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Hanyoyin alamar kusancin Enzymatic dangane da esters da aka kunna ko phenoxy radicals ana amfani da su sosai don taswirar proteomes na subcellular da masu hulɗar furotin a cikin sel masu rai.Koyaya, esters da aka kunna ba su da ƙarfi sosai, yana haifar da radius mai faɗi mai faɗi, kuma radicals phenoxy da aka samar ta hanyar jiyya ta peroxide na iya tsoma baki tare da hanyoyin redox.Anan muna ba da rahoton hanyar kusancin alamar dogaro da hoto (PDPL) wanda aka haɓaka ta hanyar haɗa furotin na miniSOG na hoto zuwa furotin mai ban sha'awa.An haifar da hasken shuɗi kuma ana sarrafa shi ta lokacin bayyanarwa, ana samar da iskar oxygen guda ɗaya sannan kuma an sami sakamako na ɗan lokaci kaɗan na ragowar histidine ta hanyar binciken aniline.Muna nuna babban amincin sa ta hanyar taswirar proteome na musamman na organelle.Kwatanta gefe-da-gefe na PDPL tare da TurboID yana nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun tsarin kariya na PDPL.Bayan haka, mun yi amfani da PDPL zuwa ga mai cutar da ke da alaƙa da BRD4 da E3 Parkin ligase kuma mun sami masu hulɗa da ba a san su ba.Ta hanyar nunin wuce gona da iri, abubuwan da ba a san su ba, Ssu72 da SNW1, an gano su don Parkin, wanda lalacewarsa ke yin sulhu ta hanyar hanyar haɓaka-proteasome.
Ingantacciyar sifa ta hanyoyin sadarwar sunadaran suna haifar da mahimman hanyoyin salon salula da yawa.Sabili da haka, ingantaccen taswirar sararin samaniya na hulɗar furotin zai samar da tushen kwayoyin halitta don tantance hanyoyin nazarin halittu, ilimin cututtuka, da tarwatsa waɗannan hulɗar don dalilai na warkewa.Don wannan, hanyoyin da za su iya gano hulɗar ɗan lokaci a cikin sel masu rai ko kyallen takarda suna da kyawawa sosai.Affinity Tsarkake Mass Spectrometry (AP-MS) a tarihi an yi amfani dashi don gano abokan haɗin gwiwar sunadaran sha'awa (POI).Tare da haɓaka hanyoyin proteomics masu ƙididdigewa, an ƙirƙiri Bioplex3.0, mafi girman bayanan cibiyoyin sadarwar furotin dangane da AP-MS.Kodayake AP-MS yana da ƙarfi sosai, ƙwayar tantanin halitta da matakan dilution a cikin aikin aiki suna karkata zuwa ga rauni da ma'amala mai ɗaurewa na wucin gadi da gabatar da kayan tarihi na bayan-lysis kamar nau'ikan ma'amala mai ban tsoro waɗanda ba su da alaƙa kafin lysis.
Don magance waɗannan batutuwa, an ƙirƙira amino acid marasa ɗabi'a (UAA) tare da ƙungiyoyi masu haɗin gwiwa da dandamalin lakabin enzymatic kusa (PL) (misali APEX da BioID)5.Kodayake an sami nasarar amfani da hanyar UAA a cikin yanayi da yawa kuma yana ba da bayanai kan mannen furotin kai tsaye, ana buƙatar haɓaka wurin shigar da UAA.Mafi mahimmanci, hanya ce ta alamar stoichiometric wacce ba ta da jujjuyawar abubuwan da suka faru ta alama.Sabanin haka, hanyoyin enzymatic PL, irin su hanyar BioID, sun haɗa biotin ligase na injiniya zuwa POI7, wanda daga baya yana kunna biotin don samar da matsakaicin biotinyl-AMP ester mai amsawa.Enzyme don haka yana haɓakawa kuma yana fitar da “girgije” biotin da aka kunna wanda ke lakafta ragowar lysine na kusa.Koyaya, BioID yana buƙatar fiye da sa'o'i 12 don samun isassun sigina mai alama, wanda ke hana amfani da shi tare da ƙudurin ɗan lokaci.Yin amfani da juyin halitta da aka ba da umarni bisa nunin yisti, TurboID an ƙirƙira shi bisa BioID don ya zama mafi inganci, yana ba da damar yin lakabi mai inganci tare da biotin cikin mintuna 10, yana ba da damar ƙarin nazarin matakai masu ƙarfi.Saboda TurboID yana aiki sosai kuma matakan biotin na endogenous sun wadatar don alamar ƙananan matakin, lakabin baya ya zama matsala mai yuwuwa lokacin da aka inganta sosai da kuma sanya lakabin lokaci ta hanyar ƙara biotin na waje.Bugu da ƙari, esters da aka kunna ba su da tasiri sosai (t1 / 2 ~ 5 min), wanda zai iya haifar da babban radiyo mai lakabi, musamman bayan jikewa na sunadaran maƙwabta tare da biotin 5. A wata hanya, haɗin kwayoyin halitta na injiniya ascorbate peroxidase (watau biotin- phenol radicals kuma yana ba da damar lakabin furotin a cikin minti daya9,10. APEX ana amfani dashi sosai don gano proteomes subcellular, membrane protein complexes, da cytosolic protein complexes11,12. salon salula tafiyar matakai.
Don haka, sabuwar hanyar da za ta iya samar da ƙarin amsawa mai suna-Radius nau'in kashe nau'ikan nau'ikan nau'ikan damfara mai girma tare da daidaiton sararin samaniya da na ɗan lokaci ba tare da ɓata mahimmancin hanyoyin salula ba zai zama muhimmin ƙari ga hanyoyin da ake da su. Daga cikin nau'ikan masu amsawa, iskar oxygen guda ɗaya ta tada hankalinmu saboda ɗan gajeren lokacin rayuwarsa da iyakataccen radius mai yaduwa (t1/2 <0.6 µs a cikin sel)13. Daga cikin nau'ikan masu amsawa, iskar oxygen guda ɗaya ta tada hankalinmu saboda ɗan gajeren lokacin rayuwarsa da iyakataccen radius mai yaduwa (t1/2 <0.6 µs a cikin sel)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии (t1/2 < 0,6 мкс в клетках)13. Daga cikin nau'ikan aiki, iskar oxygen guda ɗaya ya ja hankalinmu saboda ɗan gajeren lokacin rayuwarsa da ƙarancin watsawar radius (t1/2 <0.6 µs a cikin sel)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）命短和 1/2 <0.6 µs）而引起了我们的注意13 Среди активных форм наше внимание привлекает синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии (t1/2 < 0,6 мкс в клетках). Daga cikin nau'ikan aiki, iskar oxygen guda ɗaya yana jan hankalinmu saboda ɗan gajeren lokacin rayuwarsa da ƙarancin watsawar radius (t1/2 <0.6 μs a cikin sel).An ba da rahoton iskar oxygen guda ɗaya zuwa ga bazuwar oxidize methionine, tyrosine, histidine da tryptophan, yana mai da shi polar 14,15 don haɗawa da amine ko thiol tushen bincike16,17.Ko da yake an yi amfani da iskar oxygen guda ɗaya don lakabin sashin salula na RNA, dabarun sake dawo da alamomin kusancin POI na ƙarshe sun kasance ba a gano su ba.Anan, muna gabatar da wani dandali da ake kira lakabin kusancin kusancin hoto (PDPL), inda muke amfani da haske mai shuɗi don haskaka POIs da aka haɗe tare da miniSOG photosensitizer da kuma haifar da ƙwayar iskar oxygen guda ɗaya don oxidize ragowar kusanci, sannan gyare-gyaren da ke ɗauke da amine don oxidize binciken sinadarai zuwa cikin Matsakaicin sel masu rai..Mun gwada gungun masu binciken sinadarai don haɓaka ƙayyadaddun alamar tag da gano wuraren gyarawa ta amfani da buɗaɗɗen ayyukan haɓakar ƙwayoyin cuta.Kwatanta gefe-da-gefe na PDPL tare da TurboID yana nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun tsarin kariya na PDPL.Mun yi amfani da wannan hanyar zuwa alamomin takamaiman kwayoyin halitta na proteome na subcellular da kuma ganowar proteome gabaɗaya na abokan haɗin gwiwa don furotin da ke da alaƙa da epigenetic na furotin BRD4 da E3 ligase Parkin mai alaƙa da cutar Parkinson, wanda ya tabbatar da sanannen kuma sanannen cibiyar sadarwa na furotin. hulɗa..Ƙarfin PDPL don gane abubuwan E3 a cikin manyan rukunin furotin yana wakiltar yanayin da ake buƙatar sanin masu ɗaure kai tsaye.An tabbatar da wuraren shakatawa guda biyu waɗanda ba a san su ba waɗanda ke shiga tsakani ta hanyar ɓarna-proteasome a wurin.
Photodynamic far (PDT)19 da chromophore-assisted Laser inactivation (CALI) 20, wanda hasken haske tare da photosensitizers ke haifar da iskar oxygen guda ɗaya, na iya hana sunadaran da aka yi niyya ko haifar da mutuwar tantanin halitta.Tunda iskar oxygen guda ɗaya abu ne mai amsawa sosai tare da nisan yaduwa na kusan 70 nm, ana iya sarrafa iskar oxygen ta sararin samaniya a kusa da na'urar daukar hoto.Dangane da wannan ra'ayi, mun yanke shawarar yin amfani da iskar oxygen guda ɗaya don cimma alamar kusancin rukunin furotin a cikin sel masu rai.Mun haɓaka tsarin chemoproteomic na PDPL don cika ayyuka huɗu: (1) don haɓaka haɓakar iskar oxygen guda ɗaya mai aiki kama da tsarin PL enzymatic;(2) samar da lakabin da aka warware lokaci akan farawa haske;(3) ta hanyar canji (4) Guji yin amfani da masu haɗin gwiwa na ƙarshe (kamar biotin) don rage bango, ko amfani da manyan abubuwan da ke daɗaɗawa (kamar peroxides) don rage bayyanar tantanin halitta ga damuwa na muhalli.
Ana iya raba masu ɗaukar hoto zuwa nau'i biyu da suka haɗa da ƙananan nau'in fluorophores (misali rose bengal, methylene blue)22 da ƙananan ƙwayoyin cuta (misali miniSOG, KillerRed)23.Don cimma ƙirar ƙira, mun haɓaka dandalin PDPL na ƙarni na farko ta hanyar ƙara sunadaran photosensitizer (PS) zuwa POI24,25 (Hoto 1a).Lokacin da aka haskaka shi da shuɗi mai haske, iskar oxygen guda ɗaya yana oxidizes ragowar amino acid na nucleophilic, yana haifar da polarity mai ƙarfi wanda yake electrophilic kuma zai iya ƙara amsa tare da amine bincike nucleophiles16,17.An ƙirƙira binciken tare da riƙon alkyne don ba da damar danna sunadarai da ja ƙasa don halayyar LC/MS/MS.
