedwa (n = 3 kuyesa kwachilengedwe kodziyimira pawokha).Mayeso a Wophunzira a michira iwiri adagwiritsidwa ntchito pamagawo ophulika.HEK293T mtundu wakuthengo unagwiritsidwa ntchito ngati kuwongolera koyipa. Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> 2-fold ion intensity kusiyana). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> 2-fold ion intensity kusiyana). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> kusiyana kwa 2-fold intensity ion).显着变化的蛋白质以红色突出显示（p <0.05 和> 2 倍离子强度差异）.显着变化的蛋白质以红色突出显示（p <0.05和> 2 Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в ионной силе). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> kusiyana kwa 2-fold in ionic mphamvu).Mapuloteni ogwirizana nawo ofunikira pa HEK293T-miniSOG koma osafunikira kwa HEK293T amawonetsedwa mobiriwira.e Kuwunika kwapadera kwa dataset ya proteinomic kuchokera ku zoyeserera d.Chiwerengero chonse cha mapuloteni ofunika kwambiri mu organelle iliyonse (madontho ofiira ndi obiriwira) amalembedwa pamwamba.Ma histograms amawonetsa mapuloteni omwe amapezeka mu organelles kutengera MitoCarta 3.0, GO kusanthula ndi A. Ting et al.anthu.Olekanitsa ma dataset a mitochondria, nuclei, ndi ER.Zoyesererazi zidabwerezedwa mosayembekezeka kawiri ndi zotsatira zofanana.Zambiri zimaperekedwa m'mafayilo a data yaiwisi.
Kulimbikitsidwa ndi zotsatira za gel ndi zojambula, kuwerengera kopanda zilembo kunagwiritsidwa ntchito kuti ayese proteome yodziwika mu organelle iliyonse (Supplementary Data 2).HEK293T yopanda kachilombo idagwiritsidwa ntchito ngati njira yoletsa kuchotsa zolembera zakumbuyo. Kusanthula chiwembu cha volcano kunawonetsa mapuloteni olemera kwambiri (p <0.05 ndi> 2-fold ion intensity) komanso mapuloteni a singleton omwe amapezeka m'mizere yowonetsera miniSOG (mkuyu 3d madontho ofiira ndi obiriwira). Kusanthula chiwembu cha volcano kunawonetsa mapuloteni olemera kwambiri (p <0.05 ndi> 2-fold ion intensity) komanso mapuloteni a singleton omwe amapezeka m'mizere yowonetsera miniSOG (mkuyu 3d madontho ofiira ndi obiriwira). Анализ графика вулкана показал значительно обогащенные белки (p <0, 05 и > 2-кратная интенсивность ионов), а также одиночные белки, которые присутствуют только в линиях, экспрессирующих miniSOG (рис. 3d, красные и зеленые точки). Kusanthula kwachiwembu cha volcano kunawonetsa mapuloteni olemera kwambiri (p <0.05 ndi> 2-fold ion intensity) komanso mapuloteni amodzi omwe amapezeka mumizere yowonetsera miniSOG (Mkuyu 3d, madontho ofiira ndi obiriwira).火山图分析显示出显着富集的蛋白质（p <0.05 和>2 倍离子强度）以及仅存在精中存在纎中存在纎中存在纎中存在纎中存在纎中存在纎中存和纎miniSOG火山图 分析 的 的 蛋白质 (P <>>05 和> 2 的 在于) 在于 在于 在于 在于 在于 在于 在于 在于 Анализ графика вулкана выявил значительно обогащенные белки (p <0, 05 и> 2x ионная сила), а также отдельные белки, присутствующие только в экспрессионной линии miniSOG (красные и зеленые точки на рис. 3d). Kufufuza kwa chiwembu cha volcano kunavumbulutsa mapuloteni olemera kwambiri (p <0.05 ndi> 2x mphamvu ya ionic) komanso mapuloteni amodzi omwe amapezeka mumzere wofotokozera wa miniSOG (madontho ofiira ndi obiriwira mu Fig. 3d).Kuphatikiza izi, tidazindikira 1364, 461, ndi 911 mapuloteni ofunikira kwambiri a nyukiliya, mitochondrial, ndi ER, motsatana.Kuti tiwunike kulondola kwa organelle-localized PDPL, tinagwiritsa ntchito MitoCarta 3.0, Gene Ontology (GO) kusanthula, ndi A. Ting et al.deta set8 inagwiritsidwa ntchito kwa mitochondria, nucleus, ndi ER kuyesa ndondomeko ya organelle ya mapuloteni odziwika, ogwirizana ndi kulondola kwa 73.4, 78.5, ndi 73.0% (mkuyu 3e).Kukhazikika kwa PDPL kumatsimikizira kuti PDPL ndi chida choyenera chodziwira ma proteome enieni a organelle.Makamaka, kusanthula kwa subochondrial kwa mapuloteni odziwika a mitochondrial kunawonetsa kuti proteome yomwe idatsekeredwa idagawidwa makamaka mu matrix ndi nembanemba yamkati (226 ndi 106, motsatana), kuwerengera 91.7% (362) ya chiwerengero chonse cha mapuloteni a mitochondrial odziwika.mlingo wapamwamba wa PDPL unatsimikiziridwa (Supplementary Fig. 7a).Mofananamo, kufufuza kwa nyukiliya kunasonyeza kuti proteome yomwe inagwidwa inagawidwa makamaka mu nucleus, nucleoplasm, ndi nucleolus (Supplementary Fig. 7b).Kusanthula kwa mapuloteni a nyukiliya ndi peptide ya chizindikiro cha nyukiliya (3xNLS) kunasonyeza kulondola kofanana ndi H2B kumanga (Supplementary Fig. 7c-h).Kuti mudziwe tsatanetsatane wa chikhomo cha PDPL, nyukiliya laminin A idasankhidwa ngati msampha wodziwika bwino wa POI7.PDPL idazindikira 36 mapuloteni olemera kwambiri, omwe 12 mapuloteni (30.0% kuphatikiza lamin A) anali odziwika bwino lamin A omwe amalumikizana ndi omwe amafotokozedwa ndi database ya String, ndi kuchuluka kwakukulu kuposa njira ya BioID (mapuloteni 122) 28 a 28. , 22.9 %) 7. Njira yathu idazindikiritsa mapuloteni ochepa, mwina chifukwa cha malo ochepa olembera, omwe adatheka ndi mpweya wochuluka wa singlet.Kusanthula kwa GO kunawonetsa kuti mapuloteni ozindikirika anali makamaka mu nucleoplasm (26), nembanemba ya nyukiliya (10), nembanemba ya nyukiliya (9), ndi ma pores a nyukiliya (5).Pamodzi, mapuloteni a nyukiliya omwe ali ndi nyukiliya amakhala ndi 80% ya mapuloteni olemetsedwa, akuwonetsanso tsatanetsatane wa PDPL (Supplementary Fig. 8a-d).
Titakhazikitsa kuthekera kwa PDPL kuchita zolembera moyandikana mu organelles, tidayesa ngati PDPL ingagwiritsidwe ntchito kusanthula ma POI ogwirizana nawo.Makamaka, tidafuna kufotokozera kusanthula kwa PDPL kwa mapuloteni a cytosolic, omwe amaonedwa kuti ndizovuta kwambiri kuposa anzawo omwe ali ndi membrane-localized chifukwa cha chikhalidwe chawo champhamvu kwambiri.Puloteni ya bromodomain ndi extraterminal (BET) BRD4 yatikopa chidwi chathu chifukwa cha ntchito yake yaikulu mu matenda osiyanasiyana 35, 36.Chovuta chopangidwa ndi BRD4 ndi transcriptal coactivator komanso chandamale chofunikira chamankhwala.Poyang'anira mawu a c-myc ndi Wnt5a transcription factor, BRD4 imaganiziridwa kuti ndiyomwe imayambitsa matenda a acute myeloid leukemia (AML), multipleeloma, Burkitt's lymphoma, khansa ya m'matumbo ndi matenda otupa37,38.Kuphatikiza apo, ma virus ena amayang'ana BRD4 kuti iziwongolera zolemba zama virus ndi ma cell, monga papillomavirus, HIV, ndi SARS-CoV-236,39.
