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Mekhoa ea ho ngola ea li-enzymatic proximity e thehiloeng ho li-ester tse kentsoeng kapa li-phenoxy radicals li sebelisoa haholo ho etsa 'mapa oa liprotheine tsa subcellular le li-protein tse sebetsanang le lisele tse phelang.Leha ho le joalo, li-ester tse kentsoeng ha li sebetse hantle, 'me li etsa hore ho be le radius e pharaletseng ea li-labeling,' me phenoxy radicals e hlahisoang ke phekolo ea peroxide e ka kena-kenana le litsela tsa redox.Mona re tlaleha mokhoa oa proximity labeling dependent photoactivation (PDPL) o ntlafalitsoeng ka ho hokahanya protheine ea miniSOG photosensitizer le protheine e khahlisang.E susumetsoa ke leseli le leputsoa 'me e laoloa ke nako ea ho pepeseha, oksijene ea singlet e hlahisoa ebe ho ngoloa ho rarollotsoe ka spatiotemporally ea masala a histidine ka probe ea aniline e fihletsoeng.Re bonts'a botšepehi ba eona bo holimo ka 'mapa oa proteome oa organelle-specific.Papiso e bapileng le e 'ngoe ea PDPL le TurboID e bonts'a tšireletso e hlakileng haholoanyane ea proteomic ea PDPL.Ka mor'a moo, re ile ra sebelisa PDPL ho transcriptional coactivator e amanang le lefu BRD4 le E3 Parkin ligase 'me ra fumana batho ba neng ba sa tsejoe pele.Ka ho hlahloba ho feteletseng, li-substrates tse peli tse sa tsejoeng, Ssu72 le SNW1, li ile tsa khetholloa bakeng sa Parkin, eo ho senyeha ha eona ho kopanngoang ke tsela ea ubiquitination-proteasome.
Sebopeho se nepahetseng sa marang-rang a protheine se ipapisitse le lits'ebetso tse ngata tsa mantlha tsa cellular.Ka hona, 'mapa o nepahetseng haholo oa li-spatiotemporal oa litšebelisano tsa liprotheine o tla fana ka motheo oa limolek'hule bakeng sa ho hlalosa litsela tsa likokoana-hloko, lefu la mafu, le ho senya likamano tsena bakeng sa merero ea phekolo.Ho finyella sena, mekhoa e khonang ho lemoha ho sebelisana ha nakoana liseleng tse phelang kapa liseleng ke tse lakatsehang haholo.Affinity Purification Mass Spectrometry (AP-MS) esale e sebelisoa ho tsebahatsa balekane ba tlamang ba liprotheine tsa thahasello (POIs).Ka nts'etsopele ea mekhoa ea proteomics ea bongata, Bioplex3.0 e ile ea etsoa, setsi se seholo ka ho fetisisa sa marang-rang a protheine e thehiloeng ho AP-MS.Le hoja AP-MS e le matla haholo, mehato ea lysis le dilution tsamaisong ea mosebetsi e leeme mabapi le litšebelisano tse fokolang le tsa nakoana tse tlamang 'me li hlahisa lintho tse entsoeng ka morao-rao tse kang lipara tsa tšebelisano tse fosahetseng tse haelloang ke compartmentalization pele ho lysis.
Ho rarolla mathata ana, li-amino acid tseo e seng tsa tlhaho (UAA) tse nang le lihlopha tse kopanyang le li-platform tse haufi tsa enzymatic labeling (PL) (mohlala, APEX le BioID)5 li entsoe.Le hoja mokhoa oa UAA o sebelisitsoe ka katleho maemong a mangata 'me o fana ka tlhahisoleseding mabapi le li-adhesive tsa protheine tse tobileng, ho ntlafatsa sebaka sa ho kenya UAA ho ntse ho hlokahala.Habohlokoa le ho feta, ke mokhoa oa ho ngola stoichiometric o hlokang phetoho e matla ea liketsahalo tsa ho ngola.Ka lehlakoreng le leng, mekhoa ea enzymatic PL, joalo ka mokhoa oa BioID, e kopanya biotin ligase e entsoeng ho POI7, eo hamorao e kenyang biotin ho etsa biotinyl-AMP ester e bohareng.Ka hona, enzyme e kenya tšebetsong le ho lokolla "leru" la "biotin" le tšoaeang masala a proximal lysine.Leha ho le joalo, BioID e hloka lihora tse fetang 12 ho fumana letšoao le lekaneng le ngotsoeng, le thibelang tšebeliso ea lona ka tharollo ea nakoana.E sebelisa phetoho e laetsoeng e ipapisitseng le ponts'o ea tomoso, TurboID e entsoe ho ipapisitse le BioID hore e sebetse hantle, e lumelle ho ngola biotin ka nepo nakong ea metsotso e 10, ho lumella lits'ebetso tse matla ho ithutoa.Hobane TurboID e sebetsa haholo 'me maemo a biotin a endogenous a lekane bakeng sa ho ngolisoa ha maemo a tlase, ho ngola ka morao ho fetoha bothata bo ka bang teng ha ho ntlafatsoa haholo le ka nako ho hlokahala ka ho eketsoa ha biotin ea kantle ho naha.Ho phaella moo, li-ester tse kentsoeng ha li sebetse hantle (t1/2 ~ 5 min), e leng se ka lebisang ho radius e kholo ea li-labeling, haholo-holo ka mor'a ho tlala ha liprotheine tsa boahelani tse nang le biotin 5. Ka mokhoa o mong, fusion ea liphatsa tsa lefutso tsa enjinered ascorbate peroxidase (ke hore biotin- phenol radicals le ho lumella protheine labeling ka hare ho motsotso o le mong9, 10. APEX e sebelisoa haholo ho khetholla subcellular proteomes, membrane protein complexes, le cytosolic signaling protein complexes11, 12. Leha ho le joalo, tlhokahalo ea likhahla tse phahameng tsa peroxides e ka ama liprotheine tsa redox kapa litsela, ho senya. mekhoa ea cellular.
Ka hona, mokhoa o mocha o khonang ho hlahisa mefuta e mengata ea ho hatella e ngotsoeng-radius e nang le ts'ebetso e phahameng ea sebaka le ea nakoana ntle le ho senya litsela tsa cellular haholo e tla ba tlatsetso ea bohlokoa mekhoeng e teng. Har'a mefuta e sebetsang hantle, oksijene e le 'ngoe e ile ea tsosa tlhokomelo ea rona ka lebaka la nako e khutšoanyane ea bophelo le radius e fokolang ea ho hasana (t1 / 2 <0.6 µs liseleng)13. Har'a mefuta e sebetsang hantle, oksijene e le 'ngoe e ile ea tsosa tlhokomelo ea rona ka lebaka la nako e khutšoanyane ea bophelo le radius e fokolang ea ho hasana (t1 / 2 <0.6 µs liseleng)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии 1,3/3 (t. Har'a mefuta e sebetsang, oksijene ea singlet e ile ea hohela tlhokomelo ea rona ka lebaka la nako e khutšoanyane ea bophelo le radius e fokolang ea ho hasana (t1/2 <0.6 µs ka liseleng)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 диф16/кси , 0. Har'a mefuta e sebetsang, oksijene ea singlet e hohela tlhokomelo ea rona ka lebaka la nako e khutšoanyane ea bophelo le radius e fokolang ea ho hasana (t1/2 <0.6 μs liseleng).Ho tlalehiloe hore oksijene e le 'ngoe e na le oxidize methionine, tyrosine, histidine le tryptophan, e leng se etsang hore e be polar 14,15 bakeng sa ho khomarela amine kapa thiol based probes16,17.Leha oksijene ea singlet e sebelisitsoe ho tšoaea subcellular compartment RNA, maano a ho khutlisetsa matšoao a haufi a POI a ntse a sa hlahlojoe.Mona, re hlahisa sethala se bitsoang photoactivation-dependent proximity labeling (PDPL), moo re sebelisang leseli le leputsoa ho khantša li-POI tse kopantsoeng le miniSOG photosensitizer le ho hlahisa tlhahiso ea oksijene ea singlet ho oxidize mesaletsa ea proximal, e lateloa ke liphetoho tse nang le amine ho oxidize lik'hemik'hale tsa lik'hemik'hale. lisele tse phelang tse mahareng..Re ile ra leka sehlopha sa li-probes tsa lik'hemik'hale ho ntlafatsa boleng ba li-tag le ho tseba libaka tsa ntlafatso ka ho sebelisa mokhoa o bulehileng oa ho sebetsa oa proteomics.Papiso e bapileng le e 'ngoe ea PDPL le TurboID e bonts'a tšireletso e hlakileng haholoanyane ea proteomic ea PDPL.Re sebelisitse mokhoa ona ho li-marker tse khethehileng tsa organelle tsa subcellular proteome le proteome e akaretsang ea batho ba tlamang balekane bakeng sa protheine e amanang le mofetše e amanang le epigenetic BRD4 le lefu la Parkinson's E3 ligase Parkin, tse netefalitseng marang-rang a tsejoang le a sa tsejoeng a protheine. likamano..Bokhoni ba PDPL ba ho lemoha li-substrates tsa E3 ka har'a liprotheine tse kholo tsa liprotheine li emela boemo boo ho bona ho hlokoang li-binders tse sa tobang.Li-substrates tse peli tse sa tsejoeng tsa parkin tse kentsoeng ke ubiquitination-proteasome li netefalitsoe sebakeng seo.
Photodynamic therapy (PDT)19 le chromophore-assisted laser inactivation (CALI)20, eo ho eona khanya e khanyang e nang le photosensitizers e hlahisang oksijene e le 'ngoe, e ka koala liprotheine tse lebisitsoeng kapa tsa baka lefu la lisele.Kaha oksijene ea singlet ke ntho e sebetsang haholo e nang le sebaka sa theory e ka bang 70 nm, oxidation e fokolang ea sebaka ho potoloha photosensitizer e ka laoloa.Ho ipapisitsoe le mohopolo ona, re nkile qeto ea ho sebelisa oksijene ea singlet ho fihlela ho ngolisoa ha protheine ka har'a lisele tse phelang.Re hlahisitse mokhoa oa PDPL oa chemoproteomic ho phethahatsa mesebetsi e mene: (1) ho kenya letsoho molokong oa oksijene o sebetsang oa singlet o tšoanang le mokhoa oa PL enzymatic;(2) fana ka li-labeling tse rarollotsoeng ka nako ha ho qala leseli;(3) ka ho fetola (4) Qoba ho sebelisa li-cofactors tsa endogenous (joalo ka biotin) ho fokotsa bokamorao, kapa ho sebelisa li-reagents tse khathatsang haholo (joalo ka peroxides) ho fokotsa ho pepeseha ha lisele ho khatello ea tikoloho.
Li-photosensitizer li ka aroloa ka mekhahlelo e 'meli ho kenyelletsa le limolek'hule tse nyane tsa boima ba fluorophores (mohlala, rose bengal, methylene blue)22 le liprotheine tse nyane tse kentsoeng ka liphatsa tsa lefutso (mohlala, miniSOG, KillerRed)23.E le hore re finyelle moralo oa modular, re ntlafalitse sethaleng sa pele sa PDPL ka ho eketsa liprotheine tsa photosensitizer (PS) ho POI24,25 (Setšoantšo sa 1a).Ha e khantšitsoe ka leseli le leputsoa, oksijene ea singlet oxidizes proximal nucleophilic amino acid residues, e leng se etsang hore ho be le polarity ea umpolung e leng electrophilic 'me e ka tsoela pele ho itšoara ka amine probe nucleophiles16,17.Probe e entsoe ka letsoho la alkyne ho lumella k'hemistri ea tobetsa le ho theola bakeng sa sebopeho sa LC/MS/MS.