Misalin tsari na lakabin rukunin furotin wanda miniSOG ya shiga tsakani.Lokacin da aka fallasa su zuwa haske mai shuɗi, ƙwayoyin da ke bayyana miniSOG-POI suna haifar da iskar oxygen guda ɗaya, wanda ke canza sunadaran haɗin gwiwa amma ba sunadaran da ba su ɗaure ba.Matsakaicin samfurori na photooxidation ana kama su ta hanyar lakabin relay na binciken sinadarai na amine don samar da haɗin kai.Ƙungiyar alkynyl akan binciken sinadarai tana ba da damar danna chemistry don haɓakawa ta hanyar ja da ƙasa wanda ke biye da ƙididdigar LC-MS/MS.b Tsarin sinadarai na binciken amine 1-4.c Wakilin gel mai walƙiya mai walƙiya na mitochondrial localized miniSOG-matsakaicin alamomin proteomic ta amfani da binciken 1-4 da ƙididdige dangi dangane da densitometry na gel.An kimanta siginar-zuwa-baya na binciken sinadarai ta amfani da gwaje-gwajen sarrafawa mara kyau ban da haske mai shuɗi ko amfani da ƙwayoyin HEK293T ba tare da bayanin miniSOG ba.n = 2 samfurori masu zaman kansu na ilimin halitta.Kowace digo tana wakiltar kwafin halitta.d Wakilin ganowa da ƙididdigewa na PDPL ta amfani da ingantaccen bincike 3 a gaban ko rashi na abubuwan da aka nuna na PDPL kamar c.n = 3 samfurori masu zaman kansu na ilimin halitta.Kowace digo tana wakiltar kwafin halitta.Layukan tsakiya da wuski suna wakiltar ma'ana da ± daidaitaccen karkata.CBB: Coomassie Brilliant Blue.e Confocal Hoto na iskar oxygen guda ɗaya tare da tabon Si-DMA-ja mai nisa.Matsakaicin girman: 10µm.Hoton Gel da gwaje-gwajen ɓoye-ɓoye an maimaita kansu aƙalla sau biyu tare da sakamako iri ɗaya.
Mun fara gwada ƙarfin masu balagagge photosensitizers miniSOG26 da KillerRed23, a tsaye an bayyana su a cikin HEK293T, don daidaita alamar propargylamine na proteome azaman binciken sinadarai (Ƙarin Hoto 1a).Gel fluorescence bincike ya nuna cewa an samu duka alamar proteome ta amfani da miniSOG da iska mai haske mai shuɗi, yayin da ba a sami samfurin alamar alama tare da KillerRed ba.Don inganta siginar-zuwa-baya rabo, mun gwada saitin binciken sinadarai masu dauke da aniline (1 da 3), propylamine (2), ko benzylamine (4).Mun lura cewa ƙwayoyin HEK293T da kansu suna da siginar baya mafi girma idan aka kwatanta da babu haske mai shuɗi, mai yiwuwa saboda riboflavin photosensitizer na endogenous, flavin mononucleotide (FMN) 27. Binciken sinadarai na tushen Aniline 1 da 3 sun ba da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, tare da HEK293T a tsaye yana bayyana miniSOG a cikin mitochondria yana nuna> haɓakar ninki 8 a cikin sigina don bincike na 3, yayin binciken 2 da aka yi amfani da shi a cikin hanyar alamar RNA CAP-seq kawai yana nunawa ~ 2.5- ninka girman siginar, mai yiyuwa saboda zaɓin amsawa daban-daban tsakanin RNA da furotin (Fig. 1b, c). Binciken sinadarai na tushen Aniline 1 da 3 sun ba da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, tare da HEK293T a tsaye yana bayyana miniSOG a cikin mitochondria yana nuna> haɓakar ninki 8 a cikin sigina don bincike na 3, yayin binciken 2 da aka yi amfani da shi a cikin hanyar alamar RNA CAP-seq kawai yana nunawa ~ 2.5- ninka girman siginar, mai yiyuwa saboda zaɓin amsawa daban-daban tsakanin RNA da furotin (Fig. 1b, c).Binciken sinadarai na tushen Aniline 1 da 3 sun nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai: HEK293T, wanda ke bayyana miniSOG a cikin mitochondria, yana nuna haɓakar sigina fiye da ninki 8 don bincike na 3, yayin binciken 2, wanda aka yi amfani da shi a cikin hanyar alamar CAP-seq RNA, kawai yana nuna ~ 2.5-ninka karuwa sigina, mai yiwuwa saboda daban-daban da ake so reactivity tsakanin RNA da furotin (Fig. 1b, c).的 的 的 化学 化学 1 和 的具 更 更 好更, Hek293t 在 表达, 粒体 增加 中 RNA 标记 探针 2 探针 显示 ~ 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）。的 的 化学 化学 1 和具 更 的 的更, Hek293t 在 3 表达 于 标记 标记 标记 的 的 的 标记 标记 的 的 的 的 ~ 的 的 的 的 ~ 2.5 -倍信号增加，可能是由于RNABinciken sinadarai na tushen Aniline 1 da 3 suna da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, HEK293T da ƙarfi ya bayyana miniSOG a cikin mitochondria, kuma binciken 3 yana da haɓakar sigina sama da ninki 8, yayin da binciken 2 don hanyar lakabin CAP-seq RNA ya nuna kawai ~ 2.5 - ninka karuwa.a cikin siginar, mai yiwuwa saboda zaɓin amsawa daban-daban tsakanin RNA da furotin (Fig. 1b, c).Bugu da ƙari, an gwada binciken 3 isomers da hydrazine probes (bincike 5, 6, 7), yana tabbatar da ingantaccen bincike na 3 (Ƙarin Hoton 1b, c).Hakazalika, in-gel fluorescence bincike ya bayyana wasu ingantattun sigogi na gwaji: tsayin raƙuman raƙuman iska (460 nm), ƙaddamar da bincike na sinadarai (1 mM), da lokacin haskakawa (20 min) (Ƙarin Hoton 2a-c).Yin watsi da kowane bangare ko mataki a cikin ka'idar PDPL ya haifar da jujjuya sigina mai mahimmanci zuwa bango (Fig. 1d).Musamman ma, alamar sunadaran sun ragu sosai a gaban sodium azide ko trolox, waɗanda aka sani suna kashe iskar oxygen guda ɗaya.Kasancewar D2O, wanda aka sani don daidaita iskar oxygen guda ɗaya, yana haɓaka siginar alamar.Don bincika gudunmawar sauran nau'in oxygen mai amsawa ga lakabi, an ƙara mannitol da bitamin C don kafa hydroxyl da superoxide radical scavengers, bi da bi, 18, 29, amma ba a samo su don rage lakabi ba.Ƙarin H2O2, amma ba haske ba, bai haifar da lakabi ba (Ƙarin Hoto 3a).Fluorescence Singlet Hoto oxygen Hoto tare da binciken Si-DMA ya tabbatar da kasancewar iskar oxygen guda ɗaya a cikin waya HEK293T-miniSOG, amma ba a cikin ainihin waya HEK293T ba.Bugu da ƙari, mitoSOX Red ba zai iya gano samar da superoxide ba bayan haskakawa (Fig. 1e da Ƙarin Hoto 3b) 30. Waɗannan bayanai suna nuna karfi da cewa iskar oxygen guda ɗaya shine babban nau'in oxygen mai amsawa wanda ke da alhakin lakabin proteomic na gaba.An tantance cytotoxicity na PDPL ciki har da hasken haske mai shuɗi da binciken sinadarai, kuma ba a lura da cytotoxicity mai mahimmanci ba (Ƙarin Hoton 4a).
Domin yin nazarin tsarin yin lakabi da ba da damar gano proteomic na rukunin sunadaran gina jiki ta amfani da LC-MS/MS, da farko muna buƙatar sanin waɗanne amino acid ne aka gyaggyarawa da kuma yawan adadin alamun bincike.Methionine, histidine, tryptophan da tyrosine an ruwaito cewa an canza su ta hanyar iskar oxygen guda ɗaya14,15.Muna haɗa aikin TOP-ABPP31 tare da buɗewar buɗe ido mara son zuciya wanda dandamalin kwamfuta na FragPipe ya samar akan MSFragger32.Bayan gyare-gyaren iskar oxygen guda ɗaya da lakabin bincike na sinadarai, danna sunadarai an yi ta amfani da alamar rage biotin wanda ke ɗauke da mahaɗin da za a iya cirewa, sannan neutravidin mikewa da narkewar trypsin.peptide da aka gyara, wanda har yanzu yana daure da guduro, an ƙera hoto don nazarin LC-MS/MS (Hoto 2a da Ƙarin Bayanai 1).Yawancin gyare-gyare sun faru a ko'ina cikin proteome tare da taswirar peptide sama da 50 (PSM) da aka jera (Fig. 2b).Abin mamaki, kawai mun lura da gyare-gyare na histidine, mai yiwuwa saboda yawan maida hankali na histidine oxidized zuwa binciken aniline fiye da sauran amino acid.Dangane da tsarin da aka buga na histidine oxidation ta hanyar iskar oxygen guda ɗaya, 21,33 tsarin tsarin delta-mass na +229 Da ya dace da ƙaddamarwar bincike 3 tare da 2-oxo-histidine bayan oxidations guda biyu, yayin da +247 Da shine samfurin hydrolysis. na +229 Da (Ƙarin Hoto 5).Ƙimar bakan MS2 ya nuna babban amincin gano yawancin y da b ions, ciki har da gano gyare-gyaren ions guntu (y da b) (Fig. 2c).Binciken yanayi na jerin gida na PDPL-gyara histidines ya nuna matsakaicin fifikon fifiko don ƙananan ragowar hydrophobic a ± 1 matsayi (Ƙarin Hoto 4b).A matsakaita, an gano 1.4 histidines a kowane furotin, kuma wuraren waɗannan alamomin an ƙaddara su ta hanyar yanki mai iya samun ƙarfi (SASA) da wadatar ƙarfi mai ƙarfi (RSA) bincike (Ƙarin Hoton 4c,d).