Kupanga mapu kuyanjana kwa BRD4 pogwiritsa ntchito PDPL, tinaphatikiza miniSOG ndi N- kapena C-terminal isoform ya BRD4 yaifupi.Zotsatira za Proteomic zidawonetsa kuchuluka kwakukulu pakati pa zomanga ziwirizi (Supplementary Fig. 9a).Nyukiliya ya nyukiliya yodziwika ndi miniSOG-H2B imaphimba 77.6% ya mapuloteni omwe amagwirizana ndi BRD4 (Supplementary Fig. 9b).Kenaka, nthawi zosiyanasiyana zowunikira (2, 5, 10, 20 min) zinagwiritsidwa ntchito kuti zisinthe mawonekedwe a chizindikiro (mkuyu 4a ndi deta yowonjezera 3).Titha kunena kuti pazithunzi zazifupi, PDPL imalemba anthu omwe amamanga nawo mwachindunji, pomwe nthawi yayitali iphatikiza mapuloteni omwe amazindikiridwa panthawi yaifupi yojambula zithunzi komanso mipherezero yosalunjika m'malo olembera.Ndipotu, tinapeza kuyanjana kwakukulu pakati pa nthawi zoyandikana (84.6% kwa 2 ndi 5 min; 87.7% kwa 5 ndi 10 min; 98.7% kwa 10 ndi 20 min) (Mkuyu 4b ndi Supplementary Fig. 9c).M'magulu onse oyesera, sitinangopeza zolemba za BRD4 zokha, komanso zolinga zingapo zodziwika monga MED1, CHD8, BICRA, NIPBL, SMC1A, ndi HMGB1 zomwe zalembedwa muzosungirako zingwe.Mphamvu ya ionic ya zolingazi ndizofanana ndi nthawi yowonetsera (mkuyu 4c ndi Supplementary Fig. 9d).GO kusanthula kwa mapuloteni odziwika mu gulu la mphindi 2 kunasonyeza kuti mapuloteni odziwika anali m'kati mwa nyukiliya ndipo adakhudzidwa ndi chromatin remodeling ndi RNA polymerase function.Maselo a puloteni adapindula mu chromatin kumanga kapena transcriptal coactivation, mogwirizana ndi BRD4 ntchito (mkuyu 4d).Kusanthula kwa kuyanjana kwa mapuloteni a String database kunavumbula gawo loyamba la kuyanjana kwachindunji pakati pa BRD4 ndi HDAC zolumikizana za banja monga SIN3A, NCOR2, BCOR, ndi SAP130 (mkuyu 4e ndi Supplementary Fig. 9e), mogwirizana ndi BRD4 ndi HDAC yomanga ma histones acetylated ..Kuonjezera apo, zolinga zoimira zodziwika ndi LC-MS/MS, kuphatikizapo Sin3A, NSUN2, Fus, ndi SFPQ, zinatsimikiziridwa ndi Western blotting (Fig. 4f).Posachedwapa, isoform yaifupi ya BRD4 idanenedwa kuti ipanga ma nuclei okhala ndi madzi-liquid phase separation (LLPS).Mapuloteni omanga a RNA a Fus ndi SFPQ amalumikizana ndi LLPS yamitundu yosiyanasiyana yama cell ndipo adziwika pano ngati mapuloteni omangirira a BRD4 osalembedwa.Kugwirizana pakati pa BRD4 ndi SFPQ kunatsimikiziridwa ndi kuyesa kwa co-immunoprecipitation (co-IP) (Chithunzi 4g), kutanthauza njira ina yolekanitsa gawo lamadzimadzi lamadzimadzi la BRD4 lomwe likuyenera kufufuzidwanso.Kuphatikizidwa pamodzi, zotsatirazi zikusonyeza kuti PDPL ndi nsanja yabwino yodziwira kuyanjana kwa BRD4 komanso mapuloteni osadziwika osadziwika.
chiwonetsero cha Schematic cha miniSOG-mediated BRD4 yoyandikira chizindikiro, nthawi yowonekera: 2, 5, 10, ndi 20 min.b Kuphatikizika kwa mapuloteni odziwika panthawi zosiyanasiyana zowunikira.Kulemera kwa mapuloteni odziwika mu HEK293T-miniSOG-BRD4 kunali kofunikira poyerekeza ndi mtundu wa HEK293T wamtchire.c Kuchulukira kwa ion pakuwerengera woyimira wosalemba yemwe amadziwika kuti BRD4-binding proteins pa nthawi yodziwika.n = 3 zitsanzo zodziyimira pawokha.Deta imawonetsedwa ngati ± kupatuka kokhazikika.d Gene ontological analysis (GO) ya mapuloteni odziwika mu gulu la mphindi ziwiri.Mawu khumi oyambirira a GO alembedwa.Mivuvu imapangidwa molingana ndi gulu la GO, ndipo kukula kwa thovu kumayenderana ndi kuchuluka kwa mapuloteni omwe amapezeka patemu iliyonse.e Kusanthula kwachingwe kwa mapuloteni omwe amalumikizana ndi BRD4.Mabwalo achikasu ndi guluu wachindunji ndipo zozungulira zotuwa ndiye gawo loyamba la guluu wosalunjika.Mizere yofiyira imayimira kuyanjana koyesedwa koyeserera ndipo mizere yabuluu imayimira kuyanjana komwe kunanenedweratu.f Zolinga zomangirira za BRD4 zodziwika mu LC-MS/MS zidatsimikiziridwa ndi Western blotting.g Kuyesa kwa Co-immunoprecipitation kumatsimikizira kuyanjana pakati pa SFPQ ndi BRD4.Zoyesererazi zidabwerezedwa mosayembekezeka kawiri ndi zotsatira zofanana.Zambiri zimaperekedwa m'mafayilo a data yaiwisi.
Kuphatikiza pa kuzindikira zolinga zomwe sizinalembetsedwe ndi POI, timaganiza kuti PDPL idzakhala yoyenera kuzindikiritsa magawo a michere, zomwe zingafunike kuzindikirika kwa mapuloteni omangika osalunjika m'magulu akulu kuti afotokozere magawo omwe sanalembetsedwe.Parkin (encoded ndi PARK2) ndi E3 ligase ndipo masinthidwe mu parkin amadziwika kuti amachititsa autosomal recessive juvenile Parkinson's disease (AR-JP)42.Kuphatikiza apo, parkin yafotokozedwa kuti ndiyofunikira pa mitophagy (mitochondrial autophagy) ndikuchotsa mitundu ya okosijeni yokhazikika.Komabe, ngakhale magawo angapo a parkin adziwika, gawo la parkin mu matendawa silikudziwikabe.Kufotokozera magawo ake osadziwika, PDPL idayesedwa powonjezera miniSOG ku N- kapena C-terminus ya parkin.Maselo adathandizidwa ndi carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) kuti ayambitse parkin kudzera munjira ya PINK1-Parkin.Poyerekeza ndi zotsatira zathu za BRD4 PDPL, parkin N-terminus fusion inavumbulutsa gulu lalikulu la mapuloteni omwe amawunikira, ngakhale kuti anaphimba gawo lalikulu la C-terminus (177 kuchokera ku 210) (Chithunzi 5a, b ndi Supplementary Data 4).zotsatira zake zimagwirizana ndi malipoti oti ma tag a N-terminal amatha kuyambitsa Parkin44 molakwika.Chodabwitsa n'chakuti, munali mapuloteni 18 okha omwe amadutsana mu deta yathu ndi zotsatira zofalitsidwa za AP-MS za Parkin43, mwina chifukwa cha kusiyana pakati pa mizere ya maselo ndi ma proteomics workflows.Kuwonjezera pa mapuloteni anayi odziwika (ARDM1, HSPA8, PSMD14, ndi PSMC3) omwe amadziwika ndi njira ziwiri (mkuyu 5c) 43.Pofuna kutsimikiziranso zotsatira za LC-MS/MS, chithandizo cha PDPL ndi kutsekedwa kwa Kumadzulo kwamadzulo kunagwiritsidwa ntchito kuyerekeza zotsatira za HEK293T ma cell cell assay ndi mzere wokhazikika wa N-terminal parkin.Zolinga zosadziwika kale CDK2, DUT, CTBP1, ndi PSMC4 zinayesedwa ndi binder yodziwika, DNAJB1 (Fig. 5d).
