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Iindlela ze-Enzymatic proximity labeling ezisekelwe kwii-esters ezisebenzayo okanye i-phenoxy radicals zisetyenziswa ngokubanzi ukwenza imephu ye-subcellular proteoms kunye ne-protein interactors kwiiseli eziphilayo.Nangona kunjalo, i-esters esebenzayo ayisebenzi kangako, okukhokelela kwirediyasi yokuleyibhile ebanzi, kwaye i-phenoxy radicals eveliswa ngonyango lweperoxide inokuphazamisa iindlela ze-redox.Apha sichaza indlela esondele yokubhala i-photoactivation (PDPL) ephuhliswe ngofuzo oludibanisa i-miniSOG photosensitizer protein kwiprotein enomdla.Ukuqaliswa kukukhanya okuluhlaza kunye nokulawulwa lixesha lokuvezwa, ioksijini ye-singlet iyenziwa kwaye emva koko ukuleyibheli okusonjululwe ngokwesithuba seentsalela ze-histidine nge-aniline probe kuphunyeziwe.Sibonisa ukuthembeka kwayo okuphezulu ngokusebenzisa i-organelle-specific proteome mapping.Uthelekiso lwecala necala lwe-PDPL kunye ne-TurboID lubonisa i-proteomic coverage ethe ngqo kunye nebanzi ye-PDPL.Emva koko, safaka isicelo se-PDPL kwi-coactivator ye-transcriptional ehambelana nesifo i-BRD4 kunye ne-E3 Parkin ligase kwaye sifumene abadibanisi abangaziwa ngaphambili.Ngokujonga ngokugqithisileyo, ii-substrates ezimbini ezingaziwayo, i-Ssu72 kunye ne-SNW1, zichongiwe i-Parkin, ukuthotywa kwayo kuhlanganiswe yindlela ye-ubiquitination-proteasome.
Ukuchazwa ngokuchanekileyo kothungelwano lweprotheyini kusisiseko seenkqubo ezininzi ezisisiseko zeselula.Ke ngoko, imephu echanekileyo ye-spatiotemporal echanekileyo yokunxibelelana kweeprotheyini iya kubonelela ngesiseko semolekyuli yokucacisa iindlela zebhayoloji, izifo zesifo, kunye nokuphazamisa olu nxibelelwano ngeenjongo zonyango.Ukuza kuthi ga ngoku, iindlela ezikwaziyo ukufumanisa ukusebenzisana kwexeshana kwiiseli eziphilayo okanye izicubu zinqweneleka kakhulu.I-Affinity Purification Mass Spectrometry (AP-MS) isetyenziswe ngokwembali ukuchonga amaqabane abophezelayo kwiiprotheni ezinomdla (POIs).Ngokuphuhliswa kweendlela zeproteomics zobuninzi, i-Bioplex3.0 yadalwa, i-database enkulu yeeprotheyini zenethiwekhi ezisekelwe kwi-AP-MS.Nangona i-AP-MS inamandla kakhulu, i-cell lysis kunye ne-dilution amanyathelo ekuqhutyweni komsebenzi athambekele ekunxibelelaneni okubuthathaka kunye nokwexeshana kunye nokwazisa izinto zakudala ze-post-lysis ezifana nezibini zokusebenzisana ezikhohlisayo ezingenayo i-compartmentalization ngaphambi kwe-lysis.
Ukujongana nale miba, ii-amino acid ezingezizo ezendalo (UAA) ezinamaqela adibanisayo kunye namaqonga ee-enzymatic akufuphi okubhala ilebhile (PL) (umz. APEX kunye ne-BioID)5 aphuhlisiwe.Nangona indlela ye-UAA isetyenziswe ngempumelelo kwiimeko ezininzi kwaye inikezela ngolwazi malunga nokunamathiswa kweprotheyini ngokuthe ngqo, ukulungiswa kwendawo yokufakwa kwe-UAA kusafuneka.Okubaluleke ngakumbi, yindlela yokuleyibhela ye-stoichiometric eswele ukuguqulwa kwe-catalytic yeziganeko zokulebula.Ngokwahlukileyo, iindlela ze-enzymatic PL, ezifana nendlela ye-BioID, idibanisa i-biotin ligase eyenziwe ngobunjineli kwi-POI7, ethi emva koko isebenze i-biotin yenze i-biotinyl-AMP ester ephakathi esebenzayo.I-enzyme ke ngoko ivuselela kwaye ikhuphe i-biotin "ilifu" elisebenzayo elibhala iintsalela ze-lysine ezikufutshane.Nangona kunjalo, i-BioID idinga ngaphezu kweeyure ze-12 ukufumana uphawu olwaneleyo olubhaliweyo, oluthintela ukusetyenziswa kwayo kunye nesisombululo sexeshana.Ukusebenzisa i-evolution ekhokelwayo esekelwe kumboniso we-yeast, i-TurboID yenzelwe ngokusekelwe kwi-BioID ukuba isebenze ngakumbi, ivumela ukubhalwa ngokufanelekileyo nge-biotin ngaphakathi kwemizuzu ye-10, ivumela iinkqubo eziguquguqukayo ngakumbi ukuba zifundwe.Ngenxa yokuba i-TurboID isebenza kakhulu kwaye amanqanaba e-biotin e-endogenous anele ukuleyibhela okukwinqanaba elisezantsi, ukuleyibhelishwa okungasemva kuba yingxaki enokubakho xa uphuculo oluphezulu kunye nokufakwa kweelebhile ngexesha kuyimfuneko ngokongezwa kwe-biotin yangaphandle.Ukongezelela, i-esters esebenzayo ayisebenzi kakuhle (t1/2 ~ 5 min), nto leyo inokukhokelela kwiradiyasi enkulu yokulebula, ngakumbi emva kokuzaliswa kweeprotheyini ezingabamelwane kunye ne-biotin 5. Ngenye indlela, ukuhlanganiswa kwemfuzo ye-engineered ascorbate peroxidase (okt biotin- i-phenol radicals kwaye ivumela ukubhalwa kweprotheyini ngaphakathi komzuzu omnye 9, 10. I-APEX isetyenziselwa ngokubanzi ukuchonga i-subcellular proteomes, i-membrane protein complexes, kunye ne-cytosolic signaling protein complexes11, 12. Nangona kunjalo, imfuno ye-peroxides ephezulu inokuchaphazela iiprotheni ze-redox okanye iindlela, eziphazamisayo. iinkqubo zeselula.
Ke, indlela entsha ekwaziyo ukuvelisa iintlobo ezisebenzayo ezibhalwe-radius zokucinezelwa ngokuchaneka okuphezulu kwendawo kunye nexeshana ngaphandle kokuphazamisa kakhulu iindlela zeselula iya kuba yongezwa okubalulekileyo kwiindlela ezikhoyo. Phakathi kweentlobo ezisebenzayo, i-oksijeni ye-singlett yavusa ingqalelo yethu ngenxa yobomi bayo obufutshane kunye ne-radius limited diffusion radius (t1/2 <0.6 µs kwiiseli)13. Phakathi kweentlobo ezisebenzayo, i-oksijeni ye-singlett yavusa ingqalelo yethu ngenxa yobomi bayo obufutshane kunye ne-radius limited diffusion radius (t1/2 <0.6 µs kwiiseli)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффу2м (t.3, 3/16) Phakathi kweefom ezisebenzayo, i-oksijeni ye-singlet yatsala ingqalelo yethu ngenxa yobomi bayo obufutshane kunye ne-radius limited diffusion radius (t1/2 <0.6 µs kwiiseli)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）而引起了我們的泥3。 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/зим2. Phakathi kweefom ezisebenzayo, i-oksijeni ye-singlet itsala ingqalelo yethu ngenxa yobomi bayo obufutshane kunye ne-radius limited diffusion (t1/2 <0.6 μs kwiiseli).I-oksijeni ye-singlet iye yaxelwa ukuba i-oxidize i-methionine, i-tyrosine, i-histidine kunye ne-tryptophan, iyenze i-polar 14,15 iqhotyoshelwe kwi-amine okanye i-thiol based probes16,17.Nangona i-oksijini ye-singlet isetyenziselwe ukulebhelisha i-subcellular compartment i-RNA, izicwangciso zokuphinda ziphinde ziphinde zibonise iimpawu ezikufutshane ze-POI zihlala zingaphononongwa.Apha, sibonisa iqonga elibizwa ngokuba yi-photoactivation-dependent proximity labeling (PDPL), apho sisebenzisa ukukhanya okuluhlaza okwesibhakabhaka ukukhanyisa ii-POIs ezidityaniswe ne-miniSOG photosensitizer kwaye siqalise isizukulwana se-oksijini esisodwa ukukhupha iintsalela ezikufutshane, kulandelwa lutshintsho oluqulathe i-amine ukuze oxidize iikhemikhali probes zibe iiseli eziphilayo eziphakathi..Siye savavanya iqela leekhemikhali probes ukwandisa ukucaciswa kwethegi kunye neendawo ezichongiweyo zohlengahlengiso kusetyenziswa ukuhamba komsebenzi ovulekileyo weproteomics.Uthelekiso lwecala necala lwe-PDPL kunye ne-TurboID lubonisa i-proteomic coverage ethe ngqo kunye nebanzi ye-PDPL.Sisebenzise le ndlela kwiimpawu ezikhethekileyo ze-organelle ze-subcellular proteome kunye nokuchongwa kweproteome ngokubanzi yamaqabane abophayo kwiprotheyini yokulawula i-epigenetic ehambelana nomhlaza we-BRD4 kunye nesifo se-Parkinson esinxulumene nesifo se-E3 ligase Parkin, eqinisekise zombini inethiwekhi eyaziwayo kunye nengaziwa yeprotheni. intsebenziswano..Ikhono le-PDPL ukuqaphela i-E3 substrates kwiiprotheyini ezinkulu zeprotheyini ibonisa imeko apho ukuqatshelwa kwezibophelelo ezingathanga ngqo kuyadingeka.Ii-substrates ezimbini ezingaziwayo ze-parkin mediated yi-ubiquitination-proteasome ziqinisekisiwe kwindawo.