Papiso e hlophisitsoeng ea ho ngolisoa ha protheine complexes e kenelletseng ke miniSOG.Ha li pepesehetse leseli le leputsoa, lisele tse hlahisang miniSOG-POI li hlahisa oksijene e le 'ngoe, e fetolang liprotheine tse sebetsanang empa eseng liprotheine tse sa tlameng.Lihlahisoa tsa ka hare tsa photooxidation li amoheloa ke li-labels tsa probe ea lik'hemik'hale tsa amine ho etsa li-covalent adducts.Sehlopha sa alkynyl se probe ea k'hemistri se lumella k'hemistri ea tobetsa ho ithuisa ka ho hula fatše ho lateloa ke LC-MS/MS quantitation.b Sebopeho sa lik'hemik'hale sa li-amine probes 1-4.c Tlhahlobo ea gel e emelang fluorescent ea mitochondrial localized miniSOG-mediated proteomic markers e sebelisa li-probes 1-4 le palo e lekanyelitsoeng e thehiloeng ho gel densitometry.Karo-karolelano ea lets'oao le bokamorao ba lisebelisoa tsa lik'hemik'hale e ile ea hlahlojoa ho sebelisoa liteko tse mpe tsa taolo ntle le khanya e putsoa kapa ho sebelisa lisele tsa HEK293T ntle le polelo ea miniSOG.n = 2 disampole tse ikemetseng ka baeloji.Letheba le leng le le leng le emela setšoantšo sa baeloji.d Ho lemoha ha baemeli le palo ea PDPL ho sebelisoa probe 3 e ntlafalitsoeng boteng kapa bo le sieo ba likarolo tse bontšitsoeng tsa PDPL tse kang c.n = 3 disampole tse ikemetseng ka baeloji.Letheba le leng le le leng le emela setšoantšo sa baeloji.Bohareng le litelu li emela moelelo le ± kheloha e tloaelehileng.CBB: Coomassie Brilliant Blue.Setšoantšo sa Confocal sa oksijene ea singlet e nang le letheba le bofubelu bo bokhubelu ba Si-DMA.Scale bar: 10 µm.Liteko tsa litšoantšo tsa li-gel le li-confocal li ile tsa phetoa ka boikemelo bonyane habeli ka liphetho tse tšoanang.
Re ile ra qala ho leka bokhoni ba li-photosensitizers tse hōlileng tsebong miniSOG26 le KillerRed23, tse hlalositsoeng ka mokhoa o tsitsitseng ho HEK293T, ho buisana le propargylamine labeling ea proteome e le probe ea lik'hemik'hale (Supplementary Fig. 1a).Tlhahlobo ea Gel fluorescence e bonts'itse hore ho ngolisoa ha proteome kaofela ho fihletsoe ho sebelisoa miniSOG le mabone a mabone a maputsoa, athe ha ho sehlahisoa se bonahalang sa ho ngola se ileng sa bonoa ka KillerRed.E le ho ntlafatsa karo-karolelano ea pontšo-ho-background, re ile ra hlahloba sehlopha sa lisebelisoa tsa lik'hemik'hale tse nang le aniline (1 le 3), propylamine (2), kapa benzylamine (4).Re hlokometse hore lisele tsa HEK293T ka botsona li ne li e-na le letšoao le phahameng la mokokotlo ha le bapisoa le ho se na leseli le leputsoa, mohlomong ka lebaka la li-endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27. Lisebelisoa tsa lik'hemik'hale tse thehiloeng ho Aniline 1 le 3 li fane ka lintlha tse hlakileng haholoanyane, 'me HEK293T e hlalosa ka mokhoa o tsitsitseng miniSOG ka mitochondria e bonts'a keketseho ea makhetlo a 8 bakeng sa probe 3, ha probe 2 e sebelisoa ka mokhoa oa RNA-labeling CAP-seq feela e bontšang ~ 2.5- mena pontso eketseha, mohlomong ka lebaka la ho fapaneng reactivity dikgetho pakeng tsa RNA le protheine (sa feiga. 1b, c). Lisebelisoa tsa lik'hemik'hale tse thehiloeng ho Aniline 1 le 3 li fane ka lintlha tse hlakileng haholoanyane, 'me HEK293T e hlalosa ka mokhoa o tsitsitseng miniSOG ka mitochondria e bonts'a keketseho ea makhetlo a 8 bakeng sa probe 3, ha probe 2 e sebelisoa ka mokhoa oa RNA-labeling CAP-seq feela e bontšang ~ 2.5- mena pontso eketseha, mohlomong ka lebaka la ho fapaneng reactivity dikgetho pakeng tsa RNA le protheine (sa feiga. 1b, c).Lisebelisoa tsa lik'hemik'hale tse thehiloeng ho aniline 1 le 3 li bonts'itse lintlha tse hlakileng haholoanyane: HEK293T, e hlalosang ka mokhoa o tsitsitseng miniSOG ka mitochondria, e bontša ho feta ha 8 ho eketseha ha pontšo bakeng sa probe 3, ha probe 2, e sebelisoang ka mokhoa oa ho ngola oa CAP-seq RNA, feela. e bonts'a ~ keketseho ea matšoao a 2.5, mohlomong ka lebaka la likhetho tse fapaneng tsa reactivity pakeng tsa RNA le protheine (setšoantšo sa 1b, c).1 探针 he 的 粒体 的 的 的 的 的 的 的 的 的 用用 于 于2.5-倍信号增加，可能是由于RNA le蛋白质之间的不同反应偏好（图1b，c）.基于基于 苯胺 的 的 的 的 的 的 更 的 bel 更 更 更 更 的 倍 而 增加-倍信号增加，可能是由于RNALisebelisoa tsa lik'hemik'hale tse thehiloeng ho Aniline 1 le 3 li ne li e-na le boitsebiso bo betere, HEK293T e hlalositsoe ka mokhoa o tsitsitseng miniSOG ka mitochondria, 'me probe 3 e ne e e-na le keketseho e fetang 8 ea pontšo, ha probe 2 bakeng sa mokhoa oa ho ngola oa CAP-seq RNA e bontšitse feela ~ keketseho ea 2.5 -fold.pontsong, mohlomong ka lebaka la likhetho tse fapaneng tsa karabelo lipakeng tsa RNA le protheine (setšoantšo sa 1b, c).Ho phaella moo, probe 3 isomers le hydrazine probes (probes 5, 6, 7) e ile ea lekoa, e tiisang ho ntlafatsa probe 3 (Supplementary Fig. 1b, c).Ka mokhoa o ts'oanang, tlhahlobo ea in-gel fluorescence e senotse litekanyetso tse ling tse ntlafalitsoeng tsa liteko: wavelength ea mahlaseli (460 nm), mahloriso a lik'hemik'hale (1 mM), le nako ea mahlaseli (20 min) (Supplementary Fig. 2a-c).Ho tlohela karolo leha e le efe kapa mohato oa PDPL protocol ho ile ha fella ka phetoho e kholo ea pontšo ho ea ka morao (setšoantšo sa 1d).Haholo-holo, ho ngolisoa ha protheine ho fokotsehile haholo ha ho e-na le sodium azide kapa trolox, e tsejoang ka ho tima oksijene e le 'ngoe.Ho ba teng ha D2O, e tsejoang ka ho tsitsisa oksijene ea singlet, e matlafatsa lets'oao la ho ngola.Ho batlisisa tlatsetso ea mefuta e meng ea oksijene e sebetsang ho ngola, mannitol le vithamine C li ile tsa eketsoa ho theha hydroxyl le superoxide radical scavengers, ka ho latellana, 18, 29, empa ha lia ka tsa fumanoa li fokotsa ho ngola.Ho eketsoa ha H2O2, empa eseng khanya, ha hoa ka ha fella ka ho ngolisoa (Supplementary Fig. 3a).Fluorescence singlet imaging oxygen with Si-DMA probes e netefalitse boteng ba singlet oxygen terata ea HEK293T-miniSOG, empa eseng terata ea mantlha ea HEK293T.Ho phaella moo, mitoSOX Red e ne e sitoa ho lemoha tlhahiso ea superoxide ka mor'a ho khantša (setšoantšo sa 1e le Supplementary Fig. 3b) 30. Lintlha tsena li fana ka maikutlo a matla a hore oksijene ea singlet ke mofuta o ka sehloohong oa oksijene o sebetsang o ikarabellang bakeng sa ho ngola proteomic e latelang.Cytotoxicity ea PDPL e ile ea hlahlojoa ho kenyelletsa le mahlaseli a khanyang a buluu le lisebelisoa tsa lik'hemik'hale, 'me ha ho na cytotoxicity ea bohlokoa e ileng ea hlokomeloa (Supplementary Fig. 4a).
E le ho ithuta mokhoa oa ho ngola le ho nolofalletsa proteomic identification ea protheine complexes ho sebelisa LC-MS/MS, re lokela ho qala ka ho fumana hore na ke li-amino acid life tse fetotsoeng le delta mass ea li-labels tsa probe.Methionine, histidine, tryptophan le tyrosine li tlalehiloe hore li fetotsoe ke singlet oxygen14,15.Re kopanya mosebetsi oa TOP-ABPP31 le patlo e bulehileng e sa lebelloang e fanoeng ke sethala sa komporo sa FragPipe se thehiloeng ho MSFragger32.Ka mor'a hore ho be le phetoho ea oksijene e le 'ngoe le ho ngolisoa ha lik'hemik'hale tsa lik'hemik'hale, k'hemistri ea ho tobetsa e ile ea etsoa ho sebelisoa lengolo la phokotso ea biotin e nang le sehokelo se qhekellang, se lateloang ke ho otlolla ha neutravidin le tšilo ea trypsin.Peptide e fetotsoeng, e ntse e tlameletsoe ho resin, e ne e le photocleaved bakeng sa tlhahlobo ea LC-MS / MS (Setšoantšo sa 2a le Supplementary Data 1).Palo e kholo ea liphetoho e etsahetse ho pholletsa le proteome e nang le limmapa tse fetang 50 tsa peptide (PSM) tse thathamisitsoeng (setšoantšo sa 2b).Ho makatsang ke hore re bone feela phetoho ea histidine, mohlomong ka lebaka la ts'ebetso e phahameng ea oxidized histidine ho li-aniline probes ho feta li-amino acid tse ling.Ho ea ka mochine o hatisitsoeng oa histidine oxidation ka oksijene e le 'ngoe, 21,33 sebopeho se reriloeng sa delta-mass sa +229 Da se lumellana le tlhahiso ea probe 3 le 2-oxo-histidine ka mor'a li-oxidation tse peli, ha +247 Da ke sehlahisoa sa hydrolysis. ea +229 Da ( Tlatsetso ea setšoantšo sa 5).The tlhahlobo ea manyenyane MS2 bontša ho tšepahala ho phahameng tsa boitsebahatso ba boholo ba y le b ions, ho akarelletsa le ho khetholla ion fetotsoeng sekhechana (y le b) (sa feiga. 2c).Tlhahlobo ea moelelo oa tatellano ea sebaka sa PDPL-modified histidines e senoletse khetho e itekanetseng ea motif bakeng sa masala a manyenyane a hydrophobic libakeng tsa ± 1 (Supplementary Fig. 4b).Ka karolelano, li-histidine tsa 1.4 li ile tsa khetholloa ka protheine, 'me libaka tsa matšoao ana li ne li khethoa ke sebaka se fumanehang sa solvent (SASA) le tlhahlobo ea ho fumaneha ha solvent (RSA) (Supplementary Fig. 4c,d).