Gudun aiki mara son zuciya don nazarin zaɓin saura ta amfani da dandamalin lissafin FragPipe wanda MSFragger ke ƙarfafawa.Ana amfani da masu haɗawa da zazzagewa a cikin Click chemistry don ba da damar ɗaukar hoto na peptides da aka gyara daga resin streptavidin.An ƙaddamar da wani buɗaɗɗen bincike don gano gyare-gyare da yawa, da sauran abubuwan da suka dace.b Sanya yawan gyare-gyaren da ke faruwa a ko'ina cikin proteome.Peptide taswirar PSM.c MS2 spectral annotation of histidine sites gyare-gyare tare da bincike 3. A matsayin misali misali, wani covalent dauki tare da bincike 3 kara +229.0938 Da ga gyara amino acid.d Gwajin maye gurbi da aka yi amfani da shi don gwada alamun PDPL.PRDX3 (H155A, H225A) da PRDX1 (H10A, H81A, H169A) an canza su tare da nau'in plasmids na daji don gano anti-Flag.e An amsa peptide na roba tare da tsabtace miniSOG a gaban binciken 3 da samfurori masu dacewa tare da Δm +247 da +229 a cikin LC-MS bakan.f In vitro protein-to-protein hulɗar da aka kera tare da miniSOG-6xTag-Tag da anti-6xMaganin rigakafi.Antibiotin (streptavidin-HRP) da anti-mouse Western blot bincike na miniSOG-6xHis/anti-6xKayan rigakafin garkuwar jiki da aka yi wa lakabi da bincike 3, ya danganta da lokacin fallasa ga haske.Alamun sunadaran sunadaran guda ɗaya ana bayyana su a cikin madaidaicin nauyin kwayoyin: sarkar hasken antibody LC, sarkar antibody nauyi.An maimaita waɗannan gwaje-gwajen da kansu aƙalla sau biyu tare da sakamako iri ɗaya.
Don tantancewar sinadarai na wurin yin lakabin, PRDX3 da PRDX1 da aka gano ta hanyar yawan gani an canza su daga histidine zuwa alanine kuma idan aka kwatanta da nau'in daji a cikin gwaje-gwajen kamuwa da cuta.Sakamakon PDPL ya nuna cewa maye gurbi ya rage yawan lakabi (Fig. 2d).A halin yanzu, jerin peptide da aka gano a cikin binciken da aka buɗe an haɗa su kuma an amsa su a cikin vitro tare da ingantaccen miniSOG a gaban binciken 3 da haske mai shuɗi, samfuran samar da samfuran tare da babban motsi na + 247 da + 229 Da lokacin da LC-MS ta gano (Figure) . 2e ku).).Don gwada ko ana iya yin ma'amala da sunadaran proximal a cikin vitro don amsawa ga miniSOG photoactivation, mun tsara gwajin kusancin wucin gadi ta hanyar hulɗa tsakanin miniSOG-6xShin furotinSa da anti-Sa na monoclonal antibody in vitro (Hoto 2f).A cikin wannan binciken, muna tsammanin alamar kusanci na sarƙoƙi masu nauyi da haske tare da miniSOG.A gaskiya ma, anti-mouse (gane da nauyi da haske sarƙoƙi na anti-6xHis-labeled antibody) da streptavidin Western blots nuna karfi biotinylation na nauyi da haske sarƙoƙi.Musamman ma, mun lura da miniSOG autobiotinylation saboda alamar 6xHis da haɗe-haɗe tsakanin haske da sarƙoƙi mai nauyi, wanda zai iya danganta da rata da aka bayyana a baya tsakanin lysine da 2-oxo-histidine proximal amsa.A ƙarshe, mun kammala cewa PDPL tana canza histidine ta hanyar dogaro da kusanci.
Burinmu na gaba shine mu siffanta proteome na subcellular don gwada ƙayyadaddun alamar alamar wuri.Sabili da haka, mun bayyana miniSOG a tsaye a cikin tsakiya, mitochondrial matrix, ko ER membrane na sel HEK293T (Fig. 3a).Binciken gel fluorescence bincike ya bayyana yawan maɗaura masu lakabi a wurare uku na ƙananan ƙwayoyin cuta da kuma nau'o'in lakabi daban-daban (Fig. 3b).Binciken Hoto na Fluorescence ya nuna ƙayyadaddun ƙayyadaddun PDPL (Fig. 3c).Aikin PDPL ya biyo baya ta hanyar dannawa tare da dyes na rhodamine don ƙaddamar da proteomes na subcellular ta amfani da microscopy na fluorescence, kuma alamun PDPL sun kasance tare da DAPI, mitochondrial trackers, ko ER trackers, yana tabbatar da babban amincin PDPL.Ga wurare uku na gabobin jiki, kwatankwacin gefe-da-gefe na PDPL tare da TurboID ta amfani da avidin western blot ya nuna cewa an yi wa PDPL lakabi na musamman idan aka kwatanta da nasu sarrafawa.Ƙarƙashin sharuɗɗan PDPL, ƙarin maƙamai masu lakabi sun bayyana, suna nuna ƙarin sunadaran da aka yiwa lakabin PDPL (Ƙarin Hoton 6a-d).
wakilcin tsari na ƙayyadaddun ƙayyadaddun proteome mai tsaka-tsaki na miniSOG.miniSOG ya yi niyya ga matrix mitochondrial ta hanyar haɗuwa zuwa N-terminal 23 amino acid na COX4 (mito-miniSOG), tsakiya ta hanyar haɗuwa zuwa H2B (nucleus-miniSOG), da Sec61β ta gefen cytoplasmic na ER membrane (ER-miniSOG). ).Alamomi sun haɗa da hoton gel, hoto mai ma'ana, da ma'auni mai yawa.b Hotunan gel na wakilci na bayanan bayanan martaba na PDPL guda uku na musamman.CBB Coomassie Brilliant Blue.c Hotunan ƙwaƙƙwaran sel na sel HEK293T a tsaye suna bayyana miniSOG tare da wurare daban-daban na ƙananan ƙwayoyin cuta da aka gano ta antibody mai lakabin V5 (ja).Ana amfani da alamun subcellular don mitochondria da ER (kore).Gudun aikin PDPL ya haɗa da gano miniSOG (rawaya) masu suna proteomes na subcellular ta amfani da Cy3-azide click chemistry.Matsakaicin girman: 10µm.d Shirye-shiryen volcanic na proteomes mai alamar PDPL a cikin gabobin gabobin daban-daban waɗanda aka ƙididdige su ta hanyar ƙididdigewa mara lakabi (n = 3 gwaje-gwajen nazarin halittu masu zaman kansu).Anyi amfani da t-gwajin ɗalibi mai wutsiya biyu akan filaye masu aman wuta.An yi amfani da nau'in daji na HEK293T azaman iko mara kyau. Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> 2- ninka girman girman ion). Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> 2- ninka girman girman ion). Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в интенсивности ионов). Sunadaran sunadarai masu mahimmanci suna haskakawa a cikin ja (p <0.05 da> 2-banbanci mai tsanani a cikin ƙarfin 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). Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> bambancin ninki biyu a ƙarfin ionic).Sunadaran da ke da alaƙa masu mahimmanci don HEK293T-miniSOG amma ba mahimmanci ga HEK293T ana nuna su a cikin kore.e Nazarin ƙayyadaddun bayanan bayanan furotin daga gwaje-gwaje d.Jimlar adadin sunadaran sunadaran ƙididdiga a cikin kowace gabobin jiki (dige ja da kore) an yi alama a saman.Histograms suna nuna sunadaran da aka keɓe a cikin organelles dangane da MitoCarta 3.0, GO bincike da A. Ting et al.mutane.Rarraba saitin bayanai don mitochondria, nuclei, da ER.An maimaita waɗannan gwaje-gwajen da kansu aƙalla sau biyu tare da sakamako iri ɗaya.Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
Ƙarfafawa ta hanyar gel da sakamakon hoto, an yi amfani da ƙididdige ƙididdiga marasa alamar don ƙididdige proteome da aka gano a cikin kowace gabobin (Ƙarin Bayanan 2).An yi amfani da HEK293T mara canzawa azaman iko mara kyau don cire alamomin baya. Binciken makircin dutsen mai aman wuta ya nuna sunadaran da aka wadatar da su sosai (p <0.05 da> 2-fold ion intensity) da kuma sunadaran guda ɗaya waɗanda kawai ke cikin layin bayyana miniSOG (Fig. 3d ja da dige kore). Binciken makircin dutsen mai aman wuta ya nuna sunadaran da aka wadatar da su sosai (p <0.05 da> 2-fold ion intensity) da kuma sunadaran guda ɗaya waɗanda kawai ke cikin layin bayyana miniSOG (Fig. 3d ja da dige kore). 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). Binciken makircin dutsen mai aman wuta ya nuna sunadaran da aka wadatar da su sosai (p <0.05 da> 2-fold ion intensity) da kuma sunadaran guda ɗaya waɗanda ke cikin layin miniSOG kawai (Fig. 3d, ja da dige kore).分析 分析 出 出 显着 出 显着 显着 显着 显着 出 显着 蛋白质 蛋白质 (P <0.05 和> 2 倍 离子 的) 表达 系 在于 在于 Minisog 表达 系 3d 红色 和 绿色点).火山图分析 蛋白质 出 出 出 出 蛋白质 (p <0.05 和) 仅 存 在于 在于 单 单 的 的 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). Binciken makircin dutsen mai aman wuta ya bayyana sunadaran da aka wadatar da su sosai (p <0.05 da> 2x ƙarfin ionic) da kuma furotin guda ɗaya kawai a cikin layin magana na miniSOG (dige ja da kore a cikin siffa 3d).Hada waɗannan bayanan, mun gano 1364, 461, da 911 masu mahimmancin kididdigar makaman nukiliya, mitochondrial, da sunadaran ER na waje, bi da bi.Don nazarin daidaito na organelle-localized PDPL, mun yi amfani da MitoCarta 3.0, Gene Ontology (GO) bincike, da A. Ting et al.an yi amfani da saitin bayanai8 don mitochondria, tsakiya, da ER don gwada ƙayyadaddun kwayoyin halitta na sunadarai da aka gano, daidai da daidaitattun 73.4, 78.5, da 73.0% (Fig. 3e).Ƙayyadaddun PDPL ya tabbatar da cewa PDPL kayan aiki ne mai kyau don gano takamaiman ƙwayoyin ƙwayoyin cuta.Musamman ma, bincike-bincike na mitochondrial da aka gano sunadaran sunadaran mitochondrial sun nuna cewa an rarraba proteome da aka kama a cikin matrix da membrane na ciki (226 da 106, bi da bi), yana lissafin 91.7% (362) na adadin adadin furotin mitochondrial da aka gano.An kuma tabbatar da babban matakin PDPL (Ƙarin Hoton 7a).Hakazalika, bincike na subnuclear ya nuna cewa proteome da aka kama an rarraba shi ne a cikin tsakiya, nucleoplasm, da kuma nucleolus (Ƙarin Hoto 7b).Binciken ƙwayoyin cuta na nukiliya tare da siginar siginar siginar ƙirar nukiliya (3xNLS) ya nuna daidai daidai da ginin H2B (Ƙarin Hoton 7c-h).Don tantance ƙayyadaddun alamar alamar PDPL, an zaɓi laminin A matsayin tarkon POI7 da aka fi sani da shi.PDPL ta gano sunadaran da aka wadatar da su sosai 36, daga cikinsu sunadaran 12 (30.0% gami da lamin A) sun kasance suna da siffar lamin A hulɗar sunadaran da aka tsara ta hanyar bayanan String, tare da kaso mafi girma fiye da hanyar BioID (proteins 122) 28 na 28., 22.9 %) 7. Hanyarmu ta gano ƙananan sunadaran, mai yiyuwa saboda ƙayyadaddun wuraren lakabi, wanda ya yiwu ta hanyar iskar oxygen guda ɗaya mai aiki.Binciken GO ya nuna cewa sunadaran da aka gano galibi suna cikin nucleoplasm (26), membrane na nukiliya (10), membrane na nukiliya (9), da pores na nukiliya (5).Gaba ɗaya, waɗannan sunadaran da ke cikin makaman nukiliya sun ƙunshi 80% na furotin da aka wadatar, suna ƙara nuna ƙayyadaddun ƙimar PDPL (Ƙarin Hoton 8a-d).