Unyango lwe-Photodynamic (PDT) i-19 kunye ne-chromophore-assisted laser inactivation (CALI) 20, apho ukukhanya kwe-irradiation kunye ne-photosensitizers kuvelisa i-oksijini ye-singlett, inokwenza iiprotheni ezijoliswe kuyo zingasebenzi okanye zibangele ukufa kweeseli.Kuba ioksijini ye-singlet iyinto esebenza kakhulu enomgama wokusasazwa kwethiyori malunga ne-70 nm, i-oxidation enyiniweyo yendawo ejikeleze ifotosensitizer inokulawulwa.Ngokusekwe kolu luvo, sigqibe kwelokuba sisebenzise ioksijini ye-singlett ukufezekisa iilebhile ezikufutshane zeprotheyini kwiiseli eziphilayo.Siye saqulunqa indlela ye-PDPL ye-chemoproteomic ukuzalisekisa imisebenzi emine: (1) ukwenza i-catalyze isizukulwana se-oksijini ye-singlett esebenzayo efana ne-PL enzymatic approach;(2) ukubonelela ngeelebhile esonjululwe lixesha ekuqalisweni kokukhanya;(3) ngotshintsho (4) Kuphephe ukusebenzisa i-endogenous cofactors (efana ne-biotin) ukunciphisa imvelaphi, okanye sebenzisa ii-reagents eziphazamisayo zangaphandle (ezifana neeperoxides) ukunciphisa ukuvezwa kweeseli kuxinzelelo lokusingqongileyo.
Iifotosensitizers zingohlulwa zibe ziindidi ezimbini ezibandakanya ubunzima bemolekyuli encinci ye-fluorophore (umz. irose bengal, imethylene blue)22 kunye neeproteni ezincinci ezifakwe ngokwemfuza (umz. miniSOG, KillerRed)23.Ukufezekisa uyilo lwemodyuli, siphuhlise iqonga lokuqala le-PDPL ngokufaka iiprotheni ze-photosensitizer (PS) kwi-POI24,25 (Umfanekiso 1a).Xa ukhanyisa ukukhanya okuluhlaza okwesibhakabhaka, i-oksijini ye-singlett i-oxidizes proximal nucleophilic amino acid residues, okubangela ukuba i-polarity impolung eyi-electrophilic kwaye inokusabela ngakumbi nge-amine probe nucleophiles16,17.I-probe yenzelwe i-alkyne handle ukuvumela ukucofa i-chemistry kunye nokudiliza i-LC / MS / MS characterization.
Umzobo ocwangcisiweyo wokulebhelishwa kweeprotheyini eziyinkimbinkimbi ezilawulwa yi-miniSOG.Xa zibonakaliswe kukukhanya okuluhlaza, iiseli ezibonisa i-miniSOG-POI zivelisa ioksijini eyodwa, eguqula iiproteni ezisebenzisanayo kodwa zingabophelelanga iiproteni.Iimveliso eziPhakathi ze-photooxidation zibanjwa zireyibhile ze-amine chemical probe ukwenza i-covalent adducts.Iqela le-alkynyl kwi-chemistry probe livumela i-chemistry yokuchofoza ukutyebisa ngokutsalwa phantsi kulandelwa yi-LC-MS / MS quantitation.b Isakhiwo sekhemikhali se-amine probes 1-4.c Uhlahlelo lwejeli yefluorescent emeleyo yemitochondrial yasekhaya yeminiSOG-mediated proteomic markers usebenzisa iprobe 1-4 kunye nobungakanani obunxulumeneyo obusekwe kwijel densitometry.Umlinganiselo wesignali ukuya ngasemva weprobes yeekhemikhali wavavanywa kusetyenziswa imifuniselo yokulawula engalunganga ngaphandle kokukhanya okuluhlaza okwesibhakabhaka okanye ukusebenzisa iiseli ze-HEK293T ngaphandle kokuchazwa kwe-miniSOG.n = 2 iisampulu ezizimeleyo ngokwebhayoloji.Ichaphaza ngalinye limele i-biological replica.d Ukufunyaniswa kommeli kunye nobungakanani be-PDPL kusetyenziswa iprobe 3 ephuculweyo kubukho okanye ukungabikho kwezinto ezibonisiweyo zePDPL ezifana c.n = 3 iisampulu ezizimeleyo ngokwebhayoloji.Ichaphaza ngalinye limele i-biological replica.Imigca esembindini kunye nebhovu imele intsingiselo kunye ± ukutenxa okusemgangathweni.CBB: Coomassie Brilliant Blue.e I-confocal imaging ye-singlett oxygen enebala elibomvu le-Si-DMA.Isikali sebar: 10 µm.Imifanekiso ye-Gel kunye ne-confocal experiments zaphindwa ngokuzimeleyo ubuncinane kabini kunye neziphumo ezifanayo.
Siqale savavanya amandla e-photosensitizers aqolileyo miniSOG26 kunye ne-KillerRed23, echazwe ngokuzinzileyo kwi-HEK293T, ukulamla ukuleyibheli kwe-propargylamine yeproteome njengeprobe yekhemikhali (Fig. 1a eyongezelelweyo).Uhlalutyo lwe-Gel fluorescence lubonise ukuba ukulebhile kweproteome yonke kwaphunyezwa kusetyenziswa i-miniSOG kunye ne-irradiation yokukhanya okuluhlaza okwesibhakabhaka, ngelixa kungekho mveliso ibonakalayo yokulebhile yabonwa nge-KillerRed.Ukuphucula i-signal-background ratio, emva koko sivavanya isethi yeekhemikhali zeprobes eziqukethe i-aniline (i-1 kunye ne-3), i-propylamine (2), okanye i-benzylamine (4).Siye saqaphela ukuba iiseli ze-HEK293T ngokwazo zazinomqondiso ophezulu ongasemva xa kuthelekiswa nokukhanya okuluhlaza okwesibhakabhaka, mhlawumbi ngenxa ye-riboflavin photosensitizer engapheliyo, i-flavin mononucleotide (FMN) 27. I-Aniline-based based probes chemical probes 1 kunye ne-3 zinike ukuchaneka okungcono, kunye ne-HEK293T ebonisa ngokuzinzileyo i-miniSOG kwi-mitochondria ebonisa i> 8-fold-fold in signal for probe 3, ngelixa i-probe ye-2 isetyenziswe kwindlela ye-RNA-labeling CAP-seq kuphela ukubonisa ~ 2.5- Ukusonga kophawu lokwandisa, mhlawumbi ngenxa yeendlela ezahlukeneyo zokukhetha phakathi kwe-RNA kunye neprotheni (Umfanekiso 1b, c). I-Aniline-based based probes chemical probes 1 kunye ne-3 zinike ukuchaneka okungcono, kunye ne-HEK293T ebonisa ngokuzinzileyo i-miniSOG kwi-mitochondria ebonisa i> 8-fold-fold in signal for probe 3, ngelixa i-probe ye-2 isetyenziswe kwindlela ye-RNA-labeling CAP-seq kuphela ukubonisa ~ 2.5- Ukusonga kophawu lokwandisa, mhlawumbi ngenxa yeendlela ezahlukeneyo zokukhetha phakathi kwe-RNA kunye neprotheni (Umfanekiso 1b, c).I-Aniline-based based probes chemical probes 1 kunye ne-3 ibonise ukuchaneka okungcono: I-HEK293T, ebonisa ngokuzinzileyo i-miniSOG kwi-mitochondria, ibonisa ngaphezu kwe-8-fold-fold in signal for probe 3, ngelixa i-probe 2, isetyenziswe kwindlela yokubhala i-CAP-seq RNA, kuphela. ibonisa i- ~ 2.5-fold-fold signal ukwanda, mhlawumbi ngenxa yokukhethwa kwe-reactivity eyahlukeneyo phakathi kwe-RNA kunye neprotheni (Umfanekiso 1b, c).基于 苯胺苯胺 的 探针化学 和 3 和 更 的 的 特异性 特异性 特异性 特异性 线 线 粒体 中 中 中 中 中 中 中 中 中 中 稳定 的 方法 标记 标记 标记 I-CAQ 2.5-倍信号增加，可能是由于RNA 和蛋白贡之间的不同反应偏好（图1b，c）.