Ts'ebetso e se nang leeme bakeng sa ho ithuta khetho ea masala e sebelisang sethala sa komporo sa FragPipe se tsamaisoang ke MSFragger.Li-link tse ka khonang li sebelisoa ho Click chemistry ho lumella photocleavage ea li-peptide tse fetotsoeng ho tloha streptavidin resin.Patlisiso e bulehileng e ile ea qalisoa ho tseba liphetoho tse ngata, hammoho le masala a amehang.b Abela bongata ba liphetoho tse etsahalang ho pholletsa le proteome.'Mapa oa Peptide oa PSM.c MS2 spectral annotation ea libaka tsa histidine tse fetotsoeng ka probe 3. E le mohlala oa moemeli, karabelo ea covalent e nang le probe 3 e kentse +229.0938 Da ho amino acid e fetotsoeng.d Teko ea phetoho e sebelisoang ho lekola matšoao a PDPL.PRDX3 (H155A, H225A) le PRDX1 (H10A, H81A, H169A) li ile tsa fetisoa ka li-plasmids tsa mofuta o hlaha bakeng sa ho lemoha khahlanong le Folaga.Peptide ea maiketsetso e ile ea arajoa ka miniSOG e hloekisitsoeng ka pel'a probe 3 mme lihlahisoa tse tsamaellanang le Δm +247 le +229 li ile tsa hlokomeloa sebonong sa LC-MS.f Litšebelisano tsa protheine le protheine ea in vitro tse entsoeng ka miniSOG-6xHis-tag le anti-6xHis antibody.Antibiotin (streptavidin-HRP) le anti-mouse Western blot analysis ea miniSOG-6xHis/anti-6x Antibody complexes ea hae e ngotsoeng ka probe 3, ho itšetlehile ka nako ea ho pepesehela khanya.Lileibole tsa liprotheine ka bomong li hlahisoa ka boima ba limolek'hule tse tsamaellanang: ketane e bobebe ea LC antibody, ketane e boima ea HC.Liteko tsena li ile tsa phetoa ka boikemelo bonyane habeli ka liphetho tse tšoanang.
Bakeng sa netefatso ea lik'hemik'hale tsa sebaka sa ho ngola, PRDX3 le PRDX1 tse khetholloang ka li-mass spectrometry li fetotsoe ho tloha histidine ho ea ho alanine 'me li bapisoa le mefuta e hlaha ea liteko tsa phetisetso.Liphetho tsa PDPL li bontšitse hore phetoho e fokotsehile haholo ho ngolla (setšoantšo sa 2d).Ho sa le joalo, tatellano ea peptide e bontšitsoeng patlong e bulehileng e ile ea etsoa 'me ea sebetsa ka hare ho vitro ka miniSOG e hloekisitsoeng ka pel'a probe 3 le leseli le leputsoa, lihlahisoa tse hlahisang lihlahisoa tse nang le phetoho e kholo ea +247 le +229 Da ha li fumanoa ke LC-MS (Fig. .2e).).Ho lekola hore na liprotheine tsa proximal tse sebelisanang li ka ngoloa in vitro ho arabela miniSOG photoactivation, re thehile tlhahlobo ea haufi ea maiketsetso ka tšebelisano lipakeng tsa miniSOG-6xHis protheine le anti-His monoclonal antibody in vitro (Setšoantšo sa 2f).Tekong ena, re ne re lebelletse ho ngolisoa ha li-antibody tse boima le tse bobebe ka miniSOG.Ha e le hantle, anti-mouse (ho lemoha liketane tse boima le tse bobebe tsa anti-6xHis-labeled antibody) le streptavidin Western blots li bontšitse biotinylation e matla ea liketane tse boima le tse bobebe.Ka ho hlakileng, re hlokometse miniSOG autobiotinylation ka lebaka la tag ea 6xHis le li-cross-link pakeng tsa liketane tse bobebe le tse boima, tse ka 'nang tsa amana le lekhalo le hlalositsoeng pele pakeng tsa lysine le 2-oxo-histidine proximal response.Qetellong, re etsa qeto ea hore PDPL e fetola histidine ka mokhoa o itšetlehileng ka mokhoa o haufi.
Sepheo sa rona se latelang e ne e le ho hlahisa proteome ea subcellular ho leka ho hlaka ha in situ labeling.Ka hona, re ile ra hlalosa ka mokhoa o tsitsitseng miniSOG ka har'a nucleus, mitochondrial matrix, kapa ka ntle ho membrane ea ER ea lisele tsa HEK293T (Setšoantšo sa 3a).Tlhahlobo ea gel fluorescence e senotse lihlopha tse ngata tse ngolisitsoeng libakeng tse tharo tse nyenyane hammoho le mekhoa e fapaneng ea ho ngola (setšoantšo sa 3b).Tlhahlobo ea litšoantšo tsa Fluorescence e bontšitse tse khethehileng tse phahameng tsa PDPL (setšoantšo sa 3c).Ts'ebetso ea PDPL e ile ea lateloa ke karabelo ea ho tobetsa ka lidae tsa rhodamine ho hlahisa li-proteome tsa subcellular li sebelisa microscopy ea fluorescence, 'me matšoao a PDPL a ne a kopantsoe le DAPI, li-trackers tsa mitochondrial, kapa li-trackers tsa ER, tse tiisang botšepehi bo phahameng ba PDPL.Bakeng sa libaka tse tharo tsa organelle, papiso e lehlakoreng le e 'ngoe ea PDPL le TurboID e sebelisang avidin western blot e bontšitse hore PDPL e ngotsoe ka ho khetheha ha e bapisoa le taolo ea bona.Tlas'a maemo a PDPL, lihlopha tse ling tse ngotsoeng li ile tsa hlaha, tse bontšang liprotheine tse ngata tse ngotsoeng ka PDPL (Supplementary Fig. 6a-d).
kemelo ea Schematic ea miniSOG-mediated organelle-specific proteome labeling.miniSOG e lebisa matrix a mitochondrial ka fusion ho N-terminal 23 amino acids ea COX4 ea motho (mito-miniSOG), nucleus ka fusion ho H2B (nucleus-miniSOG), le Sec61β ka lehlakoreng la cytoplasmic la ER membrane (ER-miniSOG). ).Lipontšo li kenyelletsa litšoantšo tsa li-gel, litšoantšo tsa confocal, le mass spectrometry.b Litšoantšo tsa gel tse emelang likarolo tse tharo tse khethehileng tsa PDPL.CBB Coomassie Brilliant Blue.c Lits'oants'o tse kopaneng tsa lisele tsa HEK293T tse hlalosang ka mokhoa o tsitsitseng miniSOG ka libaka tse fapaneng tsa subcellular tse lemohuoang ke antibody e ngotsoeng V5 (e khubelu).Li-marker tsa subcellular li sebelisoa bakeng sa mitochondria le ER (e tala).Ts'ebetso ea PDPL e kenyelletsa ho fumanoa ha miniSOG (yellow) e ngotsoeng ka subcellular proteoms ho sebelisa Cy3-azide click chemistry.Scale bar: 10 µm.d Merero ea seretse se chesang ea li-proteome tse tšoailoeng ke PDPL ka har'a li-organelle tse fapaneng tse lekantsoeng ka palo e sa ngolisoang (n = liteko tse 3 tse ikemetseng tsa baeloji).Teko ea Moithuti ea nang le mehatla e 'meli e ile ea sebelisoa libakeng tsa ho foqoha ha seretse se chesang.HEK293T mofuta o hlaha o ne o sebelisoa e le taolo e mpe. Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> 2-fold ion intensity difference). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> 2-fold ion intensity difference). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> phapang ea makhetlo a 2 ka matla a 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). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> phapang ea 2 ka matla a ionic).Liprotheine tse amanang le tsona tse bohlokoa bakeng sa HEK293T-miniSOG empa eseng tsa bohlokoa bakeng sa HEK293T li bonts'oa ka botala.e Tshekatsheko ya boitsebahatso ba datha tsa proteomic ho tsoa litekong d.Palo eohle ea liprotheine tsa bohlokoa ka lipalo ho organelle ka 'ngoe (matheba a khubelu le a matala) a tšoailoe ka holimo.Li-histograms li bonts'a liprotheine tse sebakeng sa organelles tse thehiloeng ho MitoCarta 3.0, tlhahlobo ea GO le A. Ting et al.batho.Arola li-datasets tsa mitochondria, nuclei, le ER.Liteko tsena li ile tsa phetoa ka boikemelo bonyane habeli ka liphetho tse tšoanang.Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
E khothalelitsoe ke li-gel le liphetho tsa litšoantšo, quantification e se nang li-label e ne e sebelisetsoa ho lekanya proteome e khethiloeng ho organelle ka 'ngoe (Supplementary Data 2).HEK293T e sa tšoaetsanoang e ile ea sebelisoa e le taolo e mpe ho tlosa matšoao a ka morao. Tlhahlobo ea morero oa seretse se chesang e bonts'itse liprotheine tse matlafalitsoeng haholo (p <0.05 le> 2-fold ion intensity) hammoho le liprotheine tsa singleton tse teng feela meleng ea miniSOG-expressing (Fig. 3d matheba a khubelu le a matala). Tlhahlobo ea morero oa seretse se chesang e bonts'itse liprotheine tse matlafalitsoeng haholo (p <0.05 le> 2-fold ion intensity) hammoho le liprotheine tsa singleton tse teng feela meleng ea miniSOG-expressing (Fig. 3d matheba a khubelu le a matala). 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). Tlhahlobo ea morero oa seretse se chesang e bontšitse liprotheine tse matlafalitsoeng haholo (p <0.05 le> 2-fold ion intensity) hammoho le liprotheine tse le 'ngoe tse teng feela meleng e hlalosang miniSOG (setšoantšo sa 3d, matheba a khubelu le a tala).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）以及仅存在纎mini存在纎miniSOG 一亚存在纎miniSOG 一火山图火山图 显示 的 的 的 和> 2 倍 离子 在于 在于 存 存 系 系 的 的 的 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). Tlhahlobo ea morero oa seretse se chesang e ile ea senola liprotheine tse matlafalitsoeng haholo (p <0.05 le> 2x matla a ionic) hammoho le liprotheine tse le 'ngoe tse teng feela moleng oa polelo ea miniSOG (matheba a khubelu le a matala ho Fig. 3d).Ha re kopanya lintlha tsena, re khethile 1364, 461, le 911 liprotheine tsa bohlokoa tsa nyutlelie, mitochondrial, le ER ka ntle ka lipalo.Ho hlahloba ho nepahala ha organelle-localized PDPL, re sebelisitse MitoCarta 3.0, Gene Ontology (GO) analysis, le A. Ting et al.data set8 e ne e sebelisoa bakeng sa mitochondria, nucleus, le ER ho hlahloba ho khetheha ha organelle ea liprotheine tse fumanoeng, tse lumellanang le ho nepahala ha 73.4, 78.5, le 73.0% (setšoantšo sa 3e).Taba e ikhethang ea PDPL e tiisa hore PDPL ke sesebelisoa se nepahetseng sa ho khetholla li-proteome tse khethehileng tsa organelle.Haholo-holo, tlhahlobo ea subochondrial ea liprotheine tsa mitochondrial tse khethiloeng li bonts'itse hore proteome e ts'oaretsoeng e ne e ajoa haholo ka har'a matrix le membrane e ka hare (226 le 106, ka ho latellana), e leng 91.7% (362) ea palo eohle ea liprotheine tsa mitochondrial.boemo bo phahameng ba PDPL bo ile ba tiisoa ka ho eketsehileng (Supplementary Fig. 7a).Ka ho tšoanang, tlhahlobo ea li-subnuclear e bontšitse hore proteome e hapuoeng e ne e ajoa haholo-holo ka har'a nucleus, nucleoplasm le nucleolus (Supplementary Fig. 7b).Tlhahlobo ea proteomic ea nyutlelie ka peptide ea pontšo ea sebaka sa nyutlelie (3xNLS) e bontšitse ho nepahala ho ts'oanang le kaho ea H2B (Supplementary Fig. 7c–h).Ho fumana hore na lets'oao la PDPL le nepile hakae, nyutlelie laminine A e ile ea khethoa joalo ka sefi sa POI7 se fumanehang sebakeng se hlakileng haholoanyane.PDPL e ile ea tsebahatsa liprotheine tse 36 tse matlafalitsoeng haholo, tseo liprotheine tse 12 (30.0% tse kenyeletsang lamin A) e neng e le liprotheine tse sebetsang hantle tsa lamin A tse hlalositsoeng ke database ea String, tse nang le liphesente tse phahameng ho feta mokhoa oa BioID (122 protheine) 28 ea 28. , 22.9 %) 7. Mokhoa oa rona o ile oa khetholla liprotheine tse fokolang, mohlomong ka lebaka la libaka tse fokolang tsa ho ngolla, tse ileng tsa khoneha ka oksijene ea singlet e sebetsang haholoanyane.Tlhahlobo ea GO e bontšitse hore liprotheine tse khethiloeng li ne li fumaneha haholo ka har'a nucleoplasm (26), lera la nyutlelie (10), lera la nyutlelie (9), le masoba a nyutlelie (5).Ka kopanelo, liprotheine tsena tsa sebaka sa nyutlelie li ile tsa nka karolo ea 80% ea liprotheine tse ruisitsoeng, ho tsoela pele ho bontša ho khetheha ha PDPL (Supplementary Fig. 8a–d).