Bayan da aka kafa ikon PDPL don yin alamar kusanci a cikin gabobin jiki, mun gwada ko za a iya amfani da PDPL don nazarin abokan haɗin gwiwar POI.Musamman, mun nemi ma'anar nazarin PDPL na sunadaran cytosolic, waɗanda ake ɗaukar mafi wahalar hari fiye da takwarorinsu na yankin membrane saboda yanayinsu mai ƙarfi.Bromodomain da extraterminal (BET) sunadaran sunadaran BRD4 ya ja hankalin mu don muhimmiyar rawa a cikin cututtuka daban-daban 35, 36.Rukunin da BRD4 ya kirkira shine mai haɗawa da rubutu da mahimmancin maƙasudin warkewa.Ta hanyar daidaita maganganun c-myc da abubuwan rubutu na Wnt5a, ana tsammanin BRD4 shine babban mahimmin ƙayyadaddun cutar sankarar bargo ta myeloid (AML), myeloma mai yawa, lymphoma na Burkitt, ciwon hanji da cututtukan kumburi37,38.Bugu da kari, wasu ƙwayoyin cuta sun yi niyya ga BRD4 don daidaita rubutun hoto ko bidiyo mai zagaya yanar gizo da sauri, kamar su papillomavirus, HIV, da SARS-CoV-236,39.
Don taswirar hulɗar BRD4 ta amfani da PDPL, mun haɗa miniSOG tare da gajeriyar isoform N- ko C-terminal na BRD4.Sakamakon proteomic ya bayyana babban mataki na haɗuwa tsakanin gine-ginen biyu (Ƙarin Hoto 9a).Ƙwararriyar sinadarin nukiliya da aka gano tare da miniSOG-H2B yana rufe 77.6% na sunadaran da ke hulɗa da BRD4 (Ƙarin Hoto 9b).Sa'an nan kuma, an yi amfani da lokuta daban-daban na haske (2, 5, 10, 20 min) don daidaita radius mai alamar (Fig. 4a da ƙarin bayanan 3).Mun kammala cewa a cikin gajeren lokaci na lokaci-lokaci, PDPL za ta yi lakabi da abokan hulɗa kai tsaye, yayin da tsawon lokaci za su haɗa da sunadaran da aka gano a lokacin gajeren lokacin daukar hoto da kuma hari kai tsaye a cikin rukunin lakabi.A gaskiya ma, mun sami karfi mai karfi tsakanin maki masu kusa (84.6% na 2 da 5 min; 87.7% don 5 da 10 min; 98.7% don 10 da 20 min) (Fig. 4b da Ƙarin 9c).A cikin duk ƙungiyoyin gwaji, mun sami ba kawai alamar BRD4 ba, amma sanannen hari da yawa kamar MED1, CHD8, BICRA, NIPBL, SMC1A, da HMGB1 annoted a cikin bayanan kirtani.Ƙarfin ionic na waɗannan maƙasudi ya yi daidai da lokacin bayyanarwa (Fig. 4c da Ƙarin Hoto 9d).Binciken GO na sunadaran da aka gano a cikin rukuni na 2-minti ya nuna cewa an gano sunadaran da aka gano a cikin tsakiya kuma sun shiga cikin gyaran chromatin da aikin RNA polymerase.Ayyukan kwayoyin halitta na sunadaran sun wadatar da su a cikin ɗaurin chromatin ko haɗin kai na rubutu, daidai da aikin BRD4 (Fig. 4d).Binciken hulɗar furotin da aka kunna kirtani ya bayyana matakin farko na hulɗar kai tsaye tsakanin BRD4 da HDAC haɗin gwiwar iyali kamar SIN3A, NCOR2, BCOR, da SAP130 (Fig. 4e da Ƙarin Hoto 9e), daidai da BRD4 da HDAC daure acetylated histones. ..Bugu da ƙari, maƙasudin wakilcin da LC-MS/MS ya gano, ciki har da Sin3A, NSUN2, Fus, da SFPQ, an tabbatar da su ta hanyar lalata ta Yamma (Fig. 4f).Kwanan nan, an ba da rahoton gajeriyar isoform na BRD4 don samar da nuclei tare da kaddarorin rabuwa na ruwa-ruwa (LLPS).Sunadaran dauri na RNA Fus da SFPQ suna yin sulhu tsakanin LLPS na matakai daban-daban na salon salula kuma an gano su anan azaman sunadaran ɗaurin BRD4 da ba a yi rikodin su ba.An tabbatar da hulɗar tsakanin BRD4 da SFPQ ta hanyar gwaje-gwajen haɗin gwiwar rigakafi (co-IP) (Hoto 4g), yana ba da shawarar wata hanyar don rabuwa da ruwa-ruwa mai tsaka-tsakin BRD4 wanda ya cancanci ƙarin bincike.Idan aka haɗu, waɗannan sakamakon sun nuna cewa PDPL shine ingantaccen dandamali don gano sanannun hulɗar BRD4 da kuma sunadaran da ba a san su ba.
wakilcin tsari na miniSOG-mai shiga tsakani BRD4 alamar kusanci, lokutan bayyanawa: 2, 5, 10, da 20 min.b Ciwon sunadaran da aka gano a lokuta daban-daban na haske.Ingantaccen furotin da aka gano a cikin HEK293T-miniSOG-BRD4 yana da mahimmanci a ƙididdiga idan aka kwatanta da nau'in daji HEK293T.c Ion ƙarfin lokacin ƙididdige wakilai mara lakabi sanannun sunadaran sunadaran BRD4 a lokacin ƙayyadadden lokacin fallasa.n = 3 samfurori masu zaman kansu na ilimin halitta.Ana gabatar da bayanai azaman ma'anar ± daidaitaccen karkacewa.d Gene ontological analysis (GO) na sunadaran da aka gano a cikin rukunin mintuna 2.An jera sharuɗɗan GO goma na farko.An yi launin kumfa bisa ga nau'in kalmar GO, kuma girman kumfa ya yi daidai da adadin sunadaran da aka samu a kowane lokaci.e String bincike na sunadarai masu hulɗa tare da BRD4.Da'irar rawaya suna manne kai tsaye kuma masu launin toka sune farkon layin manne kai tsaye.Layukan jajayen suna wakiltar ma'amalar da aka ƙaddara ta gwaji kuma shuɗin layin suna wakiltar hulɗar da aka annabta.f Wakilin BRD4 makasudin daurin da aka gano a cikin LC-MS/MS an tabbatar da su ta hanyar lalatawar Yamma.g Gwaje-gwajen haɗin gwiwar rigakafi sun tabbatar da hulɗar tsakanin SFPQ da BRD4.An maimaita waɗannan gwaje-gwajen da kansu aƙalla sau biyu tare da sakamako iri ɗaya.Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
Bugu da ƙari ga gano maƙasudin haɗin gwiwar POI da ba a yi rajista ba, muna tsammanin cewa PDPL za ta dace don gano abubuwan da ake amfani da su don enzymes, wanda zai buƙaci halayyar sunadaran da ke ɗaure kai tsaye a cikin manyan gidaje don bayyana abubuwan da ba a yi rajista ba.Parkin (wanda PARK2 ya sanya shi) shine E3 ligase kuma an san maye gurbi a cikin wurin shakatawa yana haifar da cututtukan Parkinson na yara (AR-JP)42.Bugu da ƙari, an bayyana wurin shakatawa a matsayin mahimmanci ga mitophagy (mitochondrial autophagy) da kuma kawar da nau'in oxygen mai amsawa.Duk da haka, kodayake an gano wasu wuraren shakatawa da yawa, har yanzu ba a san rawar da fakin ke takawa a cikin wannan cuta ba.Don bayyana abubuwan da ba a bayyana su ba, an gwada PDPL ta ƙara miniSOG zuwa N- ko C-terminus na fakin.An yi amfani da kwayoyin halitta tare da jigilar carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) don kunna wurin shakatawa ta hanyar PINK1-Parkin.Idan aka kwatanta da sakamakon mu na BRD4 PDPL, fakin N-terminus fusion ya bayyana babban saitin sunadaran da ake nufi, kodayake ya rufe babban yanki na C-terminus (177 daga 210) (Hoto 5a,b da Ƙarin Bayanai 4).Sakamakon ya yi daidai da rahotannin cewa alamun N-terminal na iya kunna Parkin44 a hankali.Abin mamaki, akwai sunadaran da suka mamaye bayananmu 18 kawai tare da sakamakon AP-MS da aka buga don Parkin43, mai yuwuwa saboda bambance-bambance tsakanin layin salula da ayyukan aikin kariya.Baya ga sunadaran da aka sani guda huɗu (ARDM1, HSPA8, PSMD14, da PSMC3) waɗanda aka gano ta hanyoyi biyu (Fig. 5c)43.Don ƙara tabbatar da sakamakon LC-MS/MS, an yi amfani da jiyya na PDPL da ɓangarorin Yamma da suka biyo baya don kwatanta sakamakon HEK293T na mahaifa da kuma tsayayyen layin shakatawa na N-terminal.CDK2, DUT, CTBP1, da PSMC4 da ba a san su ba a baya an gwada su tare da sananne mai ɗaure, DNAJB1 (Fig. 5d).