基于 苯胺苯胺 的化学 探针 1 和 更 的 特异性, Hee293t 在 粒体 粒体 稳定 中 稳定 中 稳定 稳定 稳定 稳定稳定 增加增加 增加-倍信号增加，可能是由于RNAI-Aniline-based based probes chemical probes 1 kunye ne-3 yayineenkcukacha ezingcono, i-HEK293T ibonakaliswe ngokuzinzile i-miniSOG kwi-mitochondria, kwaye i-probe ye-3 yayinokwanda kwe-8-fold in signal, ngelixa i-probe ye-2 ye-CAP-seq ye-labeling ye-RNA ibonise kuphela i- ~ 2.5 -ukwanda kwe-fold.kwi-signal, mhlawumbi ngenxa yeendlela ezahlukeneyo zokusabela phakathi kwe-RNA kunye neprotheni (Umfanekiso 1b, c).Ukongezelela, i-probe ye-3 isomers kunye ne-hydrazine probes (i-probes 5, i-6, i-7) yavavanywa, iqinisekisa ukulungelelaniswa kwe-probe ye-3 (i-Supplementary Fig. 1b, c).Ngokufanayo, uhlalutyo lwe-gel fluorescence lubonise ezinye iiparameters zokulinga eziphuculweyo: i-irradiation wavelength (460 nm), i-chemical probe concentration (1 mM), kunye nexesha le-irradiation (i-20 min) (i-Supplementary Fig. 2a-c).Ukushiya nayiphi na inxalenye okanye inyathelo kwi-protocol ye-PDPL kubangele ukuguqulwa kwesignali ebalulekileyo kwimvelaphi (Umfanekiso 1d).Ngokucacileyo, iileyibhile zeprotheni zancitshiswa kakhulu kubukho be-azide ye-sodium okanye i-trolox, eyaziwa ngokucima i-oksijini eyodwa.Ubukho be-D2O, eyaziwa ngokuzinzisa i-oksijini ye-singlett, yongeza umqondiso wokulebula.Ukuphanda igalelo lezinye iintlobo ze-oksijini ezisebenzayo ekubhaleni iilebhile, i-mannitol kunye ne-vitamin C zongezwa ukuseka i-hydroxyl kunye ne-superoxide radical scavengers, ngokulandelanayo, i-18, i-29, kodwa ayizange ifunyanwe ukunciphisa ukubhalwa.Ukongezwa kwe-H2O2, kodwa kungekhona ukukhanya, akuzange kubangele ukuleyibheli (i-Supplementary Fig. 3a).I-Fluorescence singlet i-oxygen imaging kunye ne-Si-DMA probes iqinisekisile ubukho be-oksijini ye-singlett kwi-HEK293T-miniSOG ucingo, kodwa kungekhona kwi-original HEK293T wire.Ukongezelela, i-mitoSOX Ebomvu ayikwazanga ukubona ukuveliswa kwe-superoxide emva kokukhanyisa (Umfanekiso 1e kunye neFig.I-Cytotoxicity ye-PDPL yavavanywa kuquka i-irradiation yokukhanya okuluhlaza okwesibhakabhaka kunye neeprobes zekhemikhali, kwaye akukho cytotoxicity ebalulekileyo yabonwa (i-Supplementary Fig. 4a).
Ukuze sifunde indlela yokubhala kwaye sikwazi ukuchongwa kweproteomic yeeprotheyini zeprotheyini usebenzisa i-LC-MS / MS, kufuneka siqale sinqume ukuba yiyiphi i-amino acids eguqulwayo kunye ne-delta mass of probe labels.I-Methionine, i-histidine, i-tryptophan kunye ne-tyrosine ziye zaxelwa ukuba zitshintshwe yi-oksijeni ye-singlet14,15.Sidibanisa ukuhamba komsebenzi kwe-TOP-ABPP31 kunye nophendlo oluvulekileyo olungakhethi cala olunikezelwa yiFragPipe iqonga lekhompyuter elisekelwe kwi-MSFragger32.Emva kokuguqulwa kwe-oksijini ye-singlett kunye ne-chemical probe labeling, i-chemistry yokuchofoza yenziwa ngokusebenzisa ileyibhile yokunciphisa i-biotin equlethe i-linker cleavable, elandelwa yi-neutravidin yokwelula kunye ne-trypsin digestion.I-peptide eguquliweyo, isabopheleleke kwi-resin, i-photocleaved kuhlalutyo lwe-LC-MS / MS (Umfanekiso 2a kunye neDatha eyoNgezelelweyo 1).Inani elikhulu lohlengahlengiso lwenzekile kuyo yonke iproteome enemephu yepeptide engaphezulu kwe-50 (PSM) edwelisiweyo (Umfanekiso 2b).Okumangalisayo kukuba, siye sabona kuphela ukuguqulwa kwe-histidine, mhlawumbi ngenxa yokusebenza okuphezulu kwe-histidine ene-oxidized ukuya kwi-aniline probes kunezinye ii-amino acids.Ngokwendlela epapashiweyo ye-histidine oxidation nge-oksijeni eyodwa, i-21,33 i-delta-mass ecetywayo ye-229 Da ihambelana ne-adduct of probe 3 kunye ne-2-oxo-histidine emva kwe-oxidation ezimbini, ngelixa i-+247 Da yimveliso ye-hydrolysis. of +229 Da (Fig. 5 eyoNgezelelweyo).Uvavanyo lwe-spectrum ye-MS2 lubonise ukuthembeka okuphezulu kokuchongwa uninzi lwe-y kunye ne-b ions, kubandakanywa ukuchongwa kwee-ion zeqhekeza ezilungisiweyo (y kunye ne-b) (Umfanekiso we-2c).Uhlalutyo lomxholo wokulandelelana kwendawo ye-PDPL-modified histidines ibonakalise i-moderate motif ekhethwayo kwiintsalela ezincinci ze-hydrophobic kwiindawo ze-± 1 (i-Supplementary Fig. 4b).Ngokwe-avareji, i-1.4 histidines ichongiwe ngeprotheni nganye, kwaye iisayithi zezi ziphawuli zagqitywa ngummandla ofikelelekayo we-solvent (SASA) kunye nohlalutyo lokufumaneka kwe-solvent (RSA) (i-Supplementary Fig. 4c, d).
Ukuhamba komsebenzi okungakhethi cala ukufunda ukukhetha okushiyekileyo usebenzisa iFragPipe iqonga lecomputing elixhaswa nguMSFragger.Iikhonkco ezicalulwayo zisetyenziswa kwi-Click chemistry ukuvumela i-photocleavage yeepeptide ezilungisiweyo ukusuka kwi-streptavidin resin.Uphendlo oluvulekileyo lwaqaliswa ukuchonga uhlengahlengiso oluninzi, kunye neentsalela ezifanelekileyo.b Yabela ubunzima bohlengahlengiso olwenzeka kuyo yonke iproteome.Imephu yePeptide yePSM.c MS2 spectral annotation of histidine sites modified with probe 3. Njengomzekelo omeleyo, i-covalent reaction with probe 3 yongeza +229.0938 Da ukuya kwi-amino acid elungisiweyo.d Uvavanyo lokuguqula olusetyenziselwa ukuvavanya izimakishi zePDPL.I-PRDX3 (H155A, H225A) kunye ne-PRDX1 (H10A, H81A, H169A) yatshintshwa nge-plasmids ye-wild-type yokufumanisa i-anti-Flag.e I-peptide yokwenziwa yaphendulwa nge-miniSOG ecocekileyo phambi kophando lwe-3 kunye neemveliso ezihambelanayo kunye ne-Δm +247 kunye ne- +229 zaphawulwa kwi-spectrum ye-LC-MS.f In vitro protein-to-protein interactions imodeli kunye ne-miniSOG-6xHis-tag kunye ne-anti-6x yakhe ye-antibody.I-Antibiotin (i-streptavidin-HRP) kunye ne-anti-mouse uhlalutyo lwe-blot yaseNtshona ye-miniSOG-6xHis / anti-6xI-antibody complexes yakhe ebhalwe nge-probe 3, kuxhomekeke kwixesha lokuvezwa ekukhanyeni.Iileyibhile zeeproteni zomntu ngamnye zibonakaliswa kubunzima bemolekyuli obuhambelanayo: Itsheyini yokukhanya ye-LC antibody, ikhonkco elinzima le-HC antibody.Ezi zilingo ziphindwe ngokuzimeleyo ubuncinane kabini kunye neziphumo ezifanayo.
Ukuqinisekiswa kwe-biochemical yendawo yokulebula, i-PRDX3 kunye ne-PRDX1 echongwe nge-mass spectrometry yatshintshwa ukusuka kwi-histidine ukuya kwi-alanine kwaye ifaniswa nohlobo lwasendle kwiimvavanyo zokudluliselwa.Iziphumo zePDPL zibonise ukuba ukuguqulwa kwenguqu kunciphise kakhulu ukuleyibhile (Umfanekiso 2d).Ngeli xesha, ulandelelwano lwe-peptide oluchongiweyo kukhangelo oluvulekileyo lwadityaniswa kwaye lwaphendulwa kwi-vitro nge-miniSOG esulungekileyo phambi kwe-probe 3 kunye nokukhanya okuluhlaza okwesibhakabhaka, imveliso evelisayo ngokutshintsha okukhulu kwe +247 kunye +229 Da xa ifunyenwe yiLC-MS (Fig .2e).).Ukuvavanya ukuba ngaba iiprotheyini ezisebenzisanayo ezisondeleleneyo zinokubhalwa kwi-vitro ekuphenduleni ukwenziwa kwefoto ye-miniSOG, siye sayila i-assay yokusondela yokwenziwa ngokunxibelelana phakathi kwe-miniSOG-6xHis protein kunye ne-anti-His monoclonal antibody in vitro (Figure 2f).Kolu vavanyo, besilindele ukulebhelishwa okusondeleyo kwe-antibody enzima kunye namatyathanga alula nge-miniSOG.Enyanisweni, i-anti-mouse (ukuqaphela amatyathanga anzima kunye alula e-anti-6xHis-ebhalwe i-antibody) kunye ne-streptavidin Western blots ibonise i-biotinylation eqinile yamatyathanga anzima kunye alula.Ngokucacileyo, siqaphele i-miniSOG autobiotinylation ngenxa ye-tag ye-6xHis kunye ne-cross-links phakathi kwamatyathanga alula kunye namatyathanga anzima, anokuthi abhekiselele kwi-gap echazwe ngaphambili phakathi kwe-lysine kunye ne-2-oxo-histidine impendulo esondeleyo.Ukuqukumbela, sigqiba ekubeni i-PDPL iguqula i-histidine ngendlela exhomekeke kuyo.