Ha re se re thehile bokhoni ba PDPL ba ho etsa matšoao a haufi ho li-organelles, re ile ra leka hore na PDPL e ka sebelisoa ho sekaseka balekane ba tlamang ba POI.Haholo-holo, re ile ra batla ho hlalosa tlhahlobo ea PDPL ea liprotheine tsa cytosolic, tse nkoang e le liphofu tse thata ho feta li-membrane-localized tsa tsona ka lebaka la tlhaho ea tsona e matla haholo.Protheine ea bromodomain le extraterminal (BET) BRD4 e hohetse tlhokomelo ea rona bakeng sa karolo ea eona ea bohlokoa maloetseng a sa tšoaneng 35, 36 .E rarahaneng e entsoeng ke BRD4 ke transcriptional coactivator le sepheo sa bohlokoa sa phekolo.Ka ho laola polelo ea lintlha tsa c-myc le Wnt5a transcription, BRD4 ho nahanoa hore ke eona ntho e ka sehloohong e khethollang acute myeloid leukemia (AML), multiple myeloma, Burkitt's lymphoma, kankere ea mala le mafu a ho ruruha37,38.Ntle le moo, livaerase tse ling li shebile BRD4 ho laola phetiso ea vaerase le cellular, joalo ka papillomavirus, HIV, le SARS-CoV-236,39.
Ho etsa 'mapa oa tšebelisano ea BRD4 re sebelisa PDPL, re ile ra kopanya miniSOG le N- kapa C-terminal isoform e khutšoanyane ea BRD4.Liphetho tsa proteomic li ile tsa senola tekanyo e phahameng ea ho kopana pakeng tsa lihahi tse peli (Supplementary Fig. 9a).Proteome ea nyutlelie e khetholloang ka miniSOG-H2B e akaretsa 77.6% ea liprotheine tse sebetsanang le BRD4 (Supplementary Fig. 9b).Joale, linako tse fapaneng tsa khanya (2, 5, 10, 20 min) li ne li sebelisetsoa ho lokisa radius ea letšoao (setšoantšo sa 4a le lintlha tse eketsehileng tsa 3).Re etsa qeto ea hore linakong tse khuts'oane tsa lifoto, PDPL e tla fana ka mabitso a balekane ba tlamang ka kotloloho, athe nako e telele e tla kenyelletsa liprotheine tse bonts'itsoeng nakong e khuts'oane ea ho etsa lifoto hammoho le sepheo se sa tobang litsing tsa ho ngola.Ha e le hantle, re fumane ho kopana ho matla pakeng tsa lintlha tsa nako tse haufi (84.6% bakeng sa 2 le 5 min; 87.7% bakeng sa 5 le 10 min; 98.7% bakeng sa 10 le 20 min) (Fig. 4b le Supplementary Fig. 9c).Lihlopheng tsohle tsa liteko, ha rea fumana feela BRD4 ea ho ingolisa, empa lipheo tse 'maloa tse tsejoang joalo ka MED1, CHD8, BICRA, NIPBL, SMC1A, le HMGB1 li hlalositsoe polokelong ea likhoele.Matla a ionic a lipakane tsena a lekana le nako ea ho pepeseha (setšoantšo sa 4c le sa Tlatsetso sa 9d).Tlhahlobo ea GO ea liprotheine tse khethiloeng sehlopheng sa metsotso ea 2 e bontšitse hore liprotheine tse khethiloeng li ne li le sebakeng sa nucleus 'me li kentse letsoho ho lokisoeng ha chromatin le RNA polymerase.Mosebetsi oa molek'hule oa protheine o ile oa ntlafatsoa ka ho kopanya chromatin kapa transcriptal coactivation, e lumellanang le mosebetsi oa BRD4 (setšoantšo sa 4d).Tshekatsheko ya tshebedisano ya protheine e nolofaditsweng ya string database e senotse boemo ba pele ba tshebedisano e sa tobang pakeng tsa BRD4 le HDAC ya malapa a sebedisanang complexes jwalo ka SIN3A, NCOR2, BCOR, le SAP130 (Fig. 4e le Supplementary Fig. 9e), e tsamaellanang le BRD4 le HDAC e tlamang histones acetylated ..Ho phaella moo, lipakane tsa baemeli tse khetholloang ke LC-MS/MS, ho akarelletsa le Sin3A, NSUN2, Fus, le SFPQ, li tiisitsoe ke Western blotting (Fig. 4f).Haufinyane tjena, isoform e khuts'oane ea BRD4 e tlalehiloe hore e theha nuclei e nang le thepa ea ho arohana ha mokelikeli oa metsi (LLPS).Liprotheine tse tlamang tsa RNA Fus le SFPQ li hokahanya LLPS ea lits'ebetso tse fapaneng tsa cellular mme li bonts'itsoe mona e le liprotheine tse tlamang tsa BRD4 tse sa rekotiloeng.Tšebelisano pakeng tsa BRD4 le SFPQ e tiisitsoe ke liteko tsa co-immunoprecipitation (co-IP) (Figure 4g), e fanang ka maikutlo a mokhoa o mong oa karohano ea karolo ea BRD4-mediated liquid-liquid phase e lokeloang ke lipatlisiso tse eketsehileng.Ha li kopane, liphetho tsena li fana ka maikutlo a hore PDPL ke sethala se loketseng sa ho tsebahatsa BRD4 e sebelisanang hammoho le liprotheine tse sa tsejoeng tse tlamang.
kemelo ea Schematic ea miniSOG-mediated BRD4 proximty marking, linako tsa ho pepeseha: 2, 5, 10, le 20 min.b Ho kopana ha liprotheine tse khethiloeng ka linako tse fapaneng tsa khanya.Matlafatso ea liprotheine tse khethiloeng ho HEK293T-miniSOG-BRD4 e ne e le ea bohlokoa ka lipalo ha e bapisoa le HEK293T ea mofuta o hlaha.c Matla a Ion ha ho lekoa moemeli ea sa ngolisoang ea tsejoang ka liprotheine tse tlamang BRD4 nakong e behiloeng ea ho pepeseha.n = 3 disampole tse ikemetseng ka baeloji.Lintlha li hlahisoa ka mokhoa o bolelang ± kheloha e tloaelehileng.d Tlhahlobo ea liphatsa tsa lefutso (GO) ea liprotheine tse khethiloeng sehlopheng sa metsotso e 2.Ho thathamisitsoe mantsoe a leshome a pele a GO.Li-bubble li mebala ho latela sehlopha sa nako ea GO, 'me boholo ba bubble bo lekana le palo ea liprotheine tse fumanoang kotareng e' ngoe le e 'ngoe.e Tlhahlobo ea likhoele tsa liprotheine tse sebelisanang le BRD4.Li-circles tse tšehla ke sekhomaretsi se tobileng 'me li-circles tse bohlooho ke lera la pele la sekhomaretsi se sa tobang.Mela e khubelu e emela litšebelisano tse entsoeng ka liteko 'me mela e putsoa e emela litšebelisano tse boletsoeng esale pele.f Lipheo tse tlamang tsa baemeli ba BRD4 tse khethiloeng ho LC-MS/MS li netefalitsoe ke Western blotting.g Liteko tsa Co-immunoprecipitation li tiisa tšebelisano lipakeng tsa SFPQ le BRD4.Liteko tsena li ile tsa phetoa ka boikemelo bonyane habeli ka liphetho tse tšoanang.Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
Ntle le ho tsebahatsa liphofu tse sa ngolisoang tse amanang le POI, re nahana hore PDPL e tla ba e loketseng ho tsebahatsa li-substrates tsa li-enzyme, tse tla hloka hore ho be le sebopeho sa liprotheine tse tlamang tse sa tobang ka li-complexes tse kholo ho hlakisa li-substrates tse sa ngolisoang.Parkin (encoded by PARK2) ke E3 ligase mme liphetoho tsa parkin li tsejoa ho baka lefu la autosomal recessive juvenile Parkinson (AR-JP)42.Ho phaella moo, parkin e 'nile ea hlalosoa e le ea bohlokoa bakeng sa mitophagy (mitochondrial autophagy) le ho tlosoa ha mefuta ea oksijene e sebetsang.Leha ho le joalo, le hoja li-substrates tse 'maloa tsa parkin li fumanoe, karolo ea parkin lefung lena e ntse e sa hlaka.Ho hlakisa li-substrate tsa eona tse se nang sebopeho, PDPL e ile ea lekoa ka ho kenyelletsa miniSOG ho N- kapa C-terminus ea parkin.Lisele li ile tsa phekoloa ka carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) ho kenya parkin ka tsela ea PINK1-Parkin.Ha ho bapisoa le liphetho tsa rona tsa BRD4 PDPL, parkin N-terminus fusion e senotse sete e kholoanyane ea liprotheine tse lebisitsoeng, le hoja e ne e akaretsa karolo e kholoanyane ea C-terminus (177 out of 210) (Figure 5a,b le Supplementary Data 4).sephetho se lumellana le litlaleho tsa hore li-tag tsa N-terminal li ka etsa hore Parkin44 e sebetse.Ho makatsang ke hore ho ne ho e-na le liprotheine tse fetang 18 feela ho data ea rona tse nang le liphetho tsa AP-MS tse phatlalalitsoeng bakeng sa Parkin43, mohlomong ka lebaka la phapang lipakeng tsa mela ea lisele le phallo ea mosebetsi oa proteomics.Ho phaella ho liprotheine tse 'nè tse tsejoang (ARDM1, HSPA8, PSMD14, le PSMC3) tse khetholloang ka mekhoa e' meli (setšoantšo sa 5c) 43.Ho tsoela pele ho netefatsa liphetho tsa LC-MS/MS, phekolo ea PDPL le ho thijoa ha Bophirimela ho ile ha sebelisoa ho bapisa liphello tsa HEK293T ea tlhahlobo ea lisele tsa motsoali le mohala o tsitsitseng oa N-terminal parkin.Lipheo tse neng li sa tsejoe pele CDK2, DUT, CTBP1, le PSMC4 li ile tsa lekoa ka binder e tsebahalang, DNAJB1 (Fig. 5d).