Siffar dutsen mai wuta na sunadaran hulɗar fakin a cikin sel HEK293T tare da bayyana miniSOG mai ƙarfi da aka haɗe zuwa N- ko C-terminus na shakatawa (n = 3 gwaje-gwajen nazarin halittu masu zaman kansu).Anyi amfani da t-gwajin ɗalibi mai wutsiya biyu akan filaye masu aman wuta.An yi amfani da HEK293T azaman iko mara kyau. Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> 2- ninka girman girman ion). Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> 2- ninka girman girman ion). Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в интенсивности ионов). Sunadaran sunadarai masu mahimmanci suna haskakawa a cikin ja (p <0.05 da> 2-banbanci mai tsanani a cikin ƙarfin 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). Sunadaran sunadaran da aka canza suna da mahimmanci a cikin ja (p <0.05 da> bambancin ninki biyu a ƙarfin ionic).Sunadaran da ke da alaƙa masu mahimmanci don HEK293T-miniSOG amma ba mahimmanci ga HEK293T ana nuna su a cikin kore.b Zane na Venn yana nuna sunadaran da suka yi karo da juna tsakanin ginin N-terminal da C-terminal.Alamun N-terminal na iya kunna fakin cikin damuwa da haifar da ƙarin sunadaran da za a iya gane su.c zane-zane na Venn yana nuna sunadaran da ke tattare da juna tsakanin PDPL da AP-MS.An jera sanannun masu mu'amala da juna, gami da 4 na 18 sunadaran da suka mamaye juna da 11 na sunadaran 159 da aka gano musamman a cikin PDPL.d Maƙasudin wakilcin da LC-MS/MS suka gano ta hanyar lalatawar Yamma.e Ssu72 da SNW1 an gano su azaman wuraren shakatawa marasa rijista.Wadannan plasmids sunadaran sunadaran FLAG an canza su zuwa HEK293T da HEK293T-Parkin-miniSOG tare da maganin CCCP a lokuta daban-daban.Lalacewar ta fi fitowa fili a cikin layin wuce gona da iri na Parkin.f Yin amfani da mai hana proteasome MG132, an tabbatar da cewa tsarin lalacewa na Ssu72 da SNW1 ana yin sulhu ta hanyar proteasome-ubiquitination.An maimaita waɗannan gwaje-gwajen da kansu aƙalla sau biyu tare da sakamako iri ɗaya.Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
Musamman ma, sunadaran da PDPL ta gano dole ne su haɗa da sunadaran da ke ɗaure fakin da kuma abubuwan da ke cikin su.Don gano wuraren shakatawa marasa rajista, mun zaɓi sunadaran sunadaran guda bakwai da aka gano (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 da SNW1) da plasmids da aka canza don fallasa waɗannan kwayoyin halitta zuwa HEK293T na al'ada kuma a tsaye suna bayyana miniSOG-Parkin's HEK2CCCPT magani ya biyo baya.Matakan sunadaran Ssu72 da SNW1 sun ragu sosai a cikin kwanciyar hankali na miniSOG-Parkin line (Fig. 5e).Jiyya tare da CCCP na tsawon sa'o'i 12 ya haifar da mafi girman lalacewa na sassan biyu.Don bincika ko lalatawar Ssu72 da SNW1 an tsara su ta hanyar proteasome-ubiquitination, mai hanawa mai hanawa MG132 an ƙara shi don hana ayyukan proteasome, kuma a gaskiya mun gano cewa an hana tsarin lalata su (Fig. 5f).An tabbatar da ƙarin maƙasudin maƙasudin maƙasudi azaman masu mu'amala da Parkin ta amfani da lalatawar Yamma (Ƙarin Hoto 10), wanda ya nuna daidaitaccen sakamako tare da LC-MS/MS.A ƙarshe, haɗakar da aikin aiki na PDPL tare da ƙaddamar da ƙwayar furotin da aka yi niyya yana ba da damar gano abubuwan da ba a yi rajistar E3 ligase substrates.
Mun ƙirƙira dandamalin alamar kusanci na gama gari wanda ke ba ku damar gano sararin samaniya da lokacin hulɗar POIs.Dandalin yana dogara ne akan furotin na miniSOG photosensitizer, wanda kusan 12 kDa ne kawai, ƙasa da rabin girman girman APEX2 enzyme (27 kDa) da kashi ɗaya bisa uku girman TurboID (35 kDa).Karamin girman yakamata ya faɗaɗa kewayon aikace-aikace don nazarin ƙananan haɗin gwiwar furotin.Ana buƙatar ƙarin binciken ƙarin na'urorin daukar hoto, ko sunadaran sunadaran halitta ko ƙananan ƙwayoyin cuta, don ƙara yawan adadin iskar oxygen guda ɗaya da faɗaɗa hankalin wannan hanyar.Don sigar miniSOG na yanzu, ana iya samun babban ƙuduri na ɗan lokaci ta amfani da hasken shuɗi don kunna alamun kusanci.Bugu da ƙari, tsawon lokacin bayyanarwa ya fito da mafi girma "girgije" na iskar oxygen guda ɗaya, wanda ya haifar da gyare-gyare na sauran ragowar histidine mai nisa, ƙara alamar radius, da kuma ikon daidaita yanayin ƙuduri na PDPL.Mun kuma gwada gwaje-gwajen sinadarai guda bakwai don haɓaka sigina-zuwa-baya rabo kuma mun bincika tsarin kwayoyin bayan wannan hanya.Ayyukan TOP-ABPP da aka haɗa tare da bincike na budewa mara kyau ya tabbatar da cewa gyare-gyare ya faru ne kawai a cikin histidines kuma ba a lura da microenvironment mai mahimmanci don ƙara yawan gyare-gyare na histidine, sai dai ga matsakaicin fifiko ga histidines a cikin yankin madauki.
An kuma yi amfani da PDPL don siffanta proteomes na subcellular tare da ƙayyadaddun proteome da ɗaukar hoto aƙalla daidai da sauran alamar kusanci da hanyoyin binciken sinadarai na musamman na gabobin.Hakanan an sami nasarar amfani da alamun kusanci don siffata saman, lysosomal, da kuma abubuwan da ke da alaƙa da sirri46,47.Mun yi imanin cewa PDPL za ta dace da waɗannan ƙananan ƙwayoyin cuta.Bugu da kari, mun kalubalanci PDPL ta hanyar gano maƙasudi don ɗaure furotin cytosolic waɗanda suka fi rikitarwa fiye da sunadaran da aka ɗaure da membrane saboda ƙayyadaddun kaddarorinsu da shiga cikin ƙarin hulɗar ɗan lokaci.An yi amfani da PDPL zuwa sunadaran guda biyu, mai haɗin gwiwa na BRD4 da ligase E3 Parkin mai alaƙa da cuta.An zaɓi waɗannan sunadaran guda biyu ba kawai don ainihin ayyukan ilimin halitta ba, har ma don dacewarsu na asibiti da yuwuwar warkewa.Ga waɗannan POI guda biyu, an gano sanannun abokan haɗin gwiwa da kuma makasudin da ba a yi rajista ba.Musamman ma, an tabbatar da SFPQ furotin mai alaƙa da lokaci ta hanyar haɗin gwiwar IP, wanda zai iya nuna sabon tsarin da BRD4 (gajeren isoform) ke sarrafa LLPS.A lokaci guda kuma, mun yi imanin cewa gano abubuwan da ke cikin Parkin wani yanayi ne wanda ake buƙatar gano manne kai tsaye.Mun gano nau'ikan wuraren shakatawa guda biyu da ba a tantance su ba kuma mun tabbatar da lalacewarsu tare da hanyar tarwatsa-proteasome.Kwanan nan, an ƙirƙiri dabarar tarko ta hanyar dabara don gano abubuwan da ake amfani da su na hydrolase ta hanyar kama su da enzymes.Ko da yake wannan hanya ce mai ƙarfi sosai, bai dace da nazarin abubuwan da ke da hannu wajen samar da manyan gidaje ba kuma yana buƙatar samar da haɗin gwiwa tsakanin enzymes da substrate.Muna tsammanin za a iya tsawaita PDPL don yin nazarin sauran rukunin furotin da iyalai na enzyme, kamar dangin deubiquitinase da dangin metalloprotease.
Wani sabon nau'i na miniSOG, mai suna SOPP3, an haɓaka shi tare da ingantaccen samar da iskar oxygen guda ɗaya.Mun kwatanta miniSOG tare da SOPP3 kuma mun sami ingantaccen aikin yin alama, kodayake rabon siginar-zuwa-amo bai canza ba (Ƙarin Hoton 11).Mun yi hasashe cewa inganta SOPP3 (misali, ta hanyar juyin halitta da aka ba da izini) zai haifar da ingantattun sunadaran photosensitizer waɗanda ke buƙatar gajeriyar lokutan haske kuma don haka ba da damar ɗaukar matakai masu ƙarfi na salon salula.Musamman ma, sigar PDPL ta yanzu tana iyakance ga yanayin salon salula saboda tana buƙatar hasken shuɗi kuma baya iya shiga cikin kyallen takarda.Wannan fasalin ya hana amfani da shi a cikin nazarin samfurin dabba.Duk da haka, haɗuwa da optogenetics tare da PDPL zai iya ba da dama ga binciken dabba, musamman a cikin kwakwalwa.Bugu da kari, sauran injiniyoyin infrared photosensitiizers suma suna cire wannan iyakancewa.A halin yanzu ana ci gaba da bincike a wannan yanki.
An samo layin salula na HEK293T daga ATCC (CRL-3216).Layin tantanin halitta ya gwada rashin lafiya don kamuwa da cutar mycoplasma kuma an haɓaka shi a cikin DMEM (Thermo, #C11995500BT) wanda aka haɓaka da 10% ƙwayar ƙwayar ƙwayar tayi (FBS, Vistech, #SE100-B) da 1% penicillin/streptomycin (Hyclone, #SV30010).girma a.
3-Aminophenylene (samfurin 3) da (4-ethynylphenyl) methanamine (samfurin 4) an saya daga Bidepharm.An sayi Propylamine (bincike 2) daga Makamashi-sunadarai.N- (2-Aminophenyl) pent-4-ynamide (bincike 1) an haɗa shi bisa ga hanyoyin da aka buga.