Injongo yethu elandelayo yayikukubonakalisa i-subcellular proteome ukuvavanya ukuchaneka kokubhala kwi-situ.Ngoko ke, sibonise ngokuzinzileyo i-miniSOG kwi-nucleus, i-mitochondrial matrix, okanye i-membrane yangaphandle ye-ER yeeseli ze-HEK293T (Umfanekiso 3a).Uhlalutyo lwe-Gel fluorescence lubonise iibhendi ezininzi ezilebhile kwiindawo ezintathu ze-subcellular kunye neepatheni ezahlukeneyo zokulebula (Umfanekiso 3b).Uhlalutyo lwe-imaging ye-Fluorescence lubonise ukucaciswa okuphezulu kwe-PDPL (Umfanekiso we-3c).Ukuhamba komsebenzi we-PDPL kwalandelwa ngokucofa ngokucofa ngeedayi ze-rhodamine ukucacisa iiproteomes ezisezantsi zisebenzisa i-fluorescence microscopy, kunye neempawu zePDPL zahlanganiswa kunye ne-DAPI, i-mitochondrial trackers, okanye i-ER trackers, eqinisekisa ukuthembeka okuphezulu kwe-PDPL.Kwiindawo ezintathu ze-organelle, uthelekiso olusecaleni kwePDPL kunye neTurboID usebenzisa i-avidin western blot ibonise ukuba iPDPL yayibhalwe ngokuthe ngqo ngakumbi xa kuthelekiswa nolawulo lwabo.Ngaphantsi kweemeko ze-PDPL, iibhendi ezibhalwe ngaphezulu zavela, zibonisa iiprotheyini ezibhalwe nge-PDPL (i-Supplementary Fig. 6a-d).
umelo olucwangcisiweyo lwe-miniSOG-mediated organelle-specific proteome labeling.I-miniSOG ijolise kwi-matrix ye-mitochondrial ngokudibanisa kwi-N-terminal 23 amino acids yomntu COX4 (mito-miniSOG), i-nucleus nge-fusion ukuya kwi-H2B (nucleus-miniSOG), kunye ne-Sec61β ngecala le-cytoplasmic ye-ER membrane (ER-miniSOG) ).Iimpawu ziquka imifanekiso yejeli, i-confocal imaging, kunye ne-mass spectrometry.b Ijeli emele imifanekiso yeeprofayili ze-PDPL ezintathu ze-organelle.CBB Coomassie Brilliant Blue.c Imifanekiso yeconfocal emele iiseli ze-HEK293T ezibonisa ngokuzinzileyo i-miniSOG eneendawo ezahlukeneyo ze-subcellular ezichongwe li-antibody elibhalwe V5 (bomvu).Iimpawu ze-subcellular zisetyenziselwa i-mitochondria kunye ne-ER (eluhlaza).I-PDPL ye-workflow iquka ukufunyanwa kwe-miniSOG (yellow) ebizwa ngokuba yi-subcellular proteoms usebenzisa i-Cy3-azide click chemistry.Isikali sebar: 10 µm.d Iiplani ze-Volcanic ze-PDPL-tagged proteomes kwii-organelles ezahlukeneyo ezibalwe ngobungakanani obungabhalwanga (n = 3 imifuniselo yebhayoloji ezimeleyo).Uvavanyo loMfundi olunemisila emibini lusetyenziswe kwiziza zentaba-mlilo.Uhlobo lwasendle lwe-HEK293T lwasetyenziswa njengolawulo olubi. Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold ion intensity difference). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold ion intensity difference). Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold umehluko kwi-ion intensity).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). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> umehluko we-2-fold kumandla e-ionic).Iiprotheyini ezihambelanayo ezibalulekileyo kwi-HEK293T-miniSOG kodwa ezingabalulekanga kwi-HEK293T ziboniswa ngokuluhlaza.e Uhlalutyo lwento eyodwa yeeseti zedatha yeproteomic evela kwimifuniselo d.Inani elipheleleyo leeprotheyini eziphawulekayo kwi-organelle nganye (amachaphaza abomvu naluhlaza) iphawulwe phezulu.I-Histograms ibonisa iiprotheni ezibekwe kwi-organelles esekelwe kwi-MitoCarta 3.0, uhlalutyo lwe-GO kunye no-A. Ting et al.abantu.Ukwahlula iiseti zedatha ze-mitochondria, iinuclei, kunye ne-ER.Olu vavanyo lwaphindwa ngokuzimeleyo ubuncinane kabini ngeziphumo ezifanayo.Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Ukukhuthazwa yi-gel kunye neziphumo ze-imaging, i-label-free quantification yayisetyenziselwa ukulinganisa i-proteome echongiweyo kwi-organelle nganye (iDatha eyongezelelweyo 2).I-HEK293T engasulelwanga isetyenziswe njengolawulo olubi ukuthabatha iziphawuli ezingasemva. Uhlalutyo lweploti ye-volcano lubonise iiprotheni ezityetyisiweyo kakhulu (p <0.05 kunye> ne-2-fold ion intensity) kunye neeprotheni ze-singleton ezikhoyo kuphela kwimigca yokubonisa i-miniSOG (Umfanekiso we-3d obomvu kunye namachaphaza aluhlaza). Uhlalutyo lweploti ye-volcano lubonise iiprotheni ezityetyisiweyo kakhulu (p <0.05 kunye> ne-2-fold ion intensity) kunye neeprotheni ze-singleton ezikhoyo kuphela kwimigca yokubonisa i-miniSOG (Umfanekiso we-3d obomvu kunye namachaphaza aluhlaza). 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). Uhlalutyo lweploti ye-volcano lubonise iiprotheyini ezityetyisiweyo kakhulu (p <0.05 kunye> ne-2-fold ion intensity) kunye neeprotheni ezizimeleyo ezikhoyo kuphela kwimigca ye-miniSOG-ebonisa (umzobo 3d, amachaphaza abomvu naluhlaza).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).火山图 分析 显示 出 显着 的 的 的 蛋白质 的的 的的 的的 蛋白质 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). Uhlalutyo lweploti ye-volcano lubonakalise kakhulu iiprotheyini ezityetyisiweyo (p <0.05 kunye> ne-2x amandla e-ionic) kunye neeprotheni ezizimeleyo ezikhoyo kuphela kumgca wokubonisa i-miniSOG (amachaphaza abomvu kunye aluhlaza kwi-Fig. 3d).Ukudibanisa le datha, sichonge i-1364, 461, kunye ne-911 ngokwezibalo ezibalulekileyo zenyukliya, i-mitochondrial, kunye neeprotheni ze-membrane zangaphandle, ngokulandelelanayo.Ukuhlalutya ukuchaneka kwe-organelle-localized PDPL, sasebenzisa i-MitoCarta 3.0, i-Gene Ontology (GO) uhlalutyo, kunye no-A. Ting et al.isethi yedatha8 isetyenziselwa i-mitochondria, i-nucleus, kunye ne-ER ukuvavanya i-organelle ekhethekileyo yeeprotheni ezifunyenweyo, ezihambelana nokuchaneka kwe-73.4, 78.5, kunye ne-73.0% (Umfanekiso 3e).Ubume be-PDPL buqinisekisa ukuba i-PDPL sisixhobo esifanelekileyo sokuchonga i-organelle-specific proteoms.Ngokucacileyo, uhlalutyo lwe-subochondrial yeeprotheyini ze-mitochondrial ezichongiweyo zibonise ukuba iproteome ebanjiwe yayisasazwa kakhulu kwi-matrix kunye ne-membrane yangaphakathi (i-226 kunye ne-106, ngokulandelanayo), i-akhawunti ye-91.7% (362) yenani elipheleleyo leeprotheni ze-mitochondrial ezichongiweyo.inqanaba eliphezulu le-PDPL laqinisekiswa ngokongeziweyo (i-Supplementary Fig. 7a).Ngokufanayo, uhlalutyo lwe-subnuclear lubonise ukuba i-proteome ebanjwe yayisasazwa kakhulu kwi-nucleus, i-nucleoplasm, kunye ne-nucleolus (i-Supplementary Fig. 7b).Uhlalutyo lweproteomic yeNyukliya kunye ne-peptide yophawu lwenyukliya (3xNLS) lubonise ukuchaneka okufanayo kulwakhiwo lwe-H2B (Umfanekiso owongezelelweyo 7c–h).Ukumisela into ecacileyo yesiphawuli se-PDPL, i-lamin A yenyukliya iye yakhethwa njengomgibe we-POI7 obekwe ngokucace ngakumbi.I-PDPL ichonge i-36 kakhulu iiprotheni ezityetyisiweyo, apho iiprotheyini ze-12 (i-30.0% ezibandakanya i-lamin A) zibonakaliswe kakuhle i-lamin A iiprotheyini ezisebenzisanayo ezichazwe yi-String database, kunye nepesenti ephezulu kunendlela ye-BioID (iiprotheni ze-122) 28 ye-28. , 22.9 %) 7. Indlela yethu ichonge iiprotheyini ezimbalwa, mhlawumbi ngenxa yeendawo zokuleyibhile eziqingqiweyo, eziye zenziwa ukuba zenzeke ngeoksijini ye-singlett esebenzayo.Uhlalutyo lwe-GO lubonise ukuba iiproteni ezichongiweyo bezibekwe ikakhulu kwi-nucleoplasm (26), inwebu yenyukliya (10), inwebu yenyukliya (9), kunye nemingxuma yenyukliya (5).Ngokudibeneyo, ezi proteins zenyukliya-zendawo zibalwa kwi-80% yeeprotheyini ezityetyisiweyo, ezibonisa ngakumbi ukucaca kwePDPL (I-Supplementary Fig. 8a-d).