Chiwembu cha volcano cha mapuloteni ophatikizana ndi parkin m'maselo a HEK293T okhala ndi miniSOG yolongosoledwa mokhazikika atasakanikirana ndi N- kapena C-terminus ya parkin (n = 3 kuyesa kwachilengedwe kodziyimira pawokha).Mayeso a Wophunzira a michira iwiri adagwiritsidwa ntchito pamagawo ophulika.HEK293T idagwiritsidwa ntchito ngati kuwongolera koyipa. Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> 2-fold ion intensity kusiyana). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> 2-fold ion intensity kusiyana). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> kusiyana kwa 2-fold intensity ion).显着变化的蛋白质以红色突出显示（p <0.05 和> 2 倍离子强度差异）.显着变化的蛋白质以红色突出显示（p <0.05和> 2 Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в ионной силе). Mapuloteni osinthidwa kwambiri amawonetsedwa mofiira (p <0.05 ndi> kusiyana kwa 2-fold in ionic mphamvu).Mapuloteni ogwirizana nawo ofunikira pa HEK293T-miniSOG koma osafunikira kwa HEK293T amawonetsedwa mobiriwira.Chithunzi cha b Venn chowonetsa mapuloteni opitilira muyeso pakati pa N-terminal ndi C-terminal constructs.Ma tag a N-terminal amatha kuyambitsa parkin molakwika ndikupangitsa kuti pakhale mapuloteni odziwika bwino.c Chithunzi cha Venn chowonetsa mapuloteni ophatikizana pakati pa PDPL ndi AP-MS.Magulu odziwika odziwika adalembedwa, kuphatikiza 4 mwa 18 mapuloteni ophatikizika ndi mapuloteni 11 mwa 159 omwe amadziwika makamaka mu PDPL.d Zolinga zoimira zodziwika ndi LC-MS/MS zidatsimikiziridwa ndi Western blotting.e Ssu72 ndi SNW1 adadziwika kuti ndi magawo a parkin osalembetsa.Ma plasmids opangidwa ndi FLAG awa adasinthidwa kukhala HEK293T ndi HEK293T-Parkin-miniSOG ndikutsatiridwa ndi chithandizo cha CCCP panthawi zosiyanasiyana.Kuwonongekaku kudawonekera kwambiri pamzere wa Parkin overexpression.f Pogwiritsa ntchito proteasome inhibitor MG132, zinatsimikiziridwa kuti njira yowonongeka ya Ssu72 ndi SNW1 imayanjanitsidwa ndi proteasome-ubiquitination.Zoyesererazi zidabwerezedwa mosayembekezeka kawiri ndi zotsatira zofanana.Zambiri zimaperekedwa m'mafayilo a data yaiwisi.
Makamaka, mapuloteni odziwika ndi PDPL ayenera kukhala ndi mapuloteni omanga ma parkin ndi magawo ake.Kuti tizindikire magawo a parkin osalembetsa, tidasankha mapuloteni asanu ndi awiri odziwika (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 ndi SNW1) ndi ma plasmids opatsirana kuti awonetse majini awa ku HEK293T ndikufotokozera mokhazikika miniSOG-Parkin's HEK293T chithandizo.Mapuloteni a Ssu72 ndi SNW1 anachepetsedwa kwambiri mumzere wokhazikika wa miniSOG-Parkin (mkuyu 5e).Kuchiza ndi CCCP kwa maola 12 kunapangitsa kuti magawo onse awiri awonongeke kwambiri.Kuti tifufuze ngati kuwonongeka kwa Ssu72 ndi SNW1 kumayendetsedwa ndi proteasome-ubiquitination, proteasome inhibitor MG132 inawonjezeredwa kuletsa ntchito ya proteasome, ndipo kwenikweni tinapeza kuti njira yawo yowonongeka inaletsedwa (Mkuyu 5f).Zolinga zowonjezera zopanda gawo lapansi zinatsimikiziridwa ngati Parkin interactors pogwiritsa ntchito Western blotting (Supplementary Fig. 10), yomwe inasonyeza zotsatira zogwirizana ndi LC-MS / MS.Pomaliza, kuphatikizika kwa kayendetsedwe ka ntchito ya PDPL ndi kutsimikizika kwazomwe zimapangidwira mapuloteni kumalola kuzindikirika kwa magawo osalembetsedwa a E3 ligase.
Tapanga nsanja yofananira yolembera yomwe imakupatsani mwayi kuti muzindikire ma POI olumikizana mumlengalenga ndi nthawi.Pulatifomuyi imachokera ku mapuloteni a miniSOG photosensitizer, omwe ndi pafupifupi 12 kDa, osakwana theka la kukula kwa APEX2 enzyme (27 kDa) ndi gawo limodzi mwa magawo atatu a kukula kwa TurboID (35 kDa).Kukula kwakung'ono kuyenera kukulitsa kwambiri kuchuluka kwa mapulogalamu owerengera ma protein ang'onoang'ono.Kufufuza kwina kwa ma photosensitizers owonjezera, kaya ma genetic encoded protein kapena mamolekyu ang'onoang'ono, ndikofunikira kuti muwonjezere zokolola zamtundu wa singlet oxygen ndikukulitsa chidwi cha njirayi.Pa mtundu waposachedwa wa miniSOG, kusintha kwakanthawi kochepa kumatha kukwaniritsidwa pogwiritsa ntchito kuwunikira kwa buluu kuti mutsegule zolembera zoyandikira.Kuphatikiza apo, nthawi yayitali yowonekera idatulutsa "mtambo" wokulirapo wa okosijeni wa singlet, zomwe zidapangitsa kusinthidwa kwa zotsalira za distal histidine, kuchuluka kwa zilembo zolembera, komanso kuthekera kosintha mawonekedwe a PDPL.Tinayesanso ma probes asanu ndi awiri a mankhwala kuti tiwonjeze chiŵerengero cha zizindikiro ndi kumbuyo ndikufufuza njira ya maselo kumbuyo kwa njirayi.Mayendedwe a TOP-ABPP ophatikizidwa ndi kusaka kotseguka kopanda tsankho adatsimikizira kuti kusinthidwa kunachitika kokha mu histidines ndipo palibe microenvironment yokhazikika yomwe idawonedwa kuti iwonjezere kusintha kwa histidine, kupatula kukonda pang'ono kwa histidines m'chigawo cha loop.
PDPL yagwiritsidwanso ntchito kuzindikiritsa ma proteome ang'onoang'ono okhala ndi mawonekedwe a proteome komanso kufalikira pang'onopang'ono kufananiza ndi zilembo zina zapafupi ndi njira zofufuzira zamankhwala za organelle.Zolembera zoyandikira zagwiritsidwanso ntchito bwino kuwonetsa pamwamba, lysosomal, ndi ma proteoms46,47 ogwirizana ndi secretome.Tikukhulupirira kuti PDPL idzakhala yogwirizana ndi ma subcellular organelles.Kuonjezera apo, tidatsutsa PDPL pozindikira zolinga za mapuloteni a cytosolic omwe ndi ovuta kwambiri kuposa mapuloteni omangidwa ndi nembanemba chifukwa cha mphamvu zawo komanso kutenga nawo mbali pazochitika zamakono.PDPL idagwiritsidwa ntchito ku mapuloteni awiri, transcriptal coactivator BRD4 ndi ligase E3 Parkin yokhudzana ndi matenda.Mapuloteni awiriwa sanasankhidwe chifukwa cha ntchito zawo zoyambira zamoyo, komanso chifukwa cha kufunika kwawo pachipatala komanso kuthekera kwawo kuchiza.Kwa ma POI awiriwa, odziwika bwino omwe amamangiriza komanso zolinga zosalembetsa adadziwika.Chodziwika bwino, gawo lopatukana-logwirizana ndi mapuloteni a SFPQ adatsimikiziridwa ndi co-IP, zomwe zingasonyeze njira yatsopano yomwe BRD4 (short isoform) imayendetsa LLPS.Panthawi imodzimodziyo, timakhulupirira kuti chizindikiritso cha magawo a Parkin ndizochitika zomwe zimayenera kuzindikiritsa zomatira zosalunjika.Tinazindikira magawo awiri a parkin osadziwika ndikutsimikizira kuwonongeka kwawo panjira ya ubiquitination-proteasome.Posachedwapa, njira yopangira msampha yapangidwa kuti izindikire magawo a hydrolase powatsekera ndi ma enzyme.Ngakhale kuti iyi ndi njira yamphamvu kwambiri, si yoyenera kuwunikira magawo omwe amakhudzidwa ndi mapangidwe a ma complexes akuluakulu ndipo amafuna kupanga mgwirizano wa covalent pakati pa enzyme ndi gawo lapansi.Tikuyembekeza kuti PDPL ikhoza kukulitsidwa kuti iphunzire mapuloteni ena ndi mabanja a enzyme, monga mabanja a deubiquitinase ndi metalloprotease.