Sebaka sa seretse se chesang sa liprotheine tse sebetsanang le parkin liseleng tsa HEK293T tse nang le miniSOG e hlakileng e kopantsoeng le N- kapa C-terminus ea parkin (n = liteko tse 3 tse ikemetseng tsa baeloji).Teko ea Moithuti ea nang le mehatla e 'meli e ile ea sebelisoa libakeng tsa ho foqoha ha seretse se chesang.HEK293T e sebelisitsoe joalo ka taolo e mpe. Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> 2-fold ion intensity difference). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> 2-fold ion intensity difference). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> phapang ea makhetlo a 2 ka matla a 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). Liprotheine tse fetotsoeng haholo li totobatsoa ka bofubelu (p <0.05 le> phapang ea 2 ka matla a ionic).Liprotheine tse amanang le tsona tse bohlokoa bakeng sa HEK293T-miniSOG empa eseng tsa bohlokoa bakeng sa HEK293T li bonts'oa ka botala.b Setšoantšo sa Venn se bonts'ang liprotheine tse fetang pakeng tsa N-terminal le C-terminal constructs.Li-tag tsa N-terminal li ka kenya tšebetsong parkin ka mokhoa o sa tsitsang 'me tsa fella ka liprotheine tse hlokomelehang haholoanyane.c Setšoantšo sa Venn se bonts'ang liprotheine tse fetang pakeng tsa PDPL le AP-MS.Litšebelisano tse tsebahalang li thathamisitsoe, ho kenyeletsoa liprotheine tse 4 ho tse 18 le liprotheine tse 11 ho tse 159 tse khethiloeng ka ho khetheha ho PDPL.d Lipheo tsa baemeli tse khethiloeng ke LC-MS/MS li netefalitsoe ke Western blotting.e Ssu72 le SNW1 li ile tsa khetholloa e le li-parkin tse sa ngolisoang.Li-plasmids tsena tsa protheine tse tšoailoeng ke FLAG li ile tsa fetisetsoa ho HEK293T le HEK293T-Parkin-miniSOG tse lateloang ke kalafo ea CCCP ka linako tse fapaneng.Ho senyeha ho ne ho totobetse haholoanyane moleng oa Parkin overexpression.f Ho sebelisa proteasome inhibitor MG132, ho netefalitsoe hore ts'ebetso ea ho senyeha ha Ssu72 le SNW1 e kopanngoa ke proteasome-ubiquitination.Liteko tsena li ile tsa phetoa ka boikemelo bonyane habeli ka liphetho tse tšoanang.Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
Haholo-holo, liprotheine tse khethiloeng ke PDPL li tlameha ho kenyelletsa liprotheine tse tlamang parkin le likaroloana tsa tsona.Ho bona li-substrates tsa parkin tse sa ngolisoang, re ile ra khetha liprotheine tse supileng tse khethiloeng (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 le SNW1) le li-plasmids tse fetisitsoeng ho pepesa liphatsa tsena tsa lefutso ho HEK293T e tloaelehileng le ho hlalosa ka mokhoa o tsitsitseng phekolo ea miniSOG-Parkin's HEK293T ea HEK293T.Litekanyo tsa liprotheine tsa Ssu72 le SNW1 li ile tsa fokotseha haholo moleng o tsitsitseng oa miniSOG-Parkin (Fig. 5e).Kalafo ea CCCP bakeng sa lihora tse 12 e bakile ho senyeha ho hoholo ha li-substrates ka bobeli.Ho batlisisa hore na ho senyeha ha Ssu72 le SNW1 ho laoloa ke proteasome-ubiquitination, proteasome inhibitor MG132 e ile ea eketsoa ho thibela mosebetsi oa proteasome, 'me ha e le hantle re fumane hore ts'ebetso ea bona ea ho senyeha e ne e thibetsoe (Setšoantšo sa 5f).Lipheo tse ling tse se nang substrate li ile tsa tiisoa e le li-interfaces tsa Parkin tse sebelisang Western blotting (Supplementary Fig. 10), e bontšitseng liphello tse lumellanang le LC-MS / MS.Qetellong, ho kopanngoa ha mosebetsi oa PDPL le netefatso ea phetisetso ea protheine e reretsoeng ho lumella ho tsebahatsoa ha li-substrates tsa E3 ligase tse sa ngolisoang.
Re thehile sethala se tšoanang sa ho tšoaea se haufi se u lumellang ho tsebahatsa sebakeng le nako ea ho sebelisana POI.Sethala se ipapisitse le protheine ea miniSOG photosensitizer, e ka bang 12 kDa feela, e ka tlase ho halofo ea enzyme e butsoitseng ea APEX2 (27 kDa) le karolo ea boraro ea boholo ba TurboID (35 kDa).Boholo bo nyane bo lokela ho holisa haholo mefuta e mengata ea lits'ebetso tsa ho ithuta liprotheine tse nyane.Ho hlahlojoa ho eketsehileng ha li-photosensitizers tse eketsehileng, ebang ke liprotheine tse nang le liphatsa tsa lefutso kapa limolek'hule tse nyenyane, ho hlokahala ho eketsa chai ea quantum ea oksijene ea singlet le ho atolosa kutloisiso ea mokhoa ona.Bakeng sa mofuta oa hajoale oa miniSOG, tharollo e phahameng ea nakoana e ka fihlelleha ho sebelisoa khanya e putsoa ho kenya matšoao a haufi.Ho phaella moo, nako e telele ea ho pepeseha ho ile ha lokolla "leru" le leholoanyane la oksijene e le 'ngoe, e leng se ileng sa fella ka ho fetoloa ha masala a distal histidine, ho eketseha ha radius ea ho ngolla, le bokhoni ba ho lokisa qeto ea sebaka sa PDPL.Hape re ile ra leka lisebelisoa tse supileng tsa lik'hemik'hale ho eketsa tekanyo ea pontšo ho ea morao-rao 'me ra hlahloba mochine oa limolek'hule ka mor'a mokhoa ona.Ts'ebetso ea TOP-ABPP e kopantsoeng le lipatlisiso tse bulehileng tse se nang leeme li tiisitse hore liphetoho li etsahetse feela ho histidines 'me ha ho microenvironment e tsitsitseng e ileng ea hlokomeloa bakeng sa liphetoho tse eketsehileng tsa histidine, ntle le khetho e itekanetseng ea histidine sebakeng sa loop.
PDPL e boetse e sebelisitsoe ho khetholla li-proteome tsa subcellular tse nang le proteome e ikhethang le ts'ireletso bonyane tse ka bapisoang le mekhoa e meng ea ho ngola haufi le mekhoa ea ho hlahloba lik'hemik'hale e khethehileng ea organelle.Li-proximity marker li boetse li sebelisitsoe ka katleho ho khetholla bokaholimo ba liprotheine tse amanang le lysosomal, le secretome-associated46,47.Re lumela hore PDPL e tla lumellana le li-organelles tsena tsa subcellular.Ho phaella moo, re ile ra phephetsa PDPL ka ho khetholla lipheo tsa ho tlama liprotheine tsa cytosolic tse rarahaneng ho feta liprotheine tse nang le membrane ka lebaka la thepa ea tsona e matla le ho kenya letsoho likamanong tse ngata tsa nakoana.PDPL e ne e sebelisoa ho liprotheine tse peli, transcriptional coactivator BRD4 le ligase E3 Parkin e amanang le lefu.Liprotheine tsena tse peli ha lia khethoa feela bakeng sa mesebetsi ea tsona ea mantlha ea tlhaho, empa hape le molemong oa tsona oa bongaka le matla a kalafo.Bakeng sa li-POI tsena tse peli, balekane ba tsebahalang ba tlamang hammoho le lipehelo tse sa ngolisoang li ile tsa khetholloa.Ka ho hlakileng, protheine ea SFPQ e amanang le karohano e ile ea tiisoa ke co-IP, e ka 'nang ea bontša mokhoa o mocha oo BRD4 (isoform e khutšoanyane) e laolang LLPS ka eona.Ka nako e ts'oanang, re lumela hore ho tsebahatsoa ha li-substrates tsa Parkin ke boemo boo ho bona ho hlokahalang ho tsebahatsa li-adhesives tse sa tobang.Re hloaile li-substrates tse peli tsa parkin tse sa tsejoeng 'me ra tiisa ho senyeha ha tsona ka tsela ea ubiquitination-proteasome.Haufinyane tjena, ho entsoe leano la ho ts'oara ka mochini ho lemoha li-substrates tsa hydrolase ka ho li tšoasa ka li-enzyme.Le hoja ena e le mokhoa o matla haholo, ha o tšoanelehe bakeng sa tlhahlobo ea li-substrates tse amehang ho thehoa ha li-complexes tse kholo 'me li hloka ho thehoa ha likamano tse kopanetsoeng pakeng tsa enzyme le substrate.Re lebeletse hore PDPL e ka atolosoa ho ithuta liprotheine tse ling le malapa a li-enzyme, tse kang malapa a deubiquitinase le metalloprotease.
Mofuta o mocha oa miniSOG, o bitsoang SOPP3, o entsoe ka tlhahiso e ntlafalitsoeng ea oksijene ea singlet.Re ile ra bapisa miniSOG le SOPP3 mme ra fumana ts'ebetso e ntlafetseng ea ho tšoaea, le hoja tekanyo ea pontšo-ho-lerata e ile ea lula e sa fetohe (Supplementary Fig. 11).Re ile ra nahana hore optimization ea SOPP3 (mohlala, ka ho iphetola ha lintho) e tla lebisa ho liprotheine tse sebetsang hantle tsa photosensitizer tse hlokang nako e khuts'oane ea khanya mme ka hona li lumelle lits'ebetso tse matla tsa cellular hore li tšoaroe.Ka ho hlakileng, mofuta oa hajoale oa PDPL o lekanyelitsoe tikolohong ea cellular kaha o hloka leseli le leputsoa mme ha o khone ho kenella ka har'a lisele tse tebileng.Tšobotsi ena e thibela tšebeliso ea eona lithutong tsa mehlala ea liphoofolo.Leha ho le joalo, motsoako oa optogenetics le PDPL o ka fana ka monyetla oa ho etsa lipatlisiso ka liphoofolo, haholo-holo bokong.Ntle le moo, li-photosensitizer tse ling tse entsoeng ka infrared le tsona li tlosa moeli ona.Lipatlisiso li ntse li tsoela pele sebakeng sena.
Mohala oa sele oa HEK293T o fumanoe ho ATCC (CRL-3216).The cell line e ile ea fumanoa e le negative bakeng sa tšoaetso ea mycoplasma 'me e hlahisitsoe DMEM (Thermo, #C11995500BT) e tlatsitsoeng ka 10% fetal bovine serum (FBS, Vistech, #SE100-B) le 1% penicillin/streptomycin (Hyclone, #SV30010).holile ka hare.
3-Aminophenylene (sample 3) le (4-ethynylphenyl)methanamine (sample 4) li ile tsa rekoa ho Bidepharm.Propylamine (probe 2) e ile ea rekoa ho Energy-chemicals.N-(2-Aminophenyl)pent-4-ynamide (probe 1) e entsoe ho latela mekhoa e hatisitsoeng.
Tlatsetso Letlapa la 1 le thathamisa liphatsa tsa lefutso tse sebelisitsoeng thutong ena.Tatelano ea miniSOG le KillerRed e entsoe ho tsoa ho plasmid ea mpho e tsoang ho P. Zou (Univesithi ea Peking).Tatelano e lebisitsoeng ea matrix ea mitochondrial e nkiloe ho li-amino acid tse 23 tsa N-terminal tsa COX4 'me tsa etsoa ka har'a li-vector tse bontšitsoeng ho sebelisoa kopano ea Gibson (Beyotime, #D7010S).Ho shebisa lera le khubu ea endoplasmic reticulum, SEC61B DNA ea motho (NM_006808.3) (NEB, #M0491L) e matlafalitsoeng ke PCR ho tsoa laebraring ea cDNA ea lisele tsa HEK293T, le H2B DNA (e fanoeng ke D. Lin, Shenzhen Bay Laboratory) le cloned , joalokaha ho boletsoe ka holimo.Ntle le ha ho bonts'itsoe ka tsela e 'ngoe, liphatsa tsa lefutso tse ling tsa protheine tse sebelisetsoang phetisetso le kaho ea mela e tsitsitseng ea lisele li ile tsa holisoa ka PCR ho tsoa laebraring ea HEK293T cell cDNA.G3S (GGGS) le G4S (GGGGS) li ne li sebelisoa e le lihokela pakeng tsa protheine ea bait le miniSOG.Letlapa la V5 epitope (GKPIPNPLLGLDST) le kentsoe meahong ena ea fusion.Bakeng sa polelo ho liphoofolo tse anyesang le ho theha mohala o tsitsitseng oa sele, miniSOG fusion construct e ile ea kenngoa ka har'a pLX304 lentiviral vector.Bakeng sa polelo ea baktheria, miniSOG e entsoe ka har'a vector ea pET21a e ngotsoeng 6xHis ho C-terminus.