Emva kokuba siseke amandla e-PDPL ukwenza uphawu olusondeleyo kwii-organelles, siye savavanya ukuba iPDPL ingasetyenziselwa ukuhlalutya amaqabane abophezelayo e-POI.Ngokukodwa, siye safuna ukuchaza uhlalutyo lwe-PDPL yeeprotheyini ze-cytosolic, ezithathwa njengeethagethi ezinzima kune-membrane-localized counterparts ngenxa yendalo yazo enamandla kakhulu.I-bromodomain kunye ne-extraterminal (BET) iprotheni ye-BRD4 iye yatsala ingqalelo yethu ngendima yayo ephambili kwizifo ezahlukeneyo ze-35, i-36.I-complex eyenziwe yi-BRD4 yi-coactivator ye-transcript kunye nethagethi ebalulekileyo yonyango.Ngokulawula ukubonakaliswa kwezinto ze-c-myc kunye ne-Wnt5a zombhalo, i-BRD4 icingelwa ukuba iyona nto ibalulekileyo kwi-acute myeloid leukemia (AML), i-myeloma eninzi, i-lymphoma ye-Burkitt, umhlaza wekoloni kunye nezifo ezivuthayo37,38.Ukongeza, ezinye iintsholongwane zijolise kwi-BRD4 ukulawula ushicilelo lwentsholongwane kunye neselula, njenge-papillomavirus, i-HIV, kunye ne-SARS-CoV-236,39.
Ukwenza imephu yokusebenzisana kwe-BRD4 usebenzisa i-PDPL, sidibanise i-miniSOG kunye ne-N- okanye i-C-terminal isoform ye-BRD4 emfutshane.Iziphumo zeProteomic zibonakalise iqondo eliphezulu lokudityaniswa phakathi kolwakhiwo mbini (Umfanekiso owongezelelweyo 9a).I-proteome yenyukliya echongiweyo kunye ne-miniSOG-H2B igubungela i-77.6% yeeprotheni ezisebenzisana ne-BRD4 (i-Supplementary Fig. 9b).Emva koko, amaxesha ahlukeneyo okukhanyisa (i-2, i-5, i-10, i-20 min) yayisetyenziselwa ukulungelelanisa i-radius marker (Umfanekiso we-4a kunye nedatha eyongezelelweyo ye-3).Sigqibe kwelokuba kwiifoto zamaxesha amafutshane, iPDPL iza kubhala ngokuyintloko amaqabane abopha ngokuthe ngqo, ngelixa ixesha elide liza kubandakanya iiprotheyini ezichongiweyo ngexesha elifutshane lokufota kunye neethagethi ezingathanga ngqo kwiikhompleksi zokuleyibheli.Enyanisweni, sifumene ukugqithwa okunamandla phakathi kwamanqaku exesha elisondeleyo (i-84.6% ye-2 kunye ne-5 min; i-87.7% ye-5 kunye ne-10 min; i-98.7% ye-10 kunye ne-20 min) (umzobo 4b kunye ne-Supplementary Fig. 9c).Kuwo onke amaqela ovavanyo, asifumananga kuphela i-BRD4 yokuzibhala, kodwa iithagethi ezininzi ezaziwayo ezifana ne-MED1, CHD8, BICRA, NIPBL, SMC1A, kunye ne-HMGB1 echazwe kwisiseko sedatha yomtya.Amandla e-ionic kwezi thagethi alingana nexesha lokuvezwa (umzobo 4c kunye neFig. 9d).Uhlalutyo lwe-GO lweeprotheyini ezichongiweyo kwiqela le-2-minute lubonise ukuba iiprotheni ezichongiweyo zahlala kwi-nucleus kwaye zibandakanyeka kwi-chromatin remodeling kunye ne-RNA polymerase function.Umsebenzi we-molecular weprotheni wawutyetyiswe kwi-chromatin ebophezelayo okanye i-transcriptional coactivation, ehambelana nomsebenzi we-BRD4 (umzobo 4d).Uhlalutyo lokusebenzisana kweprotheyini ye-String database-enabled protein luveze inqanaba lokuqala lonxibelelwano olungathanga ngqo phakathi kwe-BRD4 kunye ne-HDAC yosapho olusebenzisanayo lwezakhiwo ezifana ne-SIN3A, NCOR2, BCOR, kunye ne-SAP130 (umzobo 4e kunye neFig. 9e eyoNgezelelweyo), ehambelana ne-BRD4 kunye ne-HDAC ebopha i-acetylated histones ..Ukongezelela, iithagethi ezimele ezichongiweyo yi-LC-MS / MS, kuquka i-Sin3A, i-NSUN2, i-Fus, kunye ne-SFPQ, iqinisekiswe yi-Western blotting (Fig. 4f).Kutshanje, i-isoform emfutshane ye-BRD4 iye yaxelwa ukuba yenze i-nuclei kunye neempawu zokuhlukaniswa kwesigaba se-liquid (LLPS).I-RNA ebopha iiprotheni i-Fus kunye ne-SFPQ zidibanisa i-LLPS yeenkqubo ezahlukeneyo zeselula kwaye zichongiwe apha njengeeprotheni ezibophezelayo ze-BRD4 ezingabhalwanga.Ukusebenzisana phakathi kwe-BRD4 kunye ne-SFPQ kwaqinisekiswa yi-co-immunoprecipitation (co-IP) imifuniselo (Figure 4g), icebisa enye indlela yokwahlulwa kwesigaba se-BRD4-mediated liquid-liquid phase efanele uphando olongezelelweyo.Zithathiwe kunye, ezi ziphumo zibonisa ukuba i-PDPL liqonga elifanelekileyo lokuchonga i-BRD4 eyaziwayo esebenzisanayo kunye neeprotheni ezibophezelayo ezingaziwayo.