Mtundu watsopano wa miniSOG, wotchedwa SOPP3, wapangidwa ndi kupanga mpweya wabwino wa singlet.Tidafanizira miniSOG ndi SOPP3 ndipo tidapeza magwiridwe antchito abwino, ngakhale chiŵerengero cha ma sign-to-noise sichinasinthe (Supplementary Fig. 11).Tidaganiza kuti kukhathamiritsa kwa SOPP3 (mwachitsanzo, kudzera mu chisinthiko chowongoka) kungapangitse mapuloteni owoneka bwino a photosensitizer omwe amafunikira nthawi yaifupi ya kuwala ndikupangitsa kuti ma cell azitha kugwidwa.Makamaka, mtundu waposachedwa wa PDPL umangotengera chilengedwe cha ma cell chifukwa umafunikira kuunikira kwa buluu ndipo sungathe kulowa m'matumbo akuya.Izi zimalepheretsa kugwiritsidwa ntchito kwake mu maphunziro a zinyama.Komabe, kuphatikiza kwa optogenetics ndi PDPL kungapereke mwayi wofufuza zinyama, makamaka mu ubongo.Kuphatikiza apo, ma infrared photosensitizer ena opangidwa ndi infrared amachotsanso izi.Kafukufuku akuchitika mderali.
Mzere wa cell wa HEK293T unapezedwa kuchokera ku ATCC (CRL-3216).Mzere wa cell udapezeka kuti alibe matenda a mycoplasma ndipo udakulitsidwa ku DMEM (Thermo, #C11995500BT) wophatikizidwa ndi 10% fetal bovine serum (FBS, Vistech, #SE100-B) ndi 1% penicillin/streptomycin (Hyclone, #SV30010).kukula mu.
3-Aminophenylene (sample 3) ndi (4-ethynylphenyl)methanamine (sample 4) anagulidwa ku Bidepharm.Propylamine (probe 2) idagulidwa ku Energy-chemicals.N-(2-Aminophenyl)pent-4-ynamide (probe 1) idapangidwa molingana ndi njira zofalitsidwa.
Supplementary Table 1 imatchula ma genetic omwe amagwiritsidwa ntchito mu kafukufukuyu.Zotsatizana za miniSOG ndi KillerRed zidapangidwa kuchokera ku plasmid yamphatso kuchokera ku P. Zou (Peking University).Mitochondrial matrix yolunjika yotsatizana idachokera ku 23 N-terminal amino acids ya COX4 ndikupangidwa m'ma vector omwe awonetsedwa pogwiritsa ntchito msonkhano wa Gibson (Beyotime, #D7010S).Kulimbana ndi nembanemba ndi phata la endoplasmic reticulum, SEC61B munthu DNA (NM_006808.3) (NEB, #M0491L) amakweza ndi PCR kuchokera cDNA laibulale ya HEK293T maselo, ndi H2B DNA (yoperekedwa ndi D. Lin, Shenzhen Bay Laboratory) ndi kupangidwa , monga tafotokozera pamwambapa.Pokhapokha zitawonetsedwa mwanjira ina, majini ena a protein omwe amagwiritsidwa ntchito popatsirana ndikumanga ma cell okhazikika anali PCR yokulitsidwa kuchokera ku library ya HEK293T cell cDNA.G3S (GGGS) ndi G4S (GGGGS) adagwiritsidwa ntchito ngati zolumikizira pakati pa mapuloteni a nyambo ndi miniSOG.V5 epitope tag (GKPIPNPLLGLDST) idawonjezedwa pamapangidwe awa.Pofotokozera zanyama zakutchire komanso kukhazikitsa mzere wokhazikika wa cell, kaphatikizidwe ka miniSOG kaphatikizidwe ka pLX304 lentiviral vector.Ponena za mabakiteriya, miniSOG idapangidwa kukhala vekitala ya pET21a yotchedwa 6xHis pa C-terminus.
Maselo a HEK293T adabzalidwa pama cell a 2.0 x 105 pachitsime chilichonse m'mbale zisanu ndi imodzi ndikusinthidwa maola 24 pambuyo pake ndi ma plasmids a lentiviral (2.4 μg pLX304) ndi ma plasmids opaka ma virus (1.5 μg psPAX2 ndi 1.2 μg00 pMD2) p. , #C0533), pafupifupi 80% kuphatikizika.Pambuyo pa kufalikira kwa usiku, sing'angayo idasinthidwa ndikuyikidwanso kwa maola ena 24.Kutoleredwa kwa kachilomboka kudachitika pambuyo pa maola 24, 48 ndi 72.Asanatenge kachilombo ka mizere ya ma cell omwe amatsata, ma virus adasefedwa kudzera mu fyuluta ya 0.8 μm (Merck, #millex-GP) ndi polybrene (Solarbio, #H8761) adawonjezeredwa ku 8 μg/ml.Pambuyo pa maola 24, ma cell adaloledwa kuchira posintha sing'anga.Maselo anasankhidwa pogwiritsa ntchito 5 μg/ml blasticidin (Solarbio, #3513-03-9) pa ndime zitatu zoyambirira monga kusankha kochepetsetsa.Kenako gwiritsani ntchito 20 μg/ml ngati njira yolimbikira m'ndime zitatu zotsatirazi.
Maselo adabzalidwa m'zipinda za 12-chitsime (Ibidi, # 81201) pa kachulukidwe ka maselo pafupifupi 20,000 pachitsime.Kuti muwongolere kumamatira kwa ma cell a HEK293T, onjezerani 50 µg/ml fibronectin (Corning, #356008) yosungunuka mu mchere wa phosphate buffered (PBS, Sangon, #B640435) pa 37°C.Zipindazo zidakonzedweratu kwa ola la 1 ndikuchotsedwa ndi PBS.Pambuyo pa 24 h, ma cell adatsukidwa kamodzi ndi PBS, yolumikizidwa ndi 1 mM probe 3 mumchere wa mchere wa Hanks watsopano (HBSS, Gibco, #14025092) kwa 1 h pa 37 ° C, kenako amapaka ndi buluu LED (460 nm. ).) adayatsidwa kwa mphindi 10 kutentha kwapakati.Pambuyo pake, maselowo adatsukidwa kawiri ndi PBS ndikukhazikika ndi 4% formaldehyde mu PBS (Sangon, #E672002) kwa mphindi 15 kutentha.Formaldehyde yochulukirapo idachotsedwa m'maselo okhazikika posamba katatu ndi PBS.Maselo adalowetsedwa ndi 0.5% Triton X-100 (Sangon, #A600198) mu PBS ndikutsuka katatu ndi PBS.Kenako chotsani chipindacho ndikuwonjezera pa chitsanzo chilichonse 25 µl cha chosakaniza chomwe chili ndi 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) ndi 0.5 mg/ml sodium ascorbate (Aladdin, no. S105024) ndi incubated kwa mphindi 30 kutentha firiji.Pambuyo pochita pang'onopang'ono, maselo amatsukidwa kasanu ndi kamodzi ndi PBS yomwe ili ndi 0.05% Tween-20 (Sangon, #A600560) (PBST) ndipo amatsekedwa ndi 5% BSA (Abcone, #B24726) mu PBST kwa mphindi 30 kutentha.