Ƙarin Tebu na 1 ya lissafa abubuwan gina jiki da aka yi amfani da su a wannan binciken.MiniSOG da KillerRed jerin an rufe su daga kyautar plasmid daga P. Zou (Jami'ar Peking).An samo jerin abubuwan da aka yi niyya na mitochondrial matrix daga 23 N-terminal amino acids na COX4 kuma an haɗa su cikin abubuwan da aka nuna ta amfani da taron Gibson (Beyotime, #D7010S).Don ƙaddamar da membrane da tsakiya na endoplasmic reticulum, SEC61B DNA na mutum (NM_006808.3) (NEB, #M0491L) ya haɓaka ta PCR daga ɗakin karatu na cDNA na kwayoyin HEK293T, da H2B DNA (wanda D. Lin ya bayar, Shenzhen Bay Laboratory) kuma cloned , kamar yadda aka ambata a sama.Sai dai in an nuna in ba haka ba, sauran ƙwayoyin furotin da aka yi amfani da su don canzawa da gina tsayayyen layukan salula an haɓaka PCR daga ɗakin karatu na tantanin halitta HEK293T.An yi amfani da G3S (GGGS) da G4S (GGGGS) azaman masu haɗin gwiwa tsakanin furotin koto da miniSOG.An ƙara alamar V5 epitope (GKPIPNPLLGLDST) zuwa waɗannan haɗin ginin.Don magana a cikin dabbobi masu shayarwa da kuma kafa tsayayyen layin salula, an sanya haɗin ginin miniSOG a cikin vector na lentiviral pLX304.Don maganganun kwayan cuta, an haɗa miniSOG a cikin pET21a vector mai lakabi 6xHis a C-terminus.
Kwayoyin HEK293T an shuka su a cikin sel 2.0 x 105 a kowace rijiya a cikin faranti guda shida kuma an canza su bayan sa'o'i 24 tare da recombinant lentiviral plasmids (2.4 μg pLX304) da plasmids fakitin hoto (1.5 μg psPAX2 da 1.2 μG) 0g pMD ta amfani da Likita. , #C0533), kusan kashi 80%.Bayan canja wurin na dare, an canza matsakaici kuma an sanya shi na tsawon sa'o'i 24.An gudanar da tarin kwayar cutar bayan sa'o'i 24, 48 da 72.Kafin kamuwa da layukan salula da aka yi niyya, an tace matsakaicin ƙwayar cuta ta hanyar tacewa 0.8 μm (Merck, #millex-GP) kuma an ƙara polybrene (Solarbio, #H8761) zuwa taro na 8 μg/ml.Bayan sa'o'i 24, an ba da izinin sel su dawo ta hanyar canza matsakaici.An zaɓi sel ta amfani da 5 μg/ml blasticidin (Solarbio, #3513-03-9) don sassa uku na farko a matsayin ƙaramin zaɓi mai ƙarfi.Sa'an nan kuma yi amfani da 20 μg/ml azaman ƙarin tsari mai tsauri don sassa uku na gaba.
An shuka sel a cikin ɗakunan rijiyoyi 12 (Ibidi, #81201) a yawan kusan sel 20,000 kowace rijiya.Don inganta mannewa na sel HEK293T, ƙara 50 µg/ml fibronectin (Corning, #356008) diluted a cikin phosphate buffered saline (PBS, Sangon, #B640435) a 37 ° C.An riga an gyara ɗakunan na 1 hour sannan an cire su tare da PBS.Bayan sa'o'i 24, an wanke sel sau ɗaya tare da PBS, an haɗa su tare da bincike na 1 mM 3 a cikin ingantaccen maganin gishiri mai daidaitaccen Hanks (HBSS, Gibco, #14025092) na 1 h a 37 ° C, sannan an haɗa shi da shuɗi mai haske (460 nm). ).) an lalata su don 10 min a cikin zafin jiki.Bayan haka, an wanke sel sau biyu tare da PBS kuma an gyara su tare da 4% formaldehyde a cikin PBS (Sangon, # E672002) na mintuna 15 a cikin zafin jiki.An cire yawan formaldehyde daga kafaffen sel ta hanyar wankewa sau uku tare da PBS.Daga nan aka lalata ƙwayoyin sel tare da 0.5% Triton X-100 (Sangon, #A600198) a cikin PBS kuma an wanke su sau 3 tare da PBS.Sa'an nan kuma cire ɗakin kuma ƙara zuwa kowane samfurin 25 µl na cakuda amsawar dannawa wanda ke dauke da 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, # A603008), 1 mM BTTAA (Confluore, #BDJ-4) da 0.5 mg / ml sodium ascorbate (Aladdin, no. S105024) da kuma sanya shi na tsawon minti 30 a dakin da zafin jiki.Bayan amsawar karyewa, an wanke sel sau shida tare da PBS mai dauke da 0.05% Tween-20 (Sangon, #A600560) (PBST) sannan kuma an toshe shi tare da 5% BSA (Abcone, # B24726) a cikin PBST na mintuna 30 a zafin jiki.
Don maganin rigakafi na colocalization, an haɗa sel tare da ƙwayoyin rigakafi na farko bisa ga yanayin da aka nuna: linzamin kwamfuta anti-V5 tag mAb (1: 500, CST, #80076), anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), zomo polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) ko zomo anti-lamin A/C monoclonal antibody (1:500; CST, #2032) a 4 °C na dare.Bayan wanke sau 3 tare da PBST, an sanya sel tare da ƙwayoyin rigakafi na biyu: goat anti-zomo Alexa Fluor 488 (Thermo, #A11034) diluted 1:1000, goat anti-mouse Alexa Fluor 594 (CST, #8889) diluted 1:1000.dilution Tsarma a dakin da zafin jiki na minti 30.Sannan an wanke sel sau 3 tare da PBST kuma an daidaita su tare da DAPI (Thermo, #D1306) a cikin PBS na mintuna 10 a zafin jiki.Bayan wanke 3 tare da PBS, an rufe sel a cikin 50% glycerol (Sangon, #A600232) a cikin PBS don hoto.An samu hotunan immunofluorescent ta amfani da na'ura mai kwakwalwa ta ZEISS LSM 900 Airyscan2 confocal microscope da software ZNE 3.5.
Don hoto guda ɗaya na iskar oxygen, an wanke sel sau biyu tare da buffer Hanks HEPES kafin ƙara 100 nM Si-DMA a cikin buffer Hanks HEPES (DOJINDO, #MT05).Bayan bayyanar da haske, an sanya ƙwayoyin sel a cikin CO2 incubator a 37 ° C na minti 45.Sannan an wanke sel sau biyu tare da buffer na Hanks'HEPES kuma an daidaita su tare da Hoechst a cikin buffer na Hanks'HEPES na mintuna 10 a zafin daki kuma an hango su ta amfani da na'urar hangen nesa ta ZEISS LSM 900., #M36008) a cikin buffer HBSS mai dauke da calcium da magnesium.Bayan bayyanar haske ko doxorubicin (MCE, #HY-15142A), an sanya sel a cikin CO2 incubator a 37 ° C. na minti 10, an wanke su sau biyu tare da buffer HBSS, kuma an sanya shi tare da Hoechst a cikin buffer HBSS a dakin da zafin jiki.mintuna.An yi amfani da Doxorubicin azaman ingantacciyar kulawar bincike inda aka bi da sel tare da 20 μM doxorubicin a cikin HBSS mai ɗauke da 1% BSA na 30 min.An samu hotunan immunofluorescent ta amfani da na'urar hangen nesa ta Zeiss LSM 900.
Kwayoyin HEK293T da ke bayyana mito-miniSOG a tsaye an shuka su a cikin nau'in nau'in kusan 30% a cikin jita-jita na 15 cm.Bayan sa'o'i 48, lokacin da ~ 80% ya kai ga haɗuwa, an wanke sel sau ɗaya tare da PBS, an haɗa su tare da 1 mM Probe 3 a cikin sabon buffer HBSS a cikin sa'a 1 a 37 ° C sa'an nan kuma haskaka da blue LED na minti 10 a daki. zafin jiki..Bayan haka, an wanke sel sau biyu tare da PBS, an goge kuma an sake dakatar da su a cikin buffer PBS mai sanyi mai sanyi wanda ya ƙunshi masu hana protease marasa EDTA (MCE, #HY-K0011).An lysed cell ta hanyar sonicating tip na minti 1 (1 seconds on da 1 seconds off a 35% amplitude).Sakamakon cakuda ya kasance a tsakiya a 15,871 xg na 10 min a 4 ° C don cire tarkace, kuma an daidaita maida hankali zuwa 4 MG / mL ta amfani da kayan gwajin furotin BCA (Beyotime, #P0009).Haɗa 1 ml na lysate na sama tare da 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), da 1 mbaMtor kasa tare da CuSO4 incuSO4. juyawa sama na awa 1 a zazzabi na ɗaki.Bayan amsawar karyewa, ƙara cakuda zuwa maganin da aka riga aka haɗa (MeOH: CHCl3: H2O = 4 ml: 1 ml: 3 ml) a cikin gilashin gilashin 10 ml.An haɗu da samfurori kuma an haɗa su a 4500 g na minti 10 a dakin da zafin jiki.An watsar da mafita na ƙasa da na sama, an wanke hazo sau biyu tare da 1 ml na methanol da centrifuged a 15871 × g na 5 min a 4 ° C.Ƙara 1 ml na 8 M urea (Aladdin, no. U111902) a cikin 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) don narkar da hazo.An sake gina samfurori tare da 10 mM dithiothreitol (Sangon, # A100281 a cikin 25 mM ABC) na minti 40 a 55 ° C sannan kuma ƙara 15 mM sabo iodoacetamide (Sangon, # A600539) a dakin da zafin jiki a cikin duhu.Alkylation a cikin minti 30..An ƙara ƙarin 5 mM dithiothreitol don dakatar da amsawa.Shirya kusan 100 µl NeutrAvidin agarose beads (Thermo, #29202) ga kowane samfurin ta hanyar wanke sau 3 tare da 1 ml PBS.Maganin proteome na sama an diluted tare da 5 ml PBS kuma an sanya shi tare da beads agarose NeutrAvidin da aka riga aka wanke don awanni 4 a zafin jiki.An wanke beads sau 3 tare da 5 ml PBS mai dauke da 0.2% SDS (Sangon, #A600485), sau 3 tare da 5 ml PBS mai dauke da urea 1M, kuma sau 3 tare da 5 ml ddH2O.An girbe beads ta hanyar centrifugation kuma an sake dakatar da su a cikin 200 μl na 25 mM ABC mai dauke da 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) da 20 ng/μl trypsin (Promega, #V5280).Trypsinize dare a 37 ° C tare da juyawa.An dakatar da amsa ta hanyar ƙara formic acid (Thermo, # A117-50) har sai pH ya kai 2-3.An wanke beads sau 3 tare da 1 ml na PBS mai dauke da 0.2% SDS, sau 3 tare da 1 ml na PBS mai dauke da urea 1 M, sannan sau 3 tare da 1 ml na ruwa mai tsabta.An saki peptides da aka gyara ta hanyar haske lysis (365 nm) na 90 min ta amfani da 200 μl na 70% MeOH.Bayan centrifugation, an tattara supernatant.An wanke beads sau ɗaya tare da 100 μl na 70% MeOH kuma an haɗa abubuwan da suka fi dacewa.An busar da samfuran a cikin na'ura mai ɗaukar hoto na Speedvac kuma an adana su a -20 ° C har sai an bincika.