ukumelwa kweSchematic ye-miniSOG-mediated BRD4 proximity marking, amaxesha okuvezwa: 2, 5, 10, kunye ne-20 min.b Ukudibana kweeprotheyini ezichongiweyo ngamaxesha ahlukeneyo okukhanyisa.Ukutyetyiswa kweprotheyini echongiweyo kwi-HEK293T-miniSOG-BRD4 yayibalulekile ngokwezibalo xa kuthelekiswa nohlobo lwasendle lwe-HEK293T.c Ukuqina kwe-Iyoni xa kubalwa ummeli ongabhalwanga owaziwayo nge-BRD4-ebopha iiproteni ngexesha elichaziweyo lokuvezwa.n = 3 iisampulu ezizimeleyo ngokwebhayoloji.Idatha inikezelwe njengentsingiselo ± ukutenxa okusemgangathweni.d Uhlalutyo lwe-Gene ontological (GO) lweeprotheni ezichongiweyo kwiqela lemizuzu emi-2.Amagama eGO alishumi okuqala adwelisiwe.Amaqamza anemibala ngokodidi lwegama elithi GO, kwaye ubukhulu beqamza bulingana nenani leeproteni ezifunyenwe kwikota nganye.e Uhlalutyo lwentambo yeeprotheni ezisebenzisana ne-BRD4.Izangqa ezityheli ziyiglue ethe ngqo kwaye izangqa ezingwevu ngumaleko wokuqala weglue engathanga ngqo.Imigca ebomvu imele ukusebenzisana okumiselwe ngokokulinga kunye nemigca eluhlaza okwesibhakabhaka imele intsebenziswano eqikelelweyo.f Iithagethi ezibophelelayo ezimele i-BRD4 ezichongwe kwi-LC-MS/MS zaqinisekiswa yi-Western blotting.g Imifuniselo ye-Co-immunoprecipitation iqinisekisa ukusebenzisana phakathi kwe-SFPQ kunye ne-BRD4.Ezi zilingo ziphindwe ngokuzimeleyo ubuncinane kabini kunye neziphumo ezifanayo.Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Ukongeza ekuchongeni iithagethi ezihambelana ne-POI ezingabhaliswanga, siqikelela ukuba i-PDPL iya kulungeleka ukuchonga i-substrates ye-enzymes, eya kufuna ukubonakaliswa kweeprotheni ezibophezelayo ezingathanga ngqo kwiikhompleksi ezinkulu ukuchasa ii-substrates ezingabhaliswanga.I-Parkin (encoded yi-PARK2) yi-E3 ligase kunye nokuguqulwa kwe-parkin kwaziwa ngokubangela isifo se-autosomal recessive juvenile Parkinson's disease (AR-JP)42.Ukongezelela, i-parkin ichazwe njengento ebalulekileyo kwi-mitophagy (i-mitochondrial autophagy) kunye nokususwa kweentlobo ze-oksijini ezisebenzayo.Nangona kunjalo, nangona ii-substrates ezininzi ze-parkin zichongiwe, indima ye-parkin kwesi sifo ayicacanga.Ukucacisa ii-substrates zayo ezingachazwanga, i-PDPL yavavanywa ngokongeza i-miniSOG kwi-N- okanye i-C-terminus ye-parkin.Iiseli ziphathwe nge-carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) ukuze isebenze i-parkin ngendlela ye-PINK1-Parkin.Xa kuthelekiswa neziphumo zethu ze-BRD4 PDPL, i-parkin N-terminus fusion ibonise isethi enkulu yeeprotheyini ezijoliswe kuyo, nangona yayigubungela inxalenye enkulu ye-C-terminus (177 ngaphandle kwe-210) (Umfanekiso 5a, b kunye neDatha eyoNgezelelweyo 4).Isiphumo siyahambelana neengxelo zokuba iithegi ze-N-terminal zinokuvula iParkin44 ngokungaqhelekanga.Okothusayo kukuba, bekukho iiproteni ezithe kratya ezili-18 kuphela kwidatha yethu eneziphumo ezipapashiweyo ze-AP-MS zeParkin43, mhlawumbi ngenxa yomahluko phakathi kwemigca yeeseli kunye nokuhamba komsebenzi weproteomics.Ukongeza kwiiprotheni ezine ezaziwayo (ARDM1, HSPA8, PSMD14, kunye ne-PSMC3) ezichongiweyo ngeendlela ezimbini (umzobo 5c) 43.Ukuqinisekisa ngakumbi iziphumo ze-LC-MS/MS, unyango lwe-PDPL kunye ne-Western blotting elandelayo zisetyenziselwe ukuthelekisa iziphumo ze-HEK293T yovavanyo lweseli yomzali kunye nomgca we-N-terminal parkin ozinzile.Iithagethi ebezingaziwa ngaphambili i-CDK2, i-DUT, i-CTBP1, kunye ne-PSMC4 zavavanywa ngesibophelelo esaziwayo, i-DNAJB1 (Umfanekiso 5d).
Iploti ye-volcano yeeprotheni ezisebenzisana ne-parkin kwiiseli ze-HEK293T ezine-miniSOG echazwe ngokuzinzileyo edityaniswe ne-N- okanye i-C-terminus ye-parkin (n = 3 imifuniselo yebhayoloji ezimeleyo).Uvavanyo loMfundi olunemisila emibini lusetyenziswe kwiziza zentaba-mlilo.I-HEK293T yasetyenziswa njengolawulo olubi. Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold ion intensity difference). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold ion intensity difference). Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> ne-2-fold umehluko kwi-ion intensity).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). Iiprotheyini ezitshintshileyo ngokuphawulekayo zigxininiswe kubomvu (p <0.05 kunye> umehluko we-2-fold kumandla e-ionic).Iiprotheyini ezihambelanayo ezibalulekileyo kwi-HEK293T-miniSOG kodwa ezingabalulekanga kwi-HEK293T ziboniswa ngokuluhlaza.b Umzobo we-Venn obonisa iiprotheni ezithelelanayo phakathi kwe-N-terminal kunye ne-C-terminal constructs.Iithegi ze-N-terminal zingenza i-parkin isebenze ngokungaqhelekanga kwaye ibangele iiprotheni ezibonakalayo ngakumbi.c Umzobo weVenn obonisa iiproteni ezithe kratya phakathi kwePDPL kunye ne-AP-MS.Abasebenzisanayo abaziwayo zidweliswe, kubandakanywa i-4 ye-18 yeeprotheni ezinqamlekileyo kunye ne-11 yeeprotheni ze-159 ezichongiweyo ngokukodwa kwi-PDPL.d Iithagethi zabameli ezichongwe yi-LC-MS/MS zaqinisekiswa yi-Western blotting.I-e Ssu72 kunye ne-SNW1 zachongwa njengeenxalenye zepaki ezingabhaliswanga.Ezi plasmids ze-FLAG-tagged protein zadluliselwa kwi-HEK293T kunye ne-HEK293T-Parkin-miniSOG elandelwa yi-CCCP yonyango ngamaxesha ahlukeneyo.Ukuthotywa kwakubonakaliswe ngakumbi kumgca weParkin overexpression line.f Ukusebenzisa i-proteasome inhibitor MG132, kwaqinisekiswa ukuba inkqubo yokuthotywa kwe-Ssu72 kunye ne-SNW1 ilawulwa yi-proteasome-ubiquitination.Ezi zilingo ziphindwe ngokuzimeleyo ubuncinane kabini kunye neziphumo ezifanayo.Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Ngokucacileyo, iiprotheyini ezichongiweyo yi-PDPL kufuneka zibandakanye iiprotheni ezibopha i-parkin kunye ne-substrates yazo.Ukubona ii-substrates ze-parkin ezingabhaliswanga, sikhethe iiprotheyini ezisixhenxe ezichongiweyo (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 kunye ne-SNW1) kunye neeplasmids ezitshintshiweyo ukuze ziveze ezi genes kwi-HEK293T eqhelekileyo kunye nokubonisa ngokuzinzileyo i-miniSOG-Parkin's unyango lwe-HEK293T.Amanqanaba e-Ssu72 kunye neeprotheni ze-SNW1 zancitshiswa kakhulu kumgca we-miniSOG-Parkin ozinzile (Umfanekiso 5e).Unyango nge-CCCP kwiiyure ze-12 kubangele ukuthotywa okuphawulekayo kwazo zombini ii-substrates.Ukuphanda ukuba ukuthotywa kwe-Ssu72 kunye ne-SNW1 kulawulwa yi-proteasome-ubiquitination, i-proteasome inhibitor MG132 yongezwa ukuvimbela umsebenzi we-proteasome, kwaye ngokwenene sifumene ukuba inkqubo yabo yokuthotywa yayinqatshelwe (umzobo 5f).Iithagethi ezongezelelweyo ezingekho phantsi kwe-substrate zaqinisekiswa njenge-Parkin interactors usebenzisa i-Western blotting (i-Supplementary Fig. 10), ebonise iziphumo ezihambelanayo kunye ne-LC-MS / MS.Ekugqibeleni, ukuhlanganiswa kwe-PDPL yokuhamba komsebenzi kunye ne-protein target transfection verification ivumela ukuchongwa kwee-substrates ze-E3 ze-ligase ezingabhaliswanga.
Siphuhlise iqonga lokumakisha elikufutshane elikuvumela ukuba uchonge kwindawo kunye nexesha elisebenzisana ne-POIs.Iqonga lisekelwe kwiprotheni ye-photosensitizer ye-miniSOG, emalunga ne-12 kDa kuphela, ngaphantsi kwesiqingatha sobukhulu be-enzyme ye-APEX2 evuthiweyo (27 kDa) kunye nesinye kwisithathu ubukhulu be-TurboID (35 kDa).Ubungakanani obuncinci kufuneka bandise kakhulu uluhlu lwezicelo zokufunda iiprotheyini ezincinci.Uphononongo olongezelelweyo lwe-photosensitizers eyongezelelweyo, nokuba ngaba iiprotheni ezifakwe kwi-genetically encoded okanye i-molecules ezincinci, ziyafuneka ukunyusa isivuno se-quantum ye-oksijini ye-singlett kunye nokwandisa uvakalelo lwale ndlela.Kuguqulelo lwangoku lwe-miniSOG, isisombululo esiphezulu sexeshana sinokufezekiswa ngokusebenzisa ukukhanya okuluhlaza okwesibhakabhaka ukuze kusebenze iimpawu ezikufutshane.Ukongeza, ixesha elide lokuvezwa likhuphe "ilifu" elikhulu le-oksijini ye-singlett, okukhokelela ekuguqulweni kweentsalela ze-histidine ezikude, ukwanda kweradiyasi yokubhala, kunye nokukwazi ukulungisa kakuhle isisombululo sendawo ye-PDPL.Siphinde savavanya iiprobes zemichiza ezisixhenxe ukunyusa umlinganiso wesignali ukuya ngasemva kwaye saphonononga indlela yeemolekyuli emva kwale ndlela.I-TOP-ABPP yokuhamba komsebenzi edibeneyo kunye nokukhangela okuvulekileyo okungakhethiyo kuqinisekisile ukuba ukuguqulwa kwenzeka kuphela kwi-histidines kwaye akukho microenvironment engaguqukiyo yabonwa ngokunyuka kokuguqulwa kwe-histidine, ngaphandle kokukhetha okuphakathi kwe-histidines kwingingqi ye-loop.