Pakuti colocalization immunostaining, maselo anali incubated ndi ma antibodies oyambirira malinga ndi mikhalidwe anasonyeza: mbewa anti-V5 tag mAb (1:500, CST, #80076), kalulu anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), kalulu polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) kapena anti-lamin A/C monoclonal antibody (1:500; CST, #2032) pa 4 °C usiku wonse.Pambuyo kutsuka katatu ndi PBST, ma cell adalumikizidwa ndi ma antibodies achiwiri: mbuzi yolimbana ndi akalulu Alexa Fluor 488 (Thermo, #A11034) yochepetsedwa 1:1000, mbuzi yotsutsa mbewa Alexa Fluor 594 (CST, #8889) idachepetsedwa 1:1000.dilution Dilute pa firiji kwa mphindi 30.Maselo adatsuka nthawi za 3 ndi PBST ndikutsutsidwa ndi DAPI (Thermo, #D1306) mu PBS kwa mphindi 10 kutentha.Pambuyo pa kusamba kwa 3 ndi PBS, maselo adasindikizidwa mu 50% glycerol (Sangon, #A600232) mu PBS kuti aganizire.Zithunzi za Immunofluorescent zinapezedwa pogwiritsa ntchito ZEISS LSM 900 Airyscan2 confocal microscope ndi pulogalamu ya ZNE 3.5.
Pakuyerekeza kwa oxygen fluorescent, ma cell adatsukidwa kawiri ndi Hanks HEPES buffer asanawonjezere 100 nM Si-DMA mu Hanks HEPES buffer (DJINDO, #MT05).Pambuyo poyatsidwa ndi kuwala, maselowo adakulungidwa mu chofungatira cha CO2 pa 37 ° C kwa mphindi 45.Maselo amatsukidwa kawiri ndi Hanks' HEPES buffer ndikutsukidwa ndi Hoechst mu Hanks' HEPES buffer kwa mphindi 10 kutentha kwa firiji ndikuwonetseredwa pogwiritsa ntchito ZEISS LSM 900 confocal microscope., #M36008) mu HBSS buffer yokhala ndi calcium ndi magnesium.Pambuyo poyang'ana kuwala kapena doxorubicin (MCE, #HY-15142A), maselo adakulungidwa mu chofungatira cha CO2 pa 37 ° C. kwa mphindi 10, amatsukidwa kawiri ndi HBSS buffer, ndikumangidwira ndi Hoechst mu HBSS buffer kutentha.mphindi.Doxorubicin idagwiritsidwa ntchito ngati njira yabwino yoyang'anira ma cell omwe amathandizidwa ndi 20 μM doxorubicin mu HBSS yokhala ndi 1% BSA kwa mphindi 30.Zithunzi za Immunofluorescent zidapezedwa pogwiritsa ntchito microscope ya Zeiss LSM 900 confocal.
Maselo a HEK293T osonyeza mito-miniSOG mokhazikika adabzalidwa pa kachulukidwe pafupifupi 30% mu mbale 15 cm.Pambuyo pa maola a 48, pamene ~ 80% confluence inafikiridwa, maselowo adatsukidwa kamodzi ndi PBS, opangidwa ndi 1 mM Probe 3 mu HBSS buffer yatsopano kwa ola la 1 pa 37 ° C ndikuwunikira ndi LED yabuluu kwa mphindi 10 m'chipinda. kutentha..Pambuyo pake, ma cell adatsukidwa kawiri ndi PBS, kukulungidwa ndikuyimitsidwanso mu ayezi wozizira wa PBS buffer wokhala ndi EDTA-free protease inhibitors (MCE, #HY-K0011).Maselo anali lysed ndi sonicating nsonga kwa mphindi 1 (1 wachiwiri ndi 1 wachiwiri pa 35% matalikidwe).Chosakaniza chotsatiracho chinali centrifuged pa 15,871 xg kwa 10 min pa 4 ° C kuchotsa zinyalala, ndipo ndende ya supernatant inasinthidwa kukhala 4 mg / mL pogwiritsa ntchito chipangizo cha BCA protein assay (Beyotime, #P0009).Phatikizani 1 ml ya lysate pamwamba ndi 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), ndi 1 mM CuSO4 incubator pansi kuzungulira kwa 1 ora kutentha kwa firiji.Mukangochita pang'onopang'ono, onjezerani kusakaniza ku njira yosakanikirana (MeOH:CHCl3:H2O = 4 ml: 1 ml: 3 ml) mu galasi la galasi la 10 ml.Zitsanzozo zinali zosakanikirana ndi centrifuged pa 4500 g kwa mphindi 10 firiji.Mayankho apansi ndi apamwamba adatayidwa, madziwo adatsukidwa kawiri ndi 1 ml ya methanol ndi centrifuged pa 15871 × g kwa 5 min pa 4 ° C.Onjezani 1 ml ya 8 M urea (Aladdin, no. U111902) mu 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) kuti musungunuke mpweya.Zitsanzo zidapangidwanso ndi 10 mM dithiothreitol (Sangon, #A100281 mu 25 mM ABC) kwa mphindi 40 pa 55°C kutsatiridwa ndi kuwonjezera kwa 15 mM iodoacetamide yatsopano (Sangon, #A600539) kutentha mumdima.Alkylation mkati mwa mphindi 30..Dithiothreitol yowonjezera 5 mM idawonjezedwa kuti ayimitse zomwe zikuchitika.Konzani pafupifupi 100 µl NeutrAvidin agarose mikanda (Thermo, #29202) pa chitsanzo chilichonse pochapa katatu ndi 1 ml PBS.Njira ya proteome yomwe ili pamwambayi idachepetsedwa ndi 5 ml PBS ndikuyikidwa ndi mikanda ya NeutrAvidin agarose yotsukidwa kale kwa maola 4 kutentha kwapakati.Mikandayo idatsukidwa nthawi 3 ndi 5 ml PBS yomwe ili ndi 0,2% SDS (Sangon, #A600485), nthawi 3 ndi 5 ml PBS yomwe ili ndi 1M urea, ndi nthawi 3 ndi 5 ml ddH2O.Mikandayo idakololedwa ndi centrifugation ndikuyimitsanso mu 200 μl ya 25 mM ABC yokhala ndi 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) ndi 20 ng/μl trypsin (Promega, #V5280).Yesani usiku wonse pa 37 ° C ndikuzungulira.Zomwe zidayimitsidwa ndikuwonjezera formic acid (Thermo, # A117-50) mpaka pH ifika 2-3.Mikanda inatsukidwa katatu ndi 1 ml ya PBS yomwe ili ndi 0.2% SDS, 3 nthawi ndi 1 ml ya PBS yomwe ili ndi 1 M urea, ndiyeno katatu ndi 1 ml ya madzi osungunuka.Ma peptides osinthidwa adatulutsidwa ndi light lysis (365 nm) kwa mphindi 90 pogwiritsa ntchito 200 μl ya 70% MeOH.Pambuyo pa centrifugation, supernatant inasonkhanitsidwa.Mikandayo idatsukidwa kamodzi ndi 100 μl ya 70% MeOH ndipo zopatsa mphamvu zidaphatikizidwa.Zitsanzo zidawumitsidwa mu Speedvac vacuum concentrator ndikusungidwa pa -20 ° C mpaka kusanthula.
Kuti mudziwe ndi kuwerengera ma peptide osinthidwa a oxygen, zitsanzo zinasungunukanso mu 0.1% formic acid ndi 1 μg ya peptides adawunikidwa pogwiritsa ntchito Orbitrap Fusion Lumos Tribrid mass spectrometer yokhala ndi gwero la nano ESI kuchokera ku Tune ndi Xcalibur kuchokera ku mapulogalamu ogulitsa 4.3.Zitsanzo zidalekanitsidwa pa 75 µm × 15 cm wodzaza mkati mwa capillary ndi 3 µm C18 zinthu (ReproSil-pur, #r13.b9.) ndikulumikizidwa ku EASY-nLC 1200 UHPLC system (Thermo).Ma peptides analekanitsidwa ndi mzere wa 95 miniti gradient chromatography kuchokera ku 8% zosungunulira B mpaka 50% zosungunulira B (A = 0.1% formic acid m'madzi, B = 0.1% formic acid mu 80% acetonitrile), kenako motsatira zidawonjezeka kufika 98% B mphindi. mu 6 min pa kuthamanga kwa 300 nl / min.Orbitrap Fusion Lumos imasonkhanitsa deta mosinthana pakati pa scan yathunthu ya MS ndi MS2 kutengera deta.Mphamvu ya sputtering idakhazikitsidwa ku 2.1 kV ndipo kutentha kwa capillary ya ion transport kunali 320 ° C.MS spectra (350-2000 m/z) anasonkhanitsidwa ndi kusamvana kwa 120,000, AGC 4 × 105, ndi nthawi yochuluka yolowera ya 150 ms.Ma precursors 10 omwe amachulukitsidwa kwambiri pa sikani iliyonse adagawika pogwiritsa ntchito HCD yokhala ndi mphamvu yakugunda ya 30%, zenera lodzipatula la quadrupole la 1.6 m/z, ndikusintha kwa 30,000.Cholinga cha AGC cha tandem mass spectrometry pogwiritsa ntchito 5 × 104 ndi nthawi yolowera kwambiri 150 ms.Kupatulapo kwamphamvu kwakhazikitsidwa masekondi 30. Ma ion omwe sanagawidwe kapena omwe ali ndi chiwongola dzanja cha 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS. Ma ion omwe sanagawidwe kapena omwe ali ndi chiwongola dzanja cha 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS. Неназначенные ионы или ионы с зарядом 1+ ndi >7+ были отклонены для МС/МС. Ma ion omwe sanagawidwe kapena ma ion okhala ndi mtengo wa 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS.未指定的离子或电荷為1+ 和>7+ 的离子被拒绝用于MS/MS.未指定的离子或电荷為1+ 和>7+ 的离子被拒绝用于MS/MS. Неуказанные ионы или ионы с зарядами 1+ и >7+ были отклонены для МС/МС. Ma ion osadziwika kapena ma ion okhala ndi milandu ya 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS.