Don ganowa da ƙididdige peptides ɗin da aka gyara guda ɗaya na oxygen, an sake narkar da samfuran a cikin 0.1% formic acid da 1 μg na peptides an bincika ta amfani da Orbitrap Fusion Lumos Tribrid mass spectrometer sanye take da tushen nano ESI daga Tune da Xcalibur daga software mai siyarwa 4.3.An raba samfurori akan 75 µm × 15 cm cike da ginshiƙi na ciki tare da kayan 3 µm C18 (ReproSil-pur, #r13.b9.) kuma an haɗa su zuwa tsarin EASY-nLC 1200 UHPLC (Thermo).An raba peptides ta hanyar chromatography na minti 95 na layi na layi daga 8% ƙarfi B zuwa 50% ƙarfi B (A = 0.1% formic acid a cikin ruwa, B = 0.1% formic acid a cikin 80% acetonitrile), sannan layi ya karu zuwa 98% B min. a cikin 6 min a saurin gudu na 300 nl / min.Orbitrap Fusion Lumos yana tattara bayanai a madadinsa tsakanin cikakken sikanin MS da duban MS2 dangane da bayanan.An saita wutar lantarki mai watsawa zuwa 2.1kV kuma zazzabi na jigilar jigilar ion shine 320 ° C.MS Spectra (350-2000 m/z) an tattara su tare da ƙuduri na 120,000, AGC 4 × 105, da matsakaicin lokacin shigarwa na 150 ms.10 mafi yawan abubuwan da aka caje da yawa a cikin kowane cikakken bincike an raba su ta amfani da HCD tare da daidaitaccen ƙarfin karo na 30%, taga keɓewar quadrupole na 1.6 m/z, da saitin ƙuduri na 30,000.Manufar AGC don tandem mass spectrometry ta amfani da 5 × 104 da matsakaicin lokacin shigarwa 150 ms.An saita keɓanta mai ƙarfi zuwa daƙiƙa 30. ions da ba a raba ko waɗanda ke da cajin 1+ da>7+ an ƙi su don MS/MS. ions da ba a raba ko waɗanda ke da cajin 1+ da>7+ an ƙi su don MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. ions ko ions mara izini tare da cajin 1+ da>7+ an ƙi su don MS/MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于MS/MS.未指定的离子或电荷为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. ions ko ions da ba a bayyana ba tare da cajin 1+ da>7+ an ƙi su don MS/MS.
Ana sarrafa danyen bayanan ta amfani da dandamalin kwamfuta na FragPipe bisa MSFragger.An ƙididdige yawan son zuciya da kuma amino acid masu dacewa ta amfani da buɗaɗɗen bincike algorithm tare da juriyar juriya na farko na -150 zuwa 500 Da.An gano peptides da aka gyara sannan aka gano ta amfani da gyare-gyare na histidine tare da yawan riba na + 229.0964 da + 247.1069 Da a cikin PD (Proteome Discoverer 2.5, Thermo).
Kwayoyin da ke bayyana haɗe-haɗen ƙwayar miniSOG an yi su a cikin jita-jita na 6 cm.Bayan isa ~80% haɗuwa, an wanke sel sau ɗaya tare da HBSS (Gibco, #14025092), sannan a sanya su tare da binciken sinadarai a cikin HBSS na awa 1 a 37 ° C kuma an haskaka su da haske mai shuɗi.10W LED na minti 20 a cikin zafin jiki.Don sanin wane nau'in nau'in oxygen mai amsawa yana cikin PDPL, 0.5 mM bitamin 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 an ƙara su zuwa sel azaman kari.Bayan wankewa tare da PBS mai sanyi, an cire sel, an tattara su a cikin tubes na 1.5 ml, kuma ana sonicated tare da tip don 1 min a cikin 200 μl na PBS tare da 1x protease inhibitor ba tare da EDTA (1 s da 1 s ba tare da, amplitude 35%).Sakamakon cakuda ya kasance a tsakiya a 15,871 × g na 10 min a 4 ° C kuma an daidaita girman girman kai zuwa 1 MG / mL ta amfani da kit ɗin furotin BCA.Kimanin 50 µl na lysate na sama an haɗa shi tare da 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, da 1 mM CuSO4 na awa 1 a dakin da zafin jiki tare da juyawa daga kasa zuwa sama.Bayan amsawar dannawa, ana aiwatar da hazo tare da acetone ta ƙara 250 μl na acetone da aka riga aka yi sanyi a cikin samfuran, a cikin -20 ° C don 20 min da centrifuging a 6010 × g na 10 min a 4 ° C.Tattara pellet ɗin kuma tafasa a cikin 50 µl na 1x Laemmli's buffer na minti 10 a 95 ° C.Sa'an nan kuma an yi nazarin samfurori akan SDS-PAGE dogayen gels kuma an hango su ta amfani da tsarin hoton Bio-rad ChemiDoc MP Touch tare da Software Lab Touch.
Bayyanawa da tsarkakewa na recombinant miniSOG-6xAn yi furotinSa kamar yadda aka bayyana a baya.A taƙaice, sel E. coli BL21 (DE3) (TransGen, # CD701-02) an canza su tare da pET21a-miniSOG-6xHis kuma an jawo maganganun furotin tare da 0.5 mM IPTG (Sangon, # A600168).Bayan tantanin halitta lysis, sunadaran suna tsarkake ta amfani da Ni-NTA agarose beads (MCE, no. 70666), dialyzed da PBS, kuma adana a -80 ° C.
Don gwajin kusancin alamar in vitro, haɗa 100 μM mai tsabta miniSOG, 1 mM bincike 3, da 1 μg anti-label linzamin kwamfuta monoclonal antibody (TransGen, #HT501-01) a cikin PBS zuwa jimlar amsawar 50 μl..Cakudawar amsa ta kasance mai haske tare da haske mai shuɗi na LED don 0, 2, 5, 10, da 20 min a zazzabi na ɗaki.An haɗu da cakuda tare da 0.1 mM biotin-PEG3-azide (Aladdin, # B122225), 1 mM TCEP, 0.1 mM TBTA ligand, da 1 mM CuSO4 na 1 h a dakin da zafin jiki a kan motsi mai motsi zuwa sama.Bayan amsawar karyewa, ƙara 4x Laemmli's buffer kai tsaye zuwa ga cakuda kuma tafasa a 95 ° C na minti 10.An yi nazarin samfurori akan gels na SDS-PAGE kuma an yi nazari ta hanyar lalatawar yamma tare da streptavidin-HRP (1: 1000, Solarbio, #SE068).
An yi amfani da peptide na roba mai ɗauke da histidine tare da C-terminal amidation (LHDALDAK-CONH2) don nazarin alamar peptide kusa da kusa.A cikin wannan binciken, 100 μM mai tsabta miniSOG, 10 mM bincike 3 da 2 μg / ml peptide synthetic sun haɗu a cikin PBS a cikin jimlar amsawar 50 μl.An kunna cakudawar amsawa tare da hasken LED mai shuɗi na awa 1 a zazzabi na ɗaki.An yi nazarin microliter ɗaya na samfurin ta amfani da tsarin LC-MS (Ruwa, SYNAPT XS Ions Motsi na Lokaci-na-Jigin Mass spectrometer tare da MassLynx spectrum analysis software).
Kwayoyin HEK293T da ke bayyana kwayar halittar miniSOG an tsara su a cikin jita-jita na 10 cm don layi tare da yanayin yanayin gabobin (Mito, ER, Nucleus) da jita-jita na 15 cm don layin Parkin-miniSOG da BRD4-miniSOG.Bayan an kai ~90% haduwa, an wanke sel sau ɗaya tare da HBSS, sannan a sanya su tare da bincike 3 a cikin HBSS na awa 1 a 37°C kuma an haskaka su da LED blue 10 W a zafin daki.Don alamar rashin tuntuɓar Parkin, 10 µM proton carbonyl cyanide m-chlorophenylhydrazone CCCP (Solarbio, #C6700) tare da bincike 3 a cikin HBSS an ƙara shi na awa 1 a 37°C.Tantanin halitta lysis, click chemistry, raguwa da matakan alkylation sun kasance daidai kamar yadda aka bayyana a sama, sai dai an ƙara 2 MG na lysate kuma an yi amfani da biotin PEG3 azide a cikin amsawar danna maimakon photodegradable biotin azide.Bayan an wadata, an wanke beads sau 3 tare da 5 ml na PBS dauke da 0.2% SDS, sau 3 tare da 5 ml na PBS dauke da 1 M urea, da sau 3 tare da 5 ml na PBS.Bayan haka, an ƙara 2 μg trypsin zuwa 300 µl 25 mM ABC mai ɗauke da urea 1 M don tsinke furotin a cikin dare a 37 ° C.An dakatar da amsa ta hanyar ƙara formic acid har sai an kai pH na 2-3.Bayan trypsinization akan beads, an zubar da maganin peptide ta amfani da ginshiƙin SOLAµ HRP (Thermo, #60209-001) kuma an bushe shi a cikin injin injin Speedvac.An sake narkar da peptides a cikin 0.1% formic acid da 500 ng na peptides an yi nazari ta hanyar amfani da na'urar gani ta Orbitrap Fusion Lumos Tribrid mai sanye da tushen nano-ESI da aka bayyana a sama.An raba Peptides akan precolumns na RP-HPLC na kasuwanci (75 μm x 2 cm) (Thermo, no. 164946) da ginshiƙan RP-HPLC na nazari (75 μm x 25 cm) (Thermo, no. 164941), dukansu sun cika da 2 μm.gradient daga 8% zuwa 35% ACN a cikin mintuna 60, sannan layi ya karu zuwa 98% B a cikin mintuna 6 a farashin 300 Nl/min.MS Spectra (350-1500 m/z) an tattara su tare da ƙuduri na 60,000, AGC 4 × 105, da matsakaicin lokacin shigarwa na 50 ms.An rarraba ions da aka zaɓa a jere ta hanyar HCD a cikin zagayowar 3 s tare da daidaitaccen ƙarfin karo na 30%, taga keɓewar quadrupole na 1.6 m/z, da ƙuduri na 15000. A 5 × 104 tandem mass spectrometer AGC manufa da iyakar lokacin allura an yi amfani da 22 ms.An saita tsayayyen cirewa zuwa 45 seconds. ions da ba a raba ko waɗanda ke da cajin 1+ da>7+ an ƙi su don MS/MS. ions da ba a raba ko waɗanda ke da cajin 1+ da>7+ an ƙi su don MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. ions ko ions mara izini tare da cajin 1+ da>7+ an ƙi su don MS/MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于MS/MS.未指定的离子或电荷为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. ions ko ions da ba a bayyana ba tare da cajin 1+ da>7+ an ƙi su don MS/MS.