I-PDPL sele isetyenziselwe ukubonakalisa i-subcellular proteomes kunye ne-proteome ethile kunye ne-inshorensi ubuncinane enokuthelekiswa nezinye iilebhile ezikufutshane kunye neendlela ze-organelle-specific chemical probe.Iziphawuli zokusondela ziye zasetyenziswa ngempumelelo ukubonisa umphezulu, i-lysosomal, kunye ne-secretome-enxulumene neproteoms46,47.Sikholelwa ukuba i-PDPL iya kuhambelana nezi organelles ezincinci.Ukongezelela, siye sacela umngeni kwi-PDPL ngokuchonga iithagethi ze-cytosolic protein dinding eziyinkimbinkimbi kune-membrane eboshiwe iiprotheni ngenxa yeempawu zabo eziguquguqukayo kunye nokubandakanyeka kwintsebenziswano yexesha elide.I-PDPL isetyenziswe kwiiprotheni ezimbini, i-coactivator ye-transcriptal BRD4 kunye ne-ligase ehambelana nesifo i-E3 Parkin.Ezi proteni zimbini azikhethwanga kuphela kwimisebenzi yazo yebhayoloji esisiseko, kodwa nangokubaluleka kwazo kweklinikhi kunye namandla onyango.Kwezi POI zimbini, amahlakani abophayo awaziwayo kwakunye nezinto ekujoliswe kuzo ezingabhaliswanga ziye zachongwa.Ngokucacileyo, iprotein ye-protein ye-SFPQ enxulumene nesigaba yaqinisekiswa yi-co-IP, enokuthi ibonise indlela entsha apho i-BRD4 (i-isoform emfutshane) ilawula i-LLPS.Ngelo xesha, sikholelwa ukuba ukuchongwa kwe-Parkin substrates yimeko apho ukuchongwa kwe-adhesives engathanga ngqo kuyadingeka.Siye sachonga ii-parkin substrates ezimbini ezingaziwayo kwaye saqinisekisa ukuthotywa kwazo ecaleni kwendlela ye-ubiquitination-proteasome.Kutshanje, kuye kwaphuhliswa isicwangciso esisekwe kumatshini wokukhangela i-hydrolase substrates ngokubambisa ngee-enzymes.Nangona le ndlela inamandla kakhulu, ayifanelekanga ukuhlalutya i-substrates ebandakanyekayo ekubunjweni kwee-complexes ezinkulu kwaye ifuna ukubunjwa kwe-covalent bonds phakathi kwe-enzyme kunye ne-substrate.Silindele ukuba i-PDPL inokwandiswa ukuba ifunde ezinye iiprotheyini eziyinkimbinkimbi kunye neentsapho ze-enzyme, ezifana ne-deubiquitinase kunye neentsapho ze-metalloprotease.
Uhlobo olutsha lwe-miniSOG, olubizwa ngokuba yi-SOPP3, luye lwaphuhliswa ngokuphuculwa kwemveliso ye-oksijini ye-singlett.Siqhathanise i-miniSOG kunye ne-SOPP3 kwaye sifumene ukusebenza okuphuculweyo kokumakisha, nangona i-signal-to-noise ratio yahlala ingatshintshi (i-Supplementary Fig. 11).Siye saqikelela ukuba ukwenziwa ngcono kwe-SOPP3 (umzekelo, ngendaleko ethe ngqo) kuya kukhokelela kwiiproteni ezisebenzayo ze-photosensitizer ezifuna amaxesha amafutshane okukhanya kwaye ngaloo ndlela zivumele iinkqubo zeselula ezinamandla ngakumbi ukuba zibanjwe.Ngokucacileyo, inguqulelo yangoku ye-PDPL ilinganiselwe kwindawo yeselula njengoko ifuna ukukhanya okuluhlaza okwesibhakabhaka kwaye ayikwazi ukungena kwiithishu ezinzulu.Olu phawu luthintela ukusetyenziswa kwayo kwizifundo zemodeli yezilwanyana.Nangona kunjalo, indibaniselwano ye-optogenetics kunye ne-PDPL inokunika ithuba lophando lwezilwanyana, ngakumbi kwingqondo.Ukongeza, ezinye iifotosensitizer ezenziwe ngobunjineli ze-infrared nazo ziyawususa lo mda.Uphando luyaqhubeka ngoku kule ndawo.
Umgca weseli we-HEK293T wafunyanwa kwi-ATCC (CRL-3216).Umgca weseli uvavanywe ukuba awunayo intsholongwane ye-mycoplasma kwaye yakhuliswa kwi-DMEM (Thermo, #C11995500BT) yongezwa nge-10% ye-fetal bovine serum (FBS, Vistech, #SE100-B) kunye ne-1% penicillin / streptomycin (Hyclone, #SV30010).ukhule kwi.
I-3-aminophenylene (isampuli 3) kunye (4-ethynylphenyl)methanamine (isampulu yesi-4) zathengwa kwi-Bidepharm.Ipropylamine (probe 2) yathengwa kwi-Energy-chemicals.I-N-(2-Aminophenyl)pent-4-ynamide (probe 1) yenziwe ngokweendlela ezipapashiweyo.
ITheyibhile yoku-1 eyoNgezelelweyo idwelisa ulwakhiwo lwemfuzo olusetyenziswe kolu phononongo.I-miniSOG kunye ne-KillerRed ulandelelwano lwenziwe kwi-plasmid yesipho esivela kwi-P. Zou (iYunivesithi yasePeking).I-matrix ye-mitochondrial ejoliswe ngokulandelelana ithathwe kwi-23 N-terminal amino acids ye-COX4 kwaye ihlanganiswe kwiivectors ezibonisiweyo zisebenzisa indibano ye-Gibson (Beyotime, #D7010S).Ukujolisa kwi-membrane kunye ne-nucleus ye-endoplasmic reticulum, i-SEC61B i-DNA yomntu (NM_006808.3) (NEB, #M0491L) inyuswe yi-PCR kwilayibrari ye-cDNA yeeseli ze-HEK293T, kunye ne-H2B DNA (enikezelwe nguD. Lin, i-Shenzhen Bay Laboratory) kwaye yenziwe , njengoko kukhankanyiwe ngasentla.Ngaphandle kokuba kuboniswe ngenye indlela, ezinye iijini zeprotheyini ezisetyenziselwa ukudluliselwa kunye nokwakhiwa kwemigca yeeseli ezizinzileyo yayiyi-PCR eyandisiweyo ukusuka kwithala leencwadi le-cDNA ye-HEK293T.I-G3S (GGGS) kunye ne-G4S (GGGGS) zisetyenziswe njengezikhonkco phakathi kweprotheni ye-bait kunye ne-miniSOG.I-V5 epitope tag (GKPIPNPLLGLDST) yongezwa kolu lwakhiwo lwe-fusion.Ukubonakaliswa kwezilwanyana ezanyisayo kunye nokuseka umgca weseli ozinzileyo, ukwakhiwa kwe-miniSOG fusion kwafakwa kwi-pLX304 lentiviral vector.Ukubonakaliswa kwebhaktheriya, i-miniSOG yahlanganiswa kwi-pET21a vector ebhalwe 6xHis kwi-C-terminus.
Iiseli ze-HEK293T zahlwayelwa kwiiseli ze-2.0 x 105 kwiqula ngalinye kwiipleyiti ezinamaqula amathandathu kwaye zadluliselwa kwiiyure ze-24 kamva kunye ne-plasmids ye-lentiviral recombinant (2.4 μg pLX304) kunye ne-plasmids yokupakisha intsholongwane (1.5 μg psPAX2 kunye ne-1.2 μg00 ye-MD2 ye-pMD2) , #C0533), malunga ne-80% fusion.Emva kosulelo ngobusuku, unxibelelwano lwatshintshwa lwaza lwafukanyelwa ezinye iiyure ezingama-24.Ukuqokelela intsholongwane kwenziwa emva kwe-24, 48 kunye ne-72 iiyure.Ngaphambi kokusuleleka kwimizila yeeseli ezijoliswe kuyo, i-viral medium yahluzwa nge-0.8 μm filter (Merck, #millex-GP) kunye ne-polybrene (i-Solarbio, # H8761) yongezwa kwi-concentration ye-8 μg / ml.Emva kweeyure ezingama-24, iiseli zavunyelwa ukuba zibuyele kwakhona ngokutshintsha okuphakathi.Iiseli zikhethwe ngokusebenzisa i-5 μg / ml i-blasticidin (i-Solarbio, #3513-03-9) kwiindinyana ezintathu zokuqala njengokhetho oluphantsi olungqongqo.Emva koko kusetyenziswe i-20 μg/ml njengerejimeni engqongqo ngakumbi kwimihlathi emithathu elandelayo.