Deta yaiwisi imakonzedwa pogwiritsa ntchito FragPipe computing platform potengera MSFragger.Kukondera kwa misa ndi ma amino acid ofanana adatsimikiziridwa pogwiritsa ntchito njira yosakira yotseguka yokhala ndi kulekerera kwamisala kwa -150 mpaka 500 Da.Ma peptides osinthidwa adadziwika pogwiritsa ntchito kusintha kwa histidine ndi phindu lalikulu la +229.0964 ndi +247.1069 Da mu PD (Proteome Discoverer 2.5, Thermo).
Maselo omwe amafotokoza mokhazikika jini ya miniSOG yosakanikirana adakutidwa mu mbale za 6 cm.Atafika ku ~ 80% confluence, maselo adatsukidwa kamodzi ndi HBSS (Gibco, #14025092), kenaka amapangidwa ndi mankhwala opangira mankhwala mu HBSS kwa ola la 1 pa 37 ° C ndikuwunikira ndi kuwala kwa buluu.10W LED kwa mphindi 20 kutentha kwa firiji.Kuti mudziwe mtundu wamtundu wa okosijeni wokhazikika womwe ukukhudzidwa ndi PDPL, 0.5 mM vitamini C (MCE, #HY-B0166), 5 mM Trolox (MCE, #HY-101445), D2O (Sigma, #7789-20-0) , 100 mM mannitol (Energy Chemical, #69-65-8), 100 μM H2O2, 10 mM NaN3 anawonjezedwa ku maselo monga zowonjezera.Pambuyo kutsuka ndi ozizira PBS, maselo scraped, anasonkhanitsa 1.5 ml centrifuge machubu, ndi sonicated ndi nsonga kwa mphindi 1 mu 200 μl wa PBS ndi 1x protease inhibitor popanda EDTA (1 s ndi 1 s popanda, matalikidwe 35%).Chosakaniza chotsatiracho chinali centrifuged pa 15,871 × g kwa 10 min pa 4 ° C ndipo ndende ya supernatant inasinthidwa kukhala 1 mg / mL pogwiritsa ntchito zida zoyesa mapuloteni a BCA.Pafupifupi 50 µl ya lysate yomwe ili pamwambayi incubated ndi 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, ndi 1 mM CuSO4 kwa ola la 1 kutentha kwa chipinda ndi kuzungulira kuchokera pansi kupita pamwamba.Pambuyo podina, mvula yokhala ndi acetone idachitika powonjezera 250 μl ya acetone yomwe isanatenthedwe ku zitsanzo, ndikumangirira pa -20 ° C kwa mphindi 20 ndi centrifuging pa 6010 × g kwa mphindi 10 pa 4 ° C.Sungani pellet ndikuphika mu 50 µl wa 1x Laemmli buffer kwa mphindi 10 pa 95 °C.Zitsanzo zidawunikidwa pa ma gels aatali a SDS-PAGE ndikuwonetsedwa pogwiritsa ntchito makina ojambulira a Bio-rad ChemiDoc MP Touch okhala ndi pulogalamu ya Image Lab Touch.
Kufotokozera ndi kuyeretsedwa kwa mapuloteni ophatikizana a miniSOG-6xHis kunachitika monga tafotokozera kale.Mwachidule, ma cell a E. coli BL21(DE3) (TransGen, #CD701-02) adasinthidwa ndi pET21a-miniSOG-6xHis ndipo mawonekedwe a protein adapangidwa ndi 0.5 mM IPTG (Sangon, #A600168).Pambuyo pa cell lysis, mapuloteni amayeretsedwa pogwiritsa ntchito mikanda ya Ni-NTA agarose (MCE, no. 70666), dialyzed motsutsana ndi PBS, ndi kusungidwa pa -80 ° C.
Kwa antibody-based in vitro label proximity assay, sakanizani 100 μM purified miniSOG, 1 mM probe 3, ndi 1 μg anti-label mouse monoclonal antibody (TransGen, #HT501-01) mu PBS kuti mumve kuchuluka kwa 50 μl..Zomwe zimasakanikirana zidayatsidwa ndi kuwala kwa buluu kwa LED kwa 0, 2, 5, 10, ndi 20 min kutentha kwapakati.Chosakanizacho chinakulungidwa ndi 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, ndi 1 mM CuSO4 kwa 1 h kutentha kwa chipinda pa chogwedeza chokwera chokwera.Pambuyo pochita pang'onopang'ono, onjezerani 4x Laemmli's buffer mwachindunji kusakaniza ndikuphika pa 95 ° C kwa 10 min.Zitsanzo zidawunikidwa pa ma gels a SDS-PAGE ndikuwunikidwa ndi blotting yakumadzulo ndi streptavidin-HRP (1: 1000, Solarbio, #SE068).
Peptide yopangidwa ndi histidine yokhala ndi C-terminal amidation (LHDALDAK-CONH2) idagwiritsidwa ntchito kusanthula zolemba za peptide zapafupi zomwe zili mu vitro.Muchiyeso ichi, 100 μM yoyeretsedwa miniSOG, 10 mM probe 3 ndi 2 μg / ml peptide yopangidwa ndi PBS mu chiwerengero chonse cha 50 μl.Zomwe zimasakanikirana zidayatsidwa ndi kuwala kwa buluu kwa ola la 1 kutentha.Chitsanzo chimodzi cha microliter chinawunikidwa pogwiritsa ntchito LC-MS system (Waters, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer ndi MassLynx spectrum analysis software).