Lisele tsa HEK293T li ile tsa jaloa liseleng tsa 2.0 x 105 selibeng ka lipoleiti tse tšeletseng tsa seliba 'me tsa fetisetsoa lihora tse 24 hamorao ka li-plasmids tsa lentiviral (2.4 μg pLX304) le li-plasmids tsa kokoana-hloko (1.5 μg psPAX2 le 1.2 μg00 pMD200 pMD) , #C0533), hoo e ka bang 80% fusion.Ka mor'a ho fetisoa ka bosiu bo le bong, mochine oo o ile oa fetoloa 'me oa kenngoa lihora tse ling tse 24.Pokello ea vaerase e entsoe kamora lihora tse 24, 48 le 72.Pele ho ts'oaetso ea lisele tse lebisitsoeng, likokoana-hloko li ne li hloekisoa ka 0.8 μm filter (Merck, #millex-GP) le polybrene (Solarbio, #H8761) e kenyelelitsoe mofuteng oa 8 μg / ml.Ka mor'a lihora tse 24, lisele li ile tsa lumelloa ho hlaphoheloa ka ho fetola seaplane.Lisele li ile tsa khethoa ho sebelisoa 5 μg / ml blasticidin (Solarbio, #3513-03-9) bakeng sa litemana tse tharo tsa pele e le khetho e tlaase e thata.Eaba ho sebelisoa 20 μg/ml e le mokhoa o thata haholoanyane litemaneng tse tharo tse latelang.
Lisele li ile tsa jaloa ka likamoreng tsa liliba tse 12 (Ibidi, #81201) ka bongata ba lisele tse ka bang 20,000 selibeng.Ho ntlafatsa ho khomarela ha lisele tsa HEK293T, eketsa 50 µg/ml fibronectin (Corning, #356008) e hlapolotsoeng ka phosphate buffered saline (PBS, Sangon, #B640435) ho 37°C.Likamore li ile tsa etsoa esale pele bakeng sa hora ea 1 ebe li tlosoa ka PBS.Ka mor'a lihora tse 24, lisele li ne li hlatsuoa hang ka PBS, li kenngoa ka 1 mM probe 3 ka har'a motsoako o hloekileng oa letsoai oa Hanks (HBSS, Gibco, #14025092) bakeng sa 1 h ho 37 ° C, ebe o kenngoa ka LED e putsoa (460 nm). ).) li ne li khantšitsoe ka metsotso e 10 mocheso oa kamore.Ka mor'a moo, lisele li ne li hlatsuoa ka makhetlo a mabeli ka PBS 'me li tsitsitsoe ka 4% formaldehyde ho PBS (Sangon, #E672002) bakeng sa metsotso ea 15 mocheso oa kamore.Formaldehyde e feteletseng e ile ea tlosoa liseleng tse tsitsitseng ka ho hlatsoa ka makhetlo a mararo ka PBS.Lisele li ile tsa kenngoa ka har'a 0.5% Triton X-100 (Sangon, #A600198) ho PBS 'me tsa hlatsuoa ka makhetlo a 3 ka PBS.Ebe o tlosa phaposi mme o kenye sampole e nngwe le e nngwe 25 µl ya motswako wa karabelo ya tobetsa o nang le 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) le 0.5 mg / ml sodium ascorbate (Aladdin, no. S105024) 'me e kenngoa ka metsotso e 30 mocheso oa kamore.Ka mor'a karabelo e potlakileng, lisele li ile tsa hlatsuoa ka makhetlo a tšeletseng ka PBS e nang le 0.05% Tween-20 (Sangon, #A600560) (PBST) ebe e thibeloa ka 5% BSA (Abcone, #B24726) ho PBST bakeng sa metsotso ea 30 mocheso oa kamore.
Bakeng sa colocalization immunostaining, lisele li ne li kenngoa ka li-antibodies tsa mantlha ho latela maemo a bontšitsoeng: mouse anti-V5 tag mAb (1: 500, CST, #80076), mmutla anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), rabbit polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) kapa mmutla anti-lamin A/C monoclonal antibody (1:500; CST, #2032) ka 4 °C bosiu bo le bong.Ka mor'a ho hlatsoa ka makhetlo a 3 ka PBST, lisele li ne li kenngoa ka li-antibodies tsa bobeli: pōli e khahlanong le mmutla Alexa Fluor 488 (Thermo, #A11034) e hlapollotsoe 1: 1000, pōli e khahlanong le mouse Alexa Fluor 594 (CST, #8889) e hlapollotsoe 1: 1000.dilution Hlapolla mocheso oa kamore bakeng sa metsotso e 30.Joale lisele li ne li hlatsuoa ka makhetlo a 3 ka PBST 'me li hanyetsanoa le DAPI (Thermo, #D1306) ho PBS bakeng sa metsotso ea 10 mocheso oa kamore.Ka mor'a hore 3 e hlatsoe ka PBS, lisele li ne li tiisitsoe ka 50% glycerol (Sangon, #A600232) ho PBS bakeng sa litšoantšo.Litšoantšo tsa Immunofluorescent li ile tsa fumanoa ho sebelisoa ZEISS LSM 900 Airyscan2 confocal microscope le software ea ZNE 3.5.
Bakeng sa litšoantšo tsa fluorescent tsa oksijene e le 'ngoe, lisele li ile tsa hlatsuoa habeli ka buffer ea Hanks HEPES pele ho kenyelletsa 100 nM Si-DMA ho Hanks HEPES buffer (DJINDO, #MT05).Ka mor'a ho pepesehela khanya, lisele li ile tsa kenngoa ka har'a seqhoqhoa sa CO2 ka 37 ° C ka metsotso e 45.Lisele li ile tsa hlatsuoa habeli ka buffer ea Hanks' HEPES 'me tsa ts'oaroa ka Hoechst ka Hanks' HEPES buffer ka metsotso e 10 ka mocheso oa kamore 'me tsa bonoa ka ZEISS LSM 900 confocal microscope., #M36008) ho HBSS buffer e nang le calcium le magnesium.Ka mor'a ho pepesehela khanya kapa doxorubicin (MCE, #HY-15142A), lisele li ne li kenngoa ka har'a incubator ea CO2 ka 37 ° C. ka metsotso e 10, li hlatsuoa habeli ka HBSS buffer, 'me li kenngoa ka Hoechst ka HBSS buffer mocheso oa kamore.metsotso.Doxorubicin e ne e sebelisoa e le taolo e ntle ea lipatlisiso moo lisele li tšoaroang ka 20 μM doxorubicin ho HBSS e nang le 1% BSA bakeng sa 30 min.Litšoantšo tsa Immunofluorescent li ile tsa fumanoa ka microscope ea Zeiss LSM 900 confocal.
Lisele tsa HEK293T tse hlalosang mito-miniSOG ka mokhoa o tsitsitseng li ile tsa lengoa ka bongata bo ka bang 30% lijaneng tse 15 cm.Ka mor'a lihora tsa 48, ha ~ 80% confluence e fihlile, lisele li ile tsa hlatsuoa hang ka PBS, li kenngoa ka 1 mM Probe 3 ka har'a buffer e ncha ea HBSS ka hora ea 1 ho 37 ° C ebe e khantšitsoe ka LED e putsoa bakeng sa metsotso ea 10 ka kamoreng. mocheso..Ka mor'a moo, lisele li ile tsa hlatsuoa habeli ka PBS, tsa hlajoa 'me tsa khutlisetsoa ka har'a leqhoa le batang la PBS le nang le EDTA-free protease inhibitors (MCE, #HY-K0011).Lisele li ne li lysed ka ho sonicating ntlha bakeng sa motsotso o le mong (motsotsoana o le mong ho ea pele le motsotsoana o le mong ho 35% amplitude).Motsoako o hlahisoang o ne o le centrifuged ho 15,871 xg bakeng sa 10 min ho 4 ° C ho tlosa litšila, 'me mahloriso a supernatant a fetotsoe ho 4 mg / mL ho sebelisa BCA protheine assay kit (Beyotime, #P0009).Kopanya 1 ml ea lysate e ka holimo le 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), le 1 mM tor CuSO4 incuba incuba Fetola hora e le 'ngoe ka mocheso oa kamore.Ka mor'a ho arabela ka potlako, eketsa motsoako ho motsoako oa pele o motsoako (MeOH:CHCl3:H2O = 4 ml: 1 ml: 3 ml) ka har'a vial ea khalase ea 10 ml.Mehlala e ne e tsoakane 'me e centrifuged ho 4500 g bakeng sa metsotso e 10 mocheso oa kamore.Litharollo tse ka tlaase le tse ka holimo li ne li lahliloe, metsi a ile a hlatsuoa habeli ka 1 ml ea methanol le centrifuged ho 15871 × g bakeng sa 5 min ho 4 ° C.Kenya 1 ml ea 8 M urea (Aladdin, no. U111902) ho 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) ho qhala pula ea pula.Mehlala e ile ea hlophisoa bocha ka 10 mM dithiothreitol (Sangon, #A100281 ho 25 mM ABC) bakeng sa metsotso e 40 ho 55°C e lateloang ke keketso ea 15 mM e ncha ea iodoacetamide (Sangon, #A600539) mocheso oa kamore lefifing.Alkylation nakong ea metsotso e 30..Ho kentsoe dithiothreitol e eketsehileng ea 5 mM ho emisa karabelo.Lokisetsa lifaha tse ka bang 100 µl NeutrAvidin agarose (Thermo, #29202) bakeng sa sampole ka 'ngoe ka ho hlatsoa makhetlo a 3 ka 1 ml PBS.Tharollo e ka holimo ea proteome e ile ea hlapolloa ka 5 ml PBS 'me ea kenngoa ka lifaha tsa agarose tse hlatsoitsoeng pele tsa NeutrAvidin lihora tse 4 mocheso oa kamore.Joale lifaha li ne li hlatsuoa ka makhetlo a 3 ka 5 ml PBS e nang le 0.2% SDS (Sangon, # A600485), makhetlo a 3 le 5 ml PBS e nang le 1M urea, le linako tse 3 tse nang le 5 ml ddH2O.Joale lifaha li ile tsa kotuloa ka centrifugation 'me tsa emisoa hape ka 200 μl ea 25 mM ABC e nang le 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) le 20 ng/μl trypsin (Promega, #V5280).Trypsinize bosiu bo le bong ho 37°C ka ho potoloha.Karabelo e ile ea emisoa ka ho eketsa formic acid (Thermo, # A117-50) ho fihlela pH e fihla ho 2-3.Lifaha li ne li hlatsuoa ka makhetlo a 3 ka 1 ml ea PBS e nang le 0.2% SDS, makhetlo a 3 ka 1 ml ea PBS e nang le 1 M urea, ebe ka makhetlo a 3 ka 1 ml ea metsi a silafetseng.Li-peptide tse fetotsoeng li ile tsa lokolloa ke light lysis (365 nm) bakeng sa 90 min ho sebelisa 200 μl ea 70% MeOH.Ka mor'a centrifugation, supernatant e ile ea bokelloa.Lifaha li ile tsa hlatsuoa hang ka 100 μl ea 70% MeOH 'me li-supernatant li ile tsa kopanngoa.Mehlala e ile ea omisoa ka har'a "Speedvac vacuum concentrator" 'me ea bolokoa ho -20 ° C ho fihlela ho hlahlojoa.
Ho tseba le ho lekanya li-peptide tse fetotsoeng tsa oksijene e le 'ngoe, lisampole li ile tsa qhaloa hape ka 0.1% formic acid le 1 μg ea li-peptide li ile tsa hlahlobjoa ho sebelisoa spectrometer ea Orbitrap Fusion Lumos Tribrid e nang le mohloli oa nano ESI ho tsoa ho Tune le Xcalibur ho tsoa ho software ea barekisi 4.3.Mehlala e ne e arotsoe ka kholomo ea 75 µm × 15 cm e kentsoeng ka hare ea capillary e nang le lisebelisoa tsa 3 µm C18 (ReproSil-pur, #r13.b9.) 'me e hokahane le tsamaiso ea EASY-nLC 1200 UHPLC (Thermo).Li-peptide li ne li arotsoe ke linear 95 minute gradient chromatography ho tloha 8% solvent B ho ea 50% solvent B (A = 0.1% formic acid ka metsing, B = 0.1% formic acid ka 80% acetonitrile), ebe e nyolohela ho 98% B min. ka metsotso e 6 ka lebelo la phallo ea 300 nl / min.Orbitrap Fusion Lumos e bokella data ka ho fapanyetsana lipakeng tsa scan e feletseng ea MS le MS2 scan ho itšetlehile ka lintlha.Matla a sputtering a ne a behiloe ho 2.1 kV mme mocheso oa capillary ea ion transport e ne e le 320 ° C.MS spectra (350-2000 m/z) e ile ea bokelloa ka qeto ea 120,000, AGC 4 × 105, le nako e ngata ea ho kenya 150 ms.Li-precursors tse 10 tse tloaelehileng tse atisang ho qosoa ka sekhahla se seng le se seng se felletseng li ile tsa aroloa ho sebelisoa HCD e nang le matla a tloaelehileng a ho thulana a 30%, fensetere ea ho itšehla thajana ea quadrupole ea 1.6 m/z, le resolution ea 30,000.Sepheo sa AGC bakeng sa tandem mass spectrometry e sebelisa 5×104 le boholo ba nako ea ho kenya 150 ms.Mokhelo o matla o behiloe ho metsotsoana e 30. Li-ion tse sa abeloang kapa tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS. Li-ion tse sa abeloang kapa tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Li-ion tse sa abeloang kapa li-ion tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa 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. Li-ion kapa li-ion tse sa hlalosoang tse nang le litefiso tsa 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS.