Matakan shirye-shiryen samfurin har zuwa haɓakar beads na NeutrAvidin sun kasance daidai da a cikin binciken LC-MS / MS da aka kwatanta a sama.An yi amfani da kusan 50 μg na lysate a matsayin shigarwa don sarrafa kaya kuma an yi amfani da 2 MG na lysate don danna halayen.Bayan haɓakawa da wankewa tare da neutravidin, sunadaran da aka ɗaure sun ɓace ta hanyar ƙara 50 μl na buffer Laemmli a cikin beads resin agarose da tafasa a 95 ° C. na minti 5.SDS-PAGE ne yayi nazari akan shigar da kayan sarrafawa da samfuran ƙwanƙwasa kuma aka tura su zuwa membranes na PVDF (Millipore, #ISEQ00010) ta daidaitattun hanyoyin ɓangarorin Yamma.An toshe membranes tare da madara 5% (Sangon, #A600669) a cikin TBS mai ƙunshe da 0.1% tween-20 (TBST) kuma an haɗa su tare da ƙwayoyin rigakafi na farko da na sakandare.An narkar da magungunan rigakafi na farko 1:1000 a cikin 5% madara a cikin TBST kuma an sanya su cikin dare a 4°C.An yi amfani da ƙwayoyin rigakafi na biyu a cikin rabo na 1:5000 kuma an sanya su na awa 1 a zafin jiki.An hango membranes ta hanyar chemiluminescence ta amfani da tsarin hoton Chemidoc MP.Duk abubuwan da ba a yanke ba na ɓangarori da gels a cikin adadi an gabatar da su azaman ɗanyen bayanai.
Magungunan rigakafi na farko da aka yi amfani da su a cikin wannan binciken sun hada da maganin anti-SFPQ monoclonal antibody (CST, no. 71992), zomo anti-FUS monoclonal antibody (CST, no. 67840), anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1- AP), zomo anti-mSin3A polyclonal antibody (Abcam, #ab3479), linzamin kwamfuta anti-tag monoclonal antibody (TransGen, #HT201-02), linzamin kwamfuta anti-β-actin monoclonal antibody (TransGen, #HC201-01), zomo antibody. -CDK2 monoclonal antibody (ABclonal, #A0094), zomo monoclonal antibody zuwa CTBP1 (ABclonal, #A11600), zomo polyclonal antibody zuwa DUT (ABclonal, #A2901), zomo polyclonal antibody zuwa PSMC4 (ABclonal, #A2505), zomo anti- DNAJB1 polyclonal antibody (ABclonal, # A5504).An yi amfani da waɗannan ƙwayoyin rigakafin a cikin 1:1000 dilution a cikin 5% na madara mai ƙima a cikin TBST.Kwayoyin rigakafi na biyu da aka yi amfani da su a cikin wannan binciken sun haɗa da anti-zomo IgG (TransGen, #HS101-01), anti-mouse IgG (TransGen, #HS201-01) a 1: 5000 dilution.
Don ci gaba da bincika ko BRD4 yana hulɗa tare da SFPQ, sel HEK293T da BRD4-miniSOG masu wuce gona da iri an sanya su cikin jita-jita na 10 cm.An wanke kwayoyin halitta tare da PBS mai sanyi kuma an sanya su a cikin 1 ml na Pierce IP lysis buffer (Thermo Fisher, #87787) tare da mai hana protease mai kyauta na EDTA na minti 30 a 4 ° C.Bayan haka, an tattara lysates a cikin bututun centrifuge na 1.5 ml kuma an sanya su a 15,871 xg na 10 min a 4 ° C.An girbe abin da ya fi girma kuma an sanya shi tare da 5 μg na anti-V5 mai lakabin linzamin kwamfuta monoclonal antibody (CST, #80076) na dare a 4°C.A wanke kusan 50 µl na furotin A/G Magnetic beads (MCE, #HY-K0202) sau biyu tare da PBS mai ɗauke da 0.5% Tween-20.Sa'an nan kuma lysates tantanin halitta an haɗa su tare da beads na maganadisu na 4 hours a 4 ° C tare da juyawa daga kasa zuwa sama.Sa'an nan kuma an wanke beads sau hudu tare da 1 ml na PBST buffer kuma a tafasa a 95 ° C na 5 min.An yi nazarin samfurori akan gels SDS-PAGE kuma an canza su zuwa membranes na PVDF ta amfani da daidaitattun hanyoyin lalata na Yamma.An toshe membranes a cikin 5% na madara mai ƙwanƙwasa a cikin TBST kuma an haɗa su tare da ƙwayoyin rigakafi na farko da na sakandare.An yi amfani da na farko Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) a cikin rabo na 1:1000 a cikin 5% madarar madara a cikin TBST kuma an sanya shi cikin dare a 4°C.An yi amfani da IgG anti-zomo a wani rabo na 1:5000 kuma an sanya shi na awa 1 a zafin jiki.An hango membranes ta hanyar chemiluminescence ta amfani da tsarin hoton Chemidoc MP.
An samo duk tsarin da aka yi amfani da shi don bincike na Yankin Rarraba Sama (SASA) daga Bankin Bayanai na Protein (PDB)52 ko DatabaseFold Protein Structure Database53.An ƙididdige cikakken SASA don kowane ragowar ta amfani da shirin FreeSASA.Cikakkun bayanan SASA masu cikakken bayani kawai na histidine da maƙwabta da aka yi amfani da su don samun ma'anar SASA ga kowane tsari.An ƙididdige damar samun damar ƙarfi na dangi (RSA) na kowane histidine ta hanyar rarraba cikakkiyar ƙimar SASA ta mafi girman maƙasudin yuwuwar ragowar farfajiyar da ke akwai ga sauran ƙarfi.Duk histidines an rarraba su azaman ɓoye idan ma'anar RSA ta kasance ƙasa da 20%, in ba haka ba fallasa56.
An bincika fayilolin da aka samo a yanayin DDA ta amfani da Proteome Discoverer (v2.5) ko MSfragger (Fragpipe v15.0) a cikin madaidaicin bayanan furotin na SwissProt wanda ya ƙunshi gurɓata gama gari.The peptides bukatar cikakken trypsin tare da biyu bace cleavage sites, carbamoyl methylation a matsayin gyarawa gyara da kuma methionine hadawan abu da iskar shaka a matsayin mai tsauri canji.An saita juriyar juriya da juzu'i zuwa 10 ppm da 0.02 Da (MS2 Orbitrap), bi da bi. An cire abubuwan gurɓatawa, kuma an tace sunadaran don samun ƙimar gano ƙarya na <1%. An cire abubuwan gurɓatawa, kuma an tace sunadaran don samun ƙimar gano ƙarya na <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэфнфить. An cire abubuwan gurɓatawa kuma an tace sunadaran don ba da ƙimar gano ƙarya na <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。 Попадания загрязняющих веществ были удалены, а белки отфильтрованы для достижения уровня лобнайрых%. An cire abubuwan gurɓatawa kuma an tace sunadaran don cimma ƙimar ƙimar ƙarya na <1%.Don ƙididdige ƙididdiga ba tare da amfani da tambari ba, an yi amfani da abun ciki na gina jiki na yau da kullun daga maimaita nazarin halittu guda uku.An gudanar da bincike na ƙananan ƙwayoyin furotin ta hanyar amfani da bincike na Gene Ontology (GO) daga DAVID Bioinformatics Resources, MitoCarta 3.0 da bayanan bayanan da ƙungiyar Alice Ting ta tattara kuma ta buga.An samo taswirar dutsen mai aman wuta daga Perseus (v1.6.15.0). An gwada canje-canje masu yawa na furotin don mahimmancin ƙididdiga ta amfani da t-gwajin mai gefe biyu, kuma an gano abubuwan furotin tare da canji mai yawa> 2 (sai dai in ba haka ba) da p darajar <0.05. An gwada canje-canje masu yawa na furotin don mahimmancin ƙididdiga ta amfani da t-gwajin mai gefe biyu, kuma an gano abubuwan furotin tare da canji mai yawa> 2 (sai dai in ba haka ba) da p darajar <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. An gwada canjin abun ciki na furotin don mahimmancin ƙididdiga ta amfani da t-test mai wutsiya biyu, kuma an gano ma'aunin furotin tare da canjin abun ciki> 2 (sai dai in an lura da shi) da ƙimar ap <0.05.测 倍数 测 测 测 测 测 测 测 测 测 测 测 测 测, 并并, 并并, 并并, 并并, 并并 的 的丰度 的 的丰度 的丰度丰度测 数 测 测 测 测 测 中 中 并 中 中 中 中 的 中 中 中 的 中 中> 2 (另另 值 <0.05. 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. An gwada mahimmancin ƙididdiga na ninka canje-canje a cikin abun ciki na furotin ta amfani da t-gwajin wutsiya biyu, kuma an ƙaddara ma'auni na furotin don canje-canjen abun ciki> 2 (sai dai in ba haka ba) da p-darajar <0.05.An yi nazarin hulɗar furotin ta amfani da bincike na GO tare da bayanan String.
An gudanar da kwafin halittu guda uku tare da sakamako iri ɗaya.An yi nazarin ƙididdiga tare da GraphPad Prism (software na GraphPad) kuma an samar da filaye masu aman wuta tare da Perseus (v1.6.15.0).Don kwatanta ƙungiyoyin biyu, an ƙaddara p-darajar ta amfani da t-gwajin ɗalibi mai wutsiya.Sunadaran guda ɗaya ne kawai aka gano aƙalla sau biyu a cikin rukunin gwaji an haɗa su a cikin filayen dutsen mai aman wuta, kuma an maye gurbin daidaitattun ƙimar da ke cikin rukunin sarrafawa tare da Perseus daga rarraba ta al'ada don a iya ƙididdige ƙimar p-darajar.Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.A cikin nazarin ƙwayoyin cuta don ƙididdigar ƙididdiga, yawancin sunadaran da suka bayyana a cikin aƙalla kwafi biyu na halitta an kiyaye su.Ba a yi amfani da hanyoyin ƙididdiga ba don tantance girman samfurin.Gwaje-gwajen ba bazuwar ba ne.Masu binciken ba su makantar da ayyukan yayin gwaji da kimanta sakamakon.
Don ƙarin bayani game da ƙira na nazari, duba Ƙididdigar Rahoton Bincike mai alaƙa da wannan labarin.
An ƙaddamar da bayanan da aka samu a cikin wannan binciken ga ProteomeXchange Consortium ta wurin ajiyar abokin tarayya na iProX57 a ƙarƙashin ID na ID PXD034811 (PDPL-MS dataset).Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.Wannan labarin yana ba da bayanan asali.
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