Maselo a HEK293T akuwonetsa jini yophatikizika ya miniSOG idabzalidwa mu mbale za 10 cm pamizere yokhala ndi malo osiyanasiyana a organelle (Mito, ER, Nucleus) ndi 15 cm mbale za Parkin-miniSOG ndi mizere ya BRD4-miniSOG.Atafika ~ 90% confluence, maselowo adatsukidwa kamodzi ndi HBSS, kenaka amaikidwa ndi probe 3 mu HBSS kwa ola la 1 pa 37 ° C ndikuwunikira ndi 10 W buluu LED kutentha.Pazolemba zosagwirizana ndi Parkin, 10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) yokhala ndi probe 3 mu HBSS idawonjezedwa kwa ola limodzi pa 37°C.The selo lysis, dinani umagwirira, kuchepetsa ndi masitepe alkylation anali chimodzimodzi monga tafotokozera pamwambapa, kupatula kuti 2 mg wa lysate anawonjezera ndi biotin PEG3 azide ntchito pitani anachita m'malo photodegradable biotin azide.Pambuyo pakulemeretsa, mikanda idatsukidwa katatu ndi 5 ml ya PBS yokhala ndi 0.2% SDS, 3 nthawi ndi 5 ml ya PBS yokhala ndi 1 M urea, ndi 3 nthawi ndi 5 ml ya PBS.Pambuyo pake, 2 µg trypsin idawonjezedwa ku 300 µl 25 mM ABC yokhala ndi 1 M urea kuti ithyole mapuloteni usiku pa 37 ° C.Zomwe zidayimitsidwa ndikuwonjezera formic acid mpaka pH ya 2-3 idafikiridwa.Pambuyo pa trypsinization pamikanda, yankho la peptide lidachotsedwa pogwiritsa ntchito gawo la SOLAµ HRP (Thermo, #60209-001) ndikuwumitsa mu chotengera cha Speedvac vacuum.Ma peptides adasungunukanso mu 0.1% formic acid ndipo 500 ng ya peptides adawunikidwa pogwiritsa ntchito Orbitrap Fusion Lumos Tribrid mass spectrometer yokhala ndi nano-ESI gwero lomwe lafotokozedwa pamwambapa.Peptides analekanitsidwa pa malonda RP-HPLC precolumns (75 μm x 2 cm) (Thermo, no. 164946) ndi analytical RP-HPLC mizati (75 μm x 25 cm) (Thermo, no. 164941), onse anadzazidwa ndi 2 μm.gradient kuchokera 8% mpaka 35% ACN mu 60 mphindi, ndiye linearly anawonjezeka kufika 98% B mu 6 mphindi pa otaya mlingo wa 300 Nl/min.MS spectra (350-1500 m/z) anasonkhanitsidwa ndi kusamvana kwa 60,000, AGC 4 × 105, ndi nthawi yolowera yochuluka ya 50 ms.Ma ion osankhidwa adagawika motsatizana ndi HCD mumayendedwe a 3s ndi mphamvu yakugunda yokhazikika ya 30%, zenera lodzipatula la quadrupole la 1.6 m / z, ndi chigamulo cha 15000. AGC chandamale cha 5 × 104 tandem mass spectrometer ndi nthawi yopitilira jekeseni. za 22 ms zidagwiritsidwa ntchito.Kupatulapo kwamphamvu kwakhazikitsidwa masekondi 45. Ma ion omwe sanagawidwe kapena omwe ali ndi chiwongola dzanja cha 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS. Ma ion omwe sanagawidwe kapena omwe ali ndi chiwongola dzanja cha 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS. Неназначенные ионы или ионы с зарядом 1+ ndi >7+ были отклонены для МС/МС. Ma ion omwe sanagawidwe kapena ma ion okhala ndi mtengo wa 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS.未指定的离子或电荷為1+ 和>7+ 的离子被拒绝用于MS/MS.未指定的离子或电荷為1+ 和>7+ 的离子被拒绝用于MS/MS. Неуказанные ионы или ионы с зарядами 1+ и >7+ были отклонены для МС/МС. Ma ion osadziwika kapena ma ion okhala ndi milandu ya 1+ ndi> 7+ adakanidwa chifukwa cha MS/MS.
Njira zokonzekera zitsanzo mpaka kupindula kwa mikanda ya NeutrAvidin zinali zofanana ndi kusanthula kwa LC-MS/MS komwe kufotokozedwa pamwambapa.Pafupifupi 50 μg ya lysate idagwiritsidwa ntchito ngati chothandizira pakuwongolera kutsitsa ndipo 2 mg ya lysate idagwiritsidwa ntchito podina.Pambuyo pakulemeretsa ndikutsuka ndi neutravidin, mapuloteni omangika adachotsedwa powonjezera 50 μl ya nkhokwe ya Laemmli ku mikanda ya agarose resin ndikuwira pa 95 ° C. kwa mphindi zisanu.Kuwongolera katundu ndi zitsanzo zolemeretsedwa za mikanda zidawunikidwa ndi SDS-PAGE ndikusamutsidwa ku ma membrane a PVDF (Millipore, #ISEQ00010) ndi njira zanthawi zonse zaku Western blot.Ma nembanemba anali otsekedwa ndi 5% mkaka wa skim (Sangon, #A600669) mu TBS yomwe ili ndi 0.1% pakati pa 20 (TBST) ndipo idalumikizidwa motsatizana ndi ma antibodies a pulayimale ndi sekondale.Ma antibodies oyambilira adasungunuka 1:1000 mu 5% mkaka wa skim mu TBST ndipo adalumikizidwa usiku wonse pa 4°C.Ma antibodies achiwiri adagwiritsidwa ntchito mu chiŵerengero cha 1: 5000 ndikuyikidwa kwa ola limodzi kutentha.Ma nembanembawa adawonedwa ndi chemiluminescence pogwiritsa ntchito makina oyerekeza a Chemidoc MP.Mawonekedwe onse osadulidwa a mabulosi ndi ma gels pachithunzichi amaperekedwa ngati data yaiwisi.
Ma antibodies oyambirira omwe amagwiritsidwa ntchito mu kafukufukuyu anaphatikizapo anti-SFPQ anti-SFPQ monoclonal antibody (CST, no. 71992), anti-FUS monoclonal antibody (CST, no. 67840), anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1- AP), anti-mSin3A polyclonal antibody (Abcam, #ab3479), mbewa anti-tag monoclonal antibody (TransGen, #HT201-02), mbewa anti-β-actin monoclonal antibody (TransGen, #HC201-01), anti-kalulu -CDK2 monoclonal antibody (ABclonal, #A0094), kalulu monoclonal antibody to CTBP1 (ABclonal, #A11600), rabbit polyclonal antibody to DUT (ABclonal, #A2901), rabbit polyclonal antibody to PSMC4 (ABclonal, #Abclonal-5), #Abclonal DNAJB1 polyclonal antibody (ABclonal, # A5504).Ma antibodies awa adagwiritsidwa ntchito pa 1: 1000 dilution mu 5% mkaka wa skim mu TBST.Ma antibodies achiwiri omwe amagwiritsidwa ntchito mu kafukufukuyu adaphatikizapo anti-rabbit IgG (TransGen, #HS101-01), anti-mouse IgG (TransGen, #HS201-01) pa 1: 5000 dilution.
Kuti mufufuzenso ngati BRD4 imalumikizana ndi SFPQ, ma cell okhazikika a HEK293T ndi BRD4-miniSOG opitilira muyeso HEK293T adayikidwa mu mbale za 10 cm.Maselo anatsukidwa ndi PBS yozizira ndi lysed mu 1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) ndi EDTA-free protease inhibitor kwa mphindi 30 pa 4 ° C.Pambuyo pake, ma lysates adasonkhanitsidwa mu machubu a 1.5 ml centrifuge ndi centrifuged pa 15,871 xg kwa mphindi 10 pa 4 ° C.Chodabwitsacho chinakololedwa ndikuyikidwa ndi 5 µg ya anti-V5 yolembedwa mbewa monoclonal antibody (CST, #80076) usiku wonse pa 4°C.Sambani pafupifupi 50 µl ya mapuloteni A/G mikanda maginito (MCE, #HY-K0202) kawiri ndi PBS yokhala ndi 0.5% Tween-20.Ndiye ma cell a lysates adakulungidwa ndi mikanda yamaginito kwa maola 4 pa 4 ° C ndikuzungulira kuchokera pansi kupita pamwamba.Kenaka mikandayo inatsukidwa kanayi ndi 1 ml ya PBST buffer ndikuphika pa 95 ° C kwa 5 min.Zitsanzo zidawunikidwa pa ma gelisi a SDS-PAGE ndikusamutsira ku nembanemba za PVDF pogwiritsa ntchito njira zodziwika bwino zaku Western blot.Ma nembanembawo adatsekedwa mu 5% mkaka wa skim mu TBST ndikulumikizidwa motsatizana ndi ma antibodies oyambirira ndi achiwiri.Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) idagwiritsidwa ntchito pa chiyerekezo cha 1:1000 mu 5% mkaka wa skim mu TBST ndipo incubated usiku wonse pa 4 ° C.Anti-kalulu IgG anagwiritsidwa ntchito pa chiŵerengero cha 1: 5000 ndi incubated kwa ola 1 kutentha firiji.Ma nembanembawa adawonedwa ndi chemiluminescence pogwiritsa ntchito makina oyerekeza a Chemidoc MP.