Lintlha tse tala li sebetsoa ho sebelisoa sethala sa komporo sa FragPipe se thehiloeng ho MSFragger.Maemo a mangata le li-amino acid tse tsamaellanang li ne li ikemiselitse ho sebelisa algorithm ea ho batla e bulehileng e nang le mamello ea boima ba precursor ea -150 ho 500 Da.Joale li-peptide tse fetotsoeng li ile tsa tsejoa ho sebelisoa liphetoho tsa histidine ka phaello ea boima ba +229.0964 le +247.1069 Da ho PD (Proteome Discoverer 2.5, Thermo).
Lisele tse hlalosang ka mokhoa o tsitsitseng mofuta oa miniSOG o kopantsoeng li kentsoe ka lijana tsa 6 cm.Ha li fihla ~ 80% confluence, lisele li ile tsa hlatsuoa hang ka HBSS (Gibco, #14025092), ebe li kenngoa ka lik'hemik'hale tsa lik'hemik'hale ho HBSS bakeng sa hora ea 1 ho 37 ° C 'me li khantšitsoe ka leseli le leputsoa.10W LED bakeng sa metsotso e 20 mocheso oa kamore.Ho fumana hore na ke mofuta ofe oa mefuta ea oksijene e sebetsang e amehang ho PDPL, 0.5 mM vithamine 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 e ile ea kenngoa liseleng e le li-supplement.Ka mor'a ho hlatsoa ka PBS e batang, lisele li ile tsa hlajoa, li bokelloa ka har'a li-tubes tsa 1.5 ml centrifuge, 'me tsa sonicated ka ntlha ea 1 min ho 200 μl ea PBS e nang le 1x protease inhibitor ntle le EDTA (1 s le 1 s ntle le, amplitude 35%).Motsoako o hlahisoang o ne o le centrifuged ho 15,871 × g bakeng sa 10 min ho 4 ° C 'me mahloriso a supernatant a fetotsoe ho 1 mg / mL ho sebelisa lisebelisoa tsa tlhahlobo ea protheine ea BCA.Hoo e ka bang 50 µl ea lysate e ka holimo e ne e kenngoa ka 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, le 1 mM CuSO4 bakeng sa hora e 1 mocheso oa kamore ka ho potoloha ho tloha tlase ho ea holimo.Kamora karabelo ea ho tobetsa, pula e nang le acetone e ile ea etsoa ka ho kenyelletsa 250 μl ea acetone e hatselitsoeng pele ho lisampole, e kenella ho -20 ° C bakeng sa metsotso e 20 le centrifuging ho 6010 × g bakeng sa metsotso e 10 ho 4 ° C.Bokella pellet 'me u belise ka 50 µl ea 1x Buffer ea Laemmli bakeng sa metsotso e 10 ka 95 °C.Joale lisampole li ile tsa hlahlobjoa ho li-gel tse telele tsa SDS-PAGE 'me tsa bonoa ho sebelisoa Bio-rad ChemiDoc MP Touch imaging system e nang le software ea Image Lab Touch.
Polelo le tlhoekiso ea protheine ea recombinant miniSOG-6xHis e entsoe joalo ka ha ho hlalositsoe pejana.Ka bokhutšoanyane, lisele tsa E. coli BL21(DE3) (TransGen, #CD701-02) li ile tsa fetoloa ka pET21a-miniSOG-6xHis le polelo ea protheine e ile ea hlahisoa ka 0.5 mM IPTG (Sangon, #A600168).Ka mor'a cell lysis, liprotheine li ile tsa hloekisoa ho sebelisoa lifaha tsa Ni-NTA agarose (MCE, no. 70666), dialyzed khahlanong le PBS, 'me li bolokiloe ho -80 ° C.
Bakeng sa tlhahlobo ea proximity ea anti-label e thehiloeng ho in vitro, kopanya 100 μM purified miniSOG, 1 mM probe 3, le 1 μg anti-label mouse monoclonal antibody (TransGen, #HT501-01) ho PBS ho kakaretso ea karabelo ea bophahamo ba 50 μl..Motsoako oa karabelo o ne o khantšitsoe ka leseli le leputsoa la LED bakeng sa 0, 2, 5, 10, le 20 min mocheso oa kamore.Motsoako ona o ile oa tšeloa ka 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, le 1 mM CuSO4 bakeng sa 1 h mocheso oa kamore ho shaker e tsamaeang holimo.Ka mor'a karabelo e potlakileng, eketsa buffer ea 4x Laemmli ka ho toba motsoako 'me u belise ka 95 ° C bakeng sa 10 min.Mehlala e ile ea hlahlojoa ka li-gel tsa SDS-PAGE 'me tsa hlahlojoa ke blotting e ka bophirimela ka streptavidin-HRP (1: 1000, Solarbio, #SE068).
Peptide ea maiketsetso e nang le histidine e nang le C-terminal amidation (LHDALDAK-CONH2) e ile ea sebelisoa ho sekaseka leibole ea in vitro e haufi le peptide.Tekong ena, 100 μM e hloekisitsoeng miniSOG, 10 mM probe 3 le 2 μg / ml peptide ea maiketsetso e ne e kopantsoe PBS ka kakaretso ea karabelo ea 50 μl.Motsoako oa karabelo o ne o khantšitsoe ka leseli le leputsoa la LED bakeng sa hora ea 1 mocheso oa kamore.Microliter e le 'ngoe ea sampole e ile ea hlahlojoa ho sebelisoa sistimi ea LC-MS (Metsi, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer ka MassLynx spectrum analysis software).
Lisele tsa HEK293T tse hlalosang ka mokhoa o tsitsitseng mofuta oa fusion ea miniSOG li ile tsa jaloa ka lijana tsa 10 cm bakeng sa mela e nang le mefuta e fapaneng ea organelle localization (Mito, ER, Nucleus) le lijana tsa 15 cm bakeng sa mela ea Parkin-miniSOG le BRD4-miniSOG.Ha li fihla ~ 90% confluence, lisele li ile tsa hlatsuoa hang ka HBSS, ebe li kenngoa ka probe 3 ho HBSS bakeng sa hora ea 1 ho 37 ° C le ho khantša ka 10 W LED e putsoa mocheso oa kamore.Bakeng sa ho ngolisoa ha Parkin ntle le khokahanyo, 10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) e nang le probe 3 ho HBSS e kentsoe hora e le 1 ka 37°C.The cell lysis, tobetsa k'hemistri, phokotso le alkylation mehato e ne e tšoana le e hlalositsoeng ka holimo, ntle le hore 2 mg ea lysate e ile ea eketsoa 'me biotin PEG3 azide e ne e sebelisoa ho tobetsa karabelo ho e-na le photodegradable biotin azide.Ka mor'a ho ntlafatsa, lifaha li ile tsa hlatsuoa ka makhetlo a 3 ka 5 ml ea PBS e nang le 0.2% SDS, makhetlo a 3 ka 5 ml ea PBS e nang le 1 M urea, le linako tse 3 ka 5 ml ea PBS.Kamora moo, 2 µg trypsin e ile ea eketsoa ho 300 µl 25 mM ABC e nang le 1 M urea ho petsola protheine ka bosiu bo le bong ho 37°C.Karabelo e ile ea emisoa ka ho eketsa formic acid ho fihlela pH ea 2-3 e fihletsoe.Kamora ho etsa trypsinization holim'a lifaha, tharollo ea peptide e ile ea hlakoloa ho sebelisoa kholomo ea SOLAµ HRP (Thermo, #60209-001) mme ea omisoa ka har'a sekontiri sa Speedvac vacuum.Li-peptide li ile tsa qhaloa hape ka 0.1% ea formic acid le 500 ng ea li-peptide li ile tsa hlahlojoa ho sebelisoa Orbitrap Fusion Lumos Tribrid mass spectrometer e nang le mohloli oa nano-ESI o hlalositsoeng ka holimo.Li-peptide li ne li arohane ka li-precolumns tsa RP-HPLC tsa khoebo (75 μm x 2 cm) (Thermo, no. 164946) le litšiea tsa RP-HPLC tsa tlhahlobo (75 μm x 25 cm) (Thermo, no. 164941), ka bobeli li tletse 2 μm.gradient ho tloha ho 8% ho ea ho 35% ACN ka metsotso ea 60, ebe e eketseha ka mokhoa o tloaelehileng ho ea ho 98% B ka metsotso ea 6 ka tekanyo ea phallo ea 300 Nl / min.MS spectra (350-1500 m/z) e ile ea bokelloa ka qeto ea 60,000, AGC 4 × 105, le nako e ngata ea ho kenya 50 ms.Li-ion tse khethiloeng li ile tsa aroloa ka tatellano ke HCD ka lipotoloho tsa 3s ka matla a tloaelehileng a ho thulana a 30%, fensetere ea quadrupole e arohaneng ea 1.6 m/z, le qeto ea 15000. Sepheo sa AGC sa 5 × 104 tandem mass spectrometer le nako e ngata ea ente. ea 22 ms e ile ea sebelisoa.Ho kenyelletsoa ka matla ho behiloe ho metsotsoana e 45. Li-ion tse sa abeloang kapa tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS. Li-ion tse sa abeloang kapa tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Li-ion tse sa abeloang kapa li-ion tse nang le tefiso ea 1+ le > 7+ li ile tsa hanoa bakeng sa 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. Li-ion kapa li-ion tse sa hlalosoang tse nang le litefiso tsa 1+ le > 7+ li ile tsa hanoa bakeng sa MS/MS.
Mehato ea ho lokisetsa mehlala ho fihlela ho ntlafatsoa ha lihlahisoa tsa NeutrAvidin li ne li tšoana le tlhahlobo ea LC-MS / MS e hlalositsoeng ka holimo.Hoo e ka bang 50 μg ea lysate e ile ea sebelisoa e le ho kenya letsoho bakeng sa taolo ea ho laela 'me 2 mg ea lysate e ne e sebelisetsoa ho arabela ka ho tobetsa.Ka mor'a ho ntlafatsa le ho hlatsoa ka neutravidin, liprotheine tse tlanngoeng li ile tsa tlosoa ka ho eketsa 50 μl ea "buffer" ea Laemmli ho lifaha tsa resin ea agarose le ho belisoa ka 95 ° C. metsotso e 5.Taolo ea ho kenya letsoho le lisampole tse ntlafalitsoeng tsa lifaha li ile tsa hlahlojoa ke SDS-PAGE mme tsa fetisetsoa ho li-membrane tsa PVDF (Millipore, #ISEQ00010) ka mekhoa e tloaelehileng ea Western blot.Li-membrane li ne li thibiloe ka 5% skim milk (Sangon, #A600669) ho TBS e nang le 0.1% pakeng tsa 20 (TBST) le ho kenngoa ka tatellano ka li-antibodies tsa mantlha le tsa bobeli.Li-antibodies tsa mantlha li ile tsa hlapolloa ka 1:1000 ho 5% ea lebese la skim ho TBST 'me tsa kenngoa bosiung ba 4°C.Li-antibodies tsa bobeli li ne li sebelisoa ka karo-karolelano ea 1: 5000 'me li kenngoa ka hora e le 1 mocheso oa kamore.Li-membrane li ile tsa bonoa ke chemiluminescence ho sebelisa mokhoa oa ho nahana oa Chemidoc MP.Lits'oants'o tsohle tse sa khaotsoang tsa li-blots le li-gels setšoantšong li hlahisoa e le data e tala.
Li-antibodies tsa mantlha tse sebelisitsoeng thutong ena li kenyelelitse 'mutla anti-SFPQ monoclonal antibody (CST, no. 71992), mmutla anti-FUS monoclonal antibody (CST, no. 67840), mmutla anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1-- AP), anti-mSin3A polyclonal antibody (Abcam, #ab3479), mouse anti-tag monoclonal antibody (TransGen, #HT201-02), mouse anti-β-actin monoclonal antibody (TransGen, #HC201-01), anti-rabbit antibody -CDK2 monoclonal antibody (ABclonal, #A0094), rabbit monoclonal antibody to CTBP1 (ABclonal, #A11600), rabbit polyclonal antibody to DUT (ABclonal, #A2901), rabbit polyclonal antibody to PSMC4 (2 anti-anti-Abclonal), #Abclonal-5 DNAJB1 polyclonal antibody (ABclonal, # A5504).Li-antibodies tsena li ile tsa sebelisoa ka 1:1000 dilution ho 5% ea lebese la skim ho TBST.Li-antibodies tsa bobeli tse sebelisitsoeng thutong ena li ne li kenyelletsa anti-rabbit IgG (TransGen, #HS101-01), anti-mouse IgG (TransGen, #HS201-01) ka 1: 5000 dilution.
Ho batlisisa ka ho eketsehileng hore na BRD4 e sebelisana le SFPQ, lisele tse tsitsitseng tsa HEK293T le BRD4-miniSOG tse fetang HEK293T li ne li kenngoa ka lijana tsa 10 cm.Lisele li ile tsa hlatsuoa ka PBS e batang 'me tsa lysed ka 1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) ka EDTA-free protease inhibitor bakeng sa metsotso e 30 ho 4°C.Ka mor'a moo, li-lysates li ile tsa bokelloa ka 1.5 ml centrifuge tubes le centrifuged ho 15,871 xg bakeng sa 10 min ho 4 ° C.Supernatant e ile ea kotuloa 'me ea kenngoa ka 5 µg ea anti-V5 e ngotsoeng mouse monoclonal antibody (CST, #80076) bosiung bo bong ka 4°C.Hlatsoa hoo e ka bang 50 µl ea lifaha tsa makenete tsa protheine A/G (MCE, #HY-K0202) habeli ka PBS e nang le 0.5% Tween-20.Joale lisele tsa lysate li ne li kenngoa ka lifaha tsa makenete bakeng sa lihora tse 4 ho 4 ° C ka ho potoloha ho tloha tlase ho ea holimo.Ebe lifaha li ne li hlatsuoa ka makhetlo a mane ka 1 ml ea PBST buffer 'me li phehiloe ka 95 ° C bakeng sa 5 min.Mehlala e ile ea hlahlojoa ho li-gel tsa SDS-PAGE 'me tsa fetisetsoa ho li-membrane tsa PVDF ho sebelisoa mekhoa e tloaelehileng ea Western blot.Li-membrane li ne li koetsoe ka 5% ea lebese la skim ho TBST le ho kenngoa ka tatellano le li-antibodies tsa mantlha le tsa bobeli.Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) e ile ea sebelisoa ka karo-karolelano ea 1:1000 ho 5% lebese la skim ho TBST le ho kenngoa bosiu bo le bong ka 4 ° C.Anti-rabbit IgG e ne e sebelisoa ka karolelano ea 1: 5000 'me e kenngoa ka hora e le 1 mocheso oa kamore.Li-membrane li ile tsa bonoa ke chemiluminescence ho sebelisa mokhoa oa ho nahana oa Chemidoc MP.
Mehaho eohle e sebelisoang bakeng sa tlhahlobo ea Solvent Accessible Surface Area (SASA) e fumanoe ho Protein Data Bank (PDB)52 kapa AlphaFold Protein Structure Database53.SASA e felletseng e ballwe bakeng sa masalla ka nngwe ho sebediswa lenaneo la FreeSASA.Ke feela data e felletseng le e sa hlakang ea SASA ea histidine e ngotsoeng le baahisani ba eona e sebelisitsoeng ho fumana SASA e bolelang bakeng sa sebopeho ka seng.Tekanyetso ea ho fumaneha ha solvent (RSA) bakeng sa histidine ka 'ngoe e ile ea baloa ka ho arola boleng bo felletseng ba SASA ka sebaka sa masala se ka khonehang se fumanehang ho solvent.Ka nako eo li-histidine kaofela li ne li khetholloa e le tse patehileng haeba RSA e bolelang e ne e le ka tlase ho 20%, ho seng joalo e pepesitsoe56.
Lifaele tse tala tse fumanoeng ka mokhoa oa DDA li ile tsa batlisisoa ho sebelisoa Proteome Discoverer (v2.5) kapa MSfragger (Fragpipe v15.0) sebakeng se nepahetseng sa polokelo ea protheine e netefalitsoeng ea SwissProt e nang le litšila tse tloaelehileng.Li-peptide li ne li hloka trypsin e felletseng e nang le libaka tse peli tse sieo tsa cleavage, carbamoyl methylation e le phetoho e tsitsitseng le methionine oxidation e le phetoho e matla.Mamello ea boima ba precursor le fragment e behiloe ho 10 ppm le 0.02 Da (MS2 Orbitrap), ka ho latellana. Likotlo tse silafetseng li ile tsa tlosoa, 'me liprotheine li ile tsa tlhotloa ho fumana tekanyo ea ho sibolloa ha bohata ea <1%. Likotlo tse silafetseng li ile tsa tlosoa, 'me liprotheine li ile tsa tlhotloa ho fumana tekanyo ea ho sibolloa ha bohata ea <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэффициент ложного обнаружения <1%. Likotlo tse silafetseng li ile tsa tlosoa 'me liprotheine tsa tlhotloa ho fana ka tekanyo ea ho lemoha e fosahetseng ea <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 ложных обнаружений <1%. Li-hits tse silafetseng li ile tsa tlosoa 'me liprotheine tsa tlhotloa ho finyella tekanyo e fosahetseng ea <1%.Bakeng sa tlhahlobo ea bongata ntle le tšebeliso ea lileibole, ho ile ha sebelisoa liprotheine tse tloaelehileng tse tsoang ho tse tharo tse pheta-phetoang.Tlhahlobo ea sebaka sa protheine ea subcellular e ile ea etsoa ho sebelisoa tlhahlobo ea Gene Ontology (GO) ho tloha ho DAVID Bioinformatics Resources, MitoCarta 3.0 le li-database tse hlophisitsoeng le ho hatisoa ke sehlopha sa Alice Ting.'Mapa oa seretse se chesang o ile oa fumanoa ho Perseus (v1.6.15.0). Liphetoho tsa bongata ba liprotheine li ile tsa lekoa bakeng sa bohlokoa ba lipalo-palo ho sebelisoa tlhahlobo ea mahlakoreng a mabeli, 'me li-protein tse otlang li ile tsa fumanoa ka phetoho e ngata> 2 (ntle le haeba ho boletsoe ka tsela e fapaneng) le p value <0.05. Liphetoho tsa bongata ba liprotheine li ile tsa lekoa bakeng sa bohlokoa ba lipalo-palo ho sebelisoa tlhahlobo ea mahlakoreng a mabeli, 'me li-protein tse otlang li ile tsa fumanoa ka phetoho e ngata> 2 (ntle le haeba ho boletsoe ka tsela e fapaneng) le 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. Liphetoho tsa liprotheine tse nang le liprotheine li ile tsa lekoa bakeng sa bohlokoa ba lipalo-palo ho sebelisoa tlhahlobo ea t-tailed e nang le mehatla e 'meli,' me lipapali tsa protheine li khetholloa ka phetoho ea litaba> 2 (ntle le haeba ho boletsoe ka tsela e 'ngoe) le boleng ba ap <0.05.1 双边使用 双边确定 的 的 说明 的 说明 的 的 和 和 的 的 和使用 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 中 中 命 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. Bohlokoa ba lipalo-palo ba liphetoho tse menahaneng tsa protheine bo ile ba lekoa ho sebelisoa tlhahlobo ea mehatla e 'meli,' me lipapali tsa protheine li ile tsa khethoa bakeng sa liphetoho tsa litaba> 2 (ntle le haeba ho boletsoe ka tsela e 'ngoe) le boleng ba p <0.05.Tlhahlobo ea tšebelisano ea protheine e entsoe ho sebelisoa tlhahlobo ea GO hammoho le database ea String.
Ho ile ha etsoa likopi tse tharo tsa baeloji ka liphetho tse tšoanang.Tlhahlobo ea lipalo-palo e entsoe ka GraphPad Prism (software ea GraphPad) le libaka tsa seretse se chesang li entsoe ka Perseus (v1.6.15.0).Ho bapisa lihlopha tsena tse peli, litekanyetso tsa p li ile tsa khethoa ho sebelisoa tlhahlobo ea t ea Moithuti e nang le mehatla e 'meli.Ke liprotheine tsa singleton feela tse bonts'itsoeng bonyane habeli sehlopheng sa liteko li kenyellelitsoe libakeng tsa seretse se chesang, 'me litekanyetso tse tsamaellanang tse sieo sehlopheng sa taolo li ile tsa nkeloa sebaka ke Perseus ho tloha kabong e tloaelehileng e le hore boleng ba p bo ka baloa.Li-barbar tsa liphoso li emela moelelo oa ± kheloha e tloaelehileng.Litlhahlobong tsa proteomic bakeng sa tlhahlobo ea lipalo-palo, bongata ba liprotheine tse hlahileng bonyane likopi tse peli tsa tlhaho li ile tsa bolokoa.Mekhoa ea lipalo-palo ha ea sebelisoa ho tseba esale pele boholo ba sampole.Liteko ha lia iketsahalla feela.Bafuputsi ba ne ba sa bone mesebetsi nakong ea liteko le tlhahlobo ea liphetho.
Bakeng sa tlhaiso-leseling e batsi mabapi le moralo oa thuto, bona Tlaleho ea Patlisiso ea Tlhaho e hokahaneng le sengoloa sena.
Lintlha tsa spectrometry tse ngata tse fumanoeng thutong ena li rometsoe ho ProteomeXchange Consortium ka polokelo ea molekane oa iProX57 tlas'a dataset ID PXD034811 (PDPL-MS dataset).Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.Sengoliloeng sena se fana ka lintlha tsa mantlha.
Gingras, AC, Abe, KT & Raught, B. Ho tseba tikoloho: ho sebelisa biotinylation e itšetlehileng ka proximity ho khetholla li-protein complexes le li-organelle tsa 'mapa. Gingras, AC, Abe, KT & Raught, B. Ho tseba tikoloho: ho sebelisa biotinylation e itšetlehileng ka proximity ho khetholla li-protein complexes le li-organelle tsa 'mapa.Gingras, AS, Abe, KT le Raut, B. Ho tloaelana le tikoloho: ho sebelisa biotinylation e itšetlehileng ka haufi ho khetholla li-protein complexes le li-organelle tsa 'mapa. 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. Ho utloisisa tikoloho: sebelisa ho itšetleha ha boahelani ka bophelo ba likokoana-hloko.Gingras, AS, Abe, KT le Raut, B. Ho utloisisa haufi: ho khetholla liprotheine tse rarahaneng le 'mapa oa li-organelles ho sebelisa biotinylation e itšetlehileng ka proximity.Hona joale.Maikutlo a ka.Lik'hemik'hale.baeloji 48, 44–54 (2019).
Geri, JB le ba bang.Ho etsa 'mapa oa tikoloho e nyane ka ho fetisetsa matla a Dexter ho lisele tsa 'mele.Saense 367, 1091–1097 (2020).
Hertling, EL le ba bang.Marang-rang a li-proteome tse peli a lemoha phetoho e khethehileng ea lisele tsa interactome ea motho.Lisele 184, 3022–3040.e3028 (2021).
Nako ea poso: Sep-15-2022