Zomangamanga zonse zomwe zimagwiritsidwa ntchito pakuwunika kwa Solvent Accessible Surface Area (SASA) zidatengedwa kuchokera ku Protein Data Bank (PDB)52 kapena AlphaFold Protein Structure Database53.Absolute SASA idawerengeredwa pa chotsalira chilichonse pogwiritsa ntchito pulogalamu ya FreeSASA.Zina zonse za SASA zolembedwa ndi histidine ndi oyandikana nawo okha ndi omwe adagwiritsidwa ntchito kupeza tanthauzo la SASA pachimake chilichonse.Wachibale zosungunulira Kufikika (RSA) kwa histidine aliyense anali masamu ndi kugawa mtheradi SASA mtengo ndi empirical pazipita zotheka zotsalira pamwamba malo kupezeka kwa zosungunulira.Ma histidine onse adasankhidwa kukhala obisika ngati RSA yapakati inali pansi pa 20%, mwina poyera56.
Mafayilo aiwisi omwe amapezedwa mumayendedwe a DDA adafufuzidwa pogwiritsa ntchito Proteome Discoverer (v2.5) kapena MSfragger (Fragpipe v15.0) mu database yoyenera ya SwissProt yotsimikizika yama protein yomwe ili ndi zowononga wamba.Ma peptides amafunikira trypsin yathunthu yokhala ndi malo awiri osowa, carbamoyl methylation ngati kusinthidwa kokhazikika komanso okosijeni wa methionine ngati kusintha kwamphamvu.Zololera za precursor ndi fragment zolemetsa zidayikidwa ku 10 ppm ndi 0.02 Da (MS2 Orbitrap), motsatana. Kugunda koyipa kunachotsedwa, ndipo mapuloteni adasefedwa kuti apeze kuchuluka kwabodza kwa <1%. Kugunda koyipa kunachotsedwa, ndipo mapuloteni adasefedwa kuti apeze kuchuluka kwabodza kwa <1%. Zolemba zapadziko lonse lapansi zimagwira ntchito bwino, komanso zopatsa chidwi, komanso zopatsa chidwi <1%. Kugunda koyipa kunachotsedwa ndipo mapuloteni amasefedwa kuti apereke chiwopsezo chabodza cha <1%.去除污染物命中，过滤蛋白质以获得<1%的错误发现率。 <1%的错误发现率. Zolemba zaposachedwa zasintha были удалены, абелки отфильтрованы для достижения уровня ложных обнаружений <1%. Kugunda koyipa kunachotsedwa ndipo mapuloteni amasefedwa kuti akwaniritse chiwopsezo chabodza cha <1%.Pakuwunika kachulukidwe popanda kugwiritsa ntchito zilembo, mapuloteni okhazikika kuchokera kubwereza katatu kwachilengedwe adagwiritsidwa ntchito.Kusanthula kwa mapuloteni amtundu wamtundu wa mapuloteni kunachitidwa pogwiritsa ntchito kusanthula kwa Gene Ontology (GO) kuchokera ku DAVID Bioinformatics Resources, MitoCarta 3.0 ndi nkhokwe zomwe zinapangidwa ndikufalitsidwa ndi gulu la Alice Ting.Mapu a phirili anatengedwa kuchokera ku Perseus (v1.6.15.0). Kusintha kwa kuchuluka kwa mapuloteni kunayesedwa kuti ndi kofunika powerengera t-test, ndipo kugunda kwa mapuloteni kunadziwika ndi kusintha kwakukulu> 2 (pokhapokha ngati tatchulidwa mwanjira ina) ndi p value <0.05. Kusintha kwa kuchuluka kwa mapuloteni kunayesedwa kuti ndi kofunika powerengera t-test, ndipo kugunda kwa mapuloteni kunadziwika ndi kusintha kwakukulu> 2 (pokhapokha ngati tatchulidwa mwanjira ina) ndi p value <0.05. Изменения кратности содержания белка были проверены на статистическую значимость с использованием двустороннего t-критерия, и совпадения белков были идентифицированы с изменением содержания> 2 (если не указано иное) и значением p <0,05. Zosintha zamapuloteni zopindika zidayesedwa kuti ziwonekere pogwiritsa ntchito mayeso a t-tailed-t-tailed, ndipo zofananira zamapuloteni zidadziwika ndi kusintha kwazinthu> 2 (pokhapokha zitadziwika) ndi mtengo wa ap <0.05.使用 双边 t 检验 测 测 的 的 显着性 的 的 的 中 中 中 中 另说明) 说明使用 双边 t 检验 测试 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 中明 0 明 星 5 Статистическую значимость кратных изменений содержания белка проверяли с использованием двустороннего t-критерия, а совпадения белков определяли для изменений содержания >2 (если не указано иное) и p-значений <0,05. Kufunika kwapang'onopang'ono kwa kusintha kwa mapuloteni kumayesedwa pogwiritsa ntchito mayeso a t-tailed-t-tailed, ndipo mafananidwe a mapuloteni adatsimikiziridwa kuti asinthe> 2 (pokhapokha atawonetsedwa mwanjira ina) ndi p-values <0.05.Kusanthula kwamapuloteni kunachitika pogwiritsa ntchito kusanthula kwa GO pamodzi ndi database ya String.
Zofananira zitatu zachilengedwe zidachitika ndi zotsatira zofanana.Kusanthula kwachiwerengero kunachitika ndi GraphPad Prism (pulogalamu ya GraphPad) ndipo ziwembu za mapiri anapangidwa ndi Perseus (v1.6.15.0).Poyerekeza magulu awiriwa, p-values idatsimikiziridwa pogwiritsa ntchito mayeso a Student a michira iwiri.Mapuloteni a singleton okha omwe adadziwika osachepera kawiri mu gulu loyesera adaphatikizidwa m'malo ophulika, ndipo zofananira zomwe zidasoweka mugulu lowongolera zidasinthidwa ndi Perseus kuchokera pakugawa wamba kuti p-value iwerengedwe.Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika.Pakuwunika kwa proteinomic pakuwunika kwa ziwerengero, kuchuluka kwa mapuloteni omwe adawonekera m'magawo awiri achilengedwe adasungidwa.Njira zowerengera sizinagwiritsidwe ntchito podziwiratu kukula kwachitsanzo.Zoyeserera sizichitika mwachisawawa.Ofufuzawo sanachite khungu pa ntchito panthawi yoyesera ndikuwunika zotsatira.
Kuti mumve zambiri pamapangidwe amaphunziro, onani Nature Research Report abstract yolumikizidwa ndi nkhaniyi.
Zomwe zapezeka mu kafukufukuyu zidatumizidwa ku ProteomeXchange Consortium kudzera pankhokwe ya abwenzi a iProX57 pansi pa dataset ID PXD034811 (PDPL-MS dataset).Zambiri zimaperekedwa m'mafayilo a data yaiwisi.Nkhaniyi imapereka deta yoyambirira.
Gingras, AC, Abe, KT & Raught, B. Kudziwa malo oyandikana nawo: kugwiritsa ntchito biotinylation yodalira pafupi kuti iwonetsere mapuloteni ndi mapu organelles. Gingras, AC, Abe, KT & Raught, B. Kudziwa malo oyandikana nawo: kugwiritsa ntchito biotinylation yodalira pafupi kuti iwonetsere mapuloteni ndi mapu organelles.Gingras, AS, Abe, KT ndi Raut, B. Kudziwana ndi malo ozungulira: kugwiritsa ntchito biotinylation yodalira pafupi kuti iwonetsere mapuloteni ndi mapu organelles. Gingras, AC, Abe, KT & Raught, B. 了解邻里：使用邻近依赖性生物素化來表征蛋白质复合物并绘制细。 Gingras, AC, Abe, KT & Raught, B. Kumvetsetsa oyandikana nawo: gwiritsani ntchito kudalira kwa oyandikana nawo pa moyo wachilengedwe.Gingras, AS, Abe, KT ndi Raut, B. Kumvetsetsa moyandikana: mawonekedwe a mapuloteni ovuta komanso mapu a organelles pogwiritsa ntchito biotinylation yodalira pafupi.Panopa.Lingaliro langa.Chemical.biology 48, 44-54 (2019).
Geri, JB et al.Kupanga mapu a microenvironment posamutsa mphamvu ya Dexter kuma cell a chitetezo.Sayansi 367, 1091-1097 (2020).
Hertling, EL et al.Ma network awiri a proteinome amazindikira kukonzanso kwapadera kwa ma cell a interactome.Maselo 184, 3022–3040.e3028 (2021).
Nthawi yotumiza: Sep-15-2022