Iiseli zafakwa kwi-12-chambers (Ibidi, #81201) ekuxininiseni malunga neeseli ze-20,000 kwiqula ngalinye.Ukuphucula ukubambelela kweeseli ze-HEK293T, yongeza i-50 µg/ml fibronectin (Corning, #356008) exutywe kwi-phosphate buffered saline (PBS, Sangon, #B640435) kwi-37 ° C.Amagumbi aye anyangwa ngeyure eyi-1 aze asuswe nge-PBS.Emva kwe-24 h, iiseli zazihlanjwa kube kanye nge-PBS, zifakwe kwi-1 mM probe 3 kwisisombululo setyuwa esilungelelanisiweyo se-Hanks (HBSS, Gibco, #14025092) ngeyure eyi-1 nge-37 ° C, kwaye emva koko ifakwe nge-LED eluhlaza (460 nm ).) zifakwe kwi-10 min kwiqondo lokushisa.Emva koko, iiseli zihlanjwe kabini nge-PBS kwaye zilungiswe nge-4% ye-formaldehyde kwi-PBS (Sangon, #E672002) imizuzu eyi-15 kwiqondo lokushisa.I-formaldehyde eyongezelelweyo isuswe kwiiseli ezisisigxina ngokuhlamba kathathu nge-PBS.Iiseli zaye zagqitywa nge-0.5% Triton X-100 (Sangon, #A600198) kwi-PBS kwaye yahlamba amaxesha e-3 nge-PBS.Emva koko susa igumbi kwaye wongeze kwisampulu nganye engama-25 µl yomxube wokucofa ukucofa oqulathe i-50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) kunye ne-0.5 mg / ml i-sodium ascorbate (i-Aladdin, no. S105024) kwaye ifakwe kwi-30 imizuzu kwiqondo lokushisa.Emva kokuphendulwa kwe-snap, iiseli zihlanjwe amaxesha amathandathu kunye ne-PBS equkethe i-0.05% Tween-20 (Sangon, #A600560) (PBST) kwaye ivalwe nge-5% BSA (Abcone, #B24726) kwi-PBST imizuzu ye-30 kwiqondo lokushisa.
Kwi-colocalization immunostaining, iiseli zifakwe kwi-antibodies eziphambili ngokwemiqathango ebonisiweyo: i-mouse anti-V5 tag mAb (1: 500, CST, #80076), umvundla anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), umvundla we-polyclonal anti-calnexin antibody (1: 500, Abcam, #ab22595) okanye umvundla we-anti-lamin A / C monoclonal antibody (1: 500; CST, #2032) kwi-4 ° C ngobusuku.Emva kokuhlamba amaxesha e-3 kunye ne-PBST, iiseli zifakwe kwi-antibodies yesibini: ibhokhwe yokulwa nomvundla i-Alexa Fluor 488 (Thermo, #A11034) ihlanjululwe 1: 1000, ibhokhwe ye-anti-mouse Alexa Fluor 594 (CST, #8889) ihlanjululwe 1: 1000.i-dilution Nciphisa kwindawo yobushushu begumbi imizuzu engama-30.Iiseli zaye zahlanjwa ngamaxesha e-3 kunye ne-PBST kwaye ziphikisana ne-DAPI (Thermo, #D1306) kwi-PBS imizuzu eyi-10 kwiqondo lokushisa.Emva kokuhlamba kwe-3 kunye ne-PBS, iiseli zavalwa kwi-50% glycerol (Sangon, #A600232) kwi-PBS yokucinga.Imifanekiso ye-Immunofluorescent ifunyenwe kusetyenziswa i-ZEISS LSM 900 Airyscan2 confocal microscope kunye ne-software ye-ZNE 3.5.
Kwi-imaging ye-oxygen fluorescent ye-singlett, iiseli zahlanjwa kabini nge-Hanks HEPES buffer ngaphambi kokongeza i-100 nM Si-DMA kwi-Hanks HEPES buffer (DAJINDO, #MT05).Emva kokuvezwa ekukhanyeni, iiseli zifakwe kwi-incubator ye-CO2 kwi-37 ° C imizuzu engama-45.Iiseli zaye zahlanjwa kabini nge-Hanks 'HEPES buffer kwaye zachaswa kunye ne-Hoechst kwi-Hanks' HEPES buffer imizuzu eyi-10 kwiqondo lobushushu begumbi kwaye zibonwa kusetyenziswa i-ZEISS LSM 900 ye-confocal microscope., #M36008) kwi-HBSS buffer equkethe i-calcium kunye ne-magnesium.Emva kokuvezwa kukukhanya okanye i-doxorubicin (MCE, #HY-15142A), iiseli zifakwe kwi-CO2 incubator kwi-37 ° C. imizuzu eyi-10, zihlanjwe kabini nge-HBSS buffer, kwaye zifakwe kwi-Hoechst kwi-HBSS buffer kwiqondo lokushisa.imizuzu.I-Doxorubicin isetyenziswe njengolawulo olulungileyo lweprobe apho iiseli ziphathwa nge-20 μM doxorubicin kwi-HBSS equlethe i-1% ye-BSA ye-30 min.Imifanekiso ye-Immunofluorescent yafunyanwa kusetyenziswa i-Zeiss LSM 900 confocal microscope.
Iiseli ze-HEK293T ezibonisa ngokuzinzileyo i-mito-miniSOG zaye zatyalwa kuxinaniso olumalunga nama-30% kwizitya eziyi-15 cm.Emva kweeyure ze-48, xa i-~80% ye-confluence ifikelelwe, iiseli zahlanjwa kanye nge-PBS, zifakwe kwi-1 mM Probe 3 kwi-buffer entsha ye-HBSS kwi-1 iyure kwi-37 ° C kwaye emva koko yakhanyiswa nge-LED eluhlaza okwemizuzu eyi-10 kwigumbi. ubushushu..Emva koko, iiseli zahlanjwa kabini nge-PBS, zikhutshiwe kwaye zaphinda zamiswa kwi-ice-cold PBS buffer equkethe i-EDTA-free protease inhibitors (MCE, #HY-K0011).Iiseli zaye zahlanjululwa ngokufaka i-sonicating i-tip ye-1 ngomzuzu (i-1 yesibini kunye ne-1 yesibini kwi-35% amplitude).Umxube obangelwayo wawuyi-centrifuged kwi-15,871 xg ye-10 min kwi-4 ° C ukususa i-debris, kwaye i-concentration ye-supernatant yalungiswa kwi-4 mg / mL usebenzisa i-BCA protein assay kit (Beyotime, #P0009).Dibanisa i-1 ml ye-lysate engasentla kunye ne-0.1 mM i-biotin aside eyonakaliswayo (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), kunye ne-1 mM ye-CuSO4 ye-incuba ukujikeleza ukuya kwi-1 iyure kwiqondo lokushisa.Emva kokuphendula ngokukhawuleza, yongeza umxube kwisisombululo esixutywe ngaphambili (MeOH: CHCl3: H2O = 4 ml: 1 ml: 3 ml) kwi-vial yeglasi eyi-10 ml.Iisampuli zixutywe kwaye zixutywe kwi-4500 g imizuzu eyi-10 kwindawo yokushisa.Izisombululo ezisezantsi neziphezulu zalahlwa, i-precipitate ihlanjwe kabini nge-1 ml ye-methanol kunye ne-centrifuged kwi-15871 × g ye-5 min kwi-4 ° C.Yongeza i-1 ml ye-8 M urea (Aladdin, no. U111902) kwi-25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) ukunyibilikisa imvula.Iisampulu zaphinda zamiselwa nge-10 mM dithiothreitol (Sangon, #A100281 kwi-25 mM ABC) imizuzu engama-40 kwi-55°C ilandelwa kukongezwa kwe-15 mM entsha iodoacetamide (Sangon, #A600539) kwiqondo lobushushu begumbi ebumnyameni.Alkylation ngaphakathi kwemizuzu engama-30..I-5 mM eyongezelelweyo i-dithiothreitol yongezwa ukumisa ukusabela.Lungiselela malunga ne-100 µl amaso e-NeutrAvidin agarose (Thermo, #29202) kwisampulu nganye ngokuhlamba amaxesha ama-3 nge-1 ml ye-PBS.Isisombululo seproteome esingentla sihlanjululwe nge-5 ml ye-PBS kwaye yafukanyelwa ngamaso e-agarose e-NeutrAvidin ehlanjwe ngaphambili kwiiyure ezi-4 kwiqondo lokushisa.Emva koko amaso ahlanjwe ngamaxesha e-3 kunye ne-5 ml ye-PBS equkethe i-0.2% SDS (Sangon, # A600485), amaxesha e-3 kunye ne-5 ml ye-PBS equkethe i-1M urea, kunye namaxesha e-3 kunye ne-5 ml ddH2O.Amaso aye avunwa nge-centrifugation kwaye amiswa kwakhona kwi-200 μl ye-25 mM ABC equlethe i-1 M urea, 1 mM CaCl 2 (Macklin, #C805228) kunye ne-20 ng/μl trypsin (Promega, #V5280).Zama i-Trypsinize ngobusuku kwi-37°C ngokujikeleza.Ukusabela kwamiswa ngokongeza i-asidi ye-formic (i-Thermo, # A117-50) de kube i-pH ifikelele kwi-2-3.Ubuhlalu buhlanjwe ngamaxesha e-3 kunye ne-1 ml ye-PBS equkethe i-0.2% ye-SDS, amaxesha e-3 kunye ne-1 ml ye-PBS equkethe i-1 M urea, kwaye emva koko amaxesha ama-3 nge-1 ml yamanzi adibeneyo.Iipeptides ezilungisiweyo zikhutshwe yi-light lysis (365 nm) kwimizuzu engama-90 kusetyenziswa i-200 μl ye-70% MeOH.Emva kwe-centrifugation, i-supernatant yaqokelelwa.Amaso aye ahlanjwa kube kanye nge-100 μl ye-70% ye-MeOH kwaye amandla amakhulu adityaniswa.Iisampulu zomisiwe kwi-Speedvac vacuum concentrator kwaye zigcinwe kwi -20 ° C de kube kuhlalutya.
Ukuchonga kunye nokulinganisa i-peptide ye-oxygen e-singlett eguqulweyo, iisampulu zachithwa kwakhona kwi-0.1% ye-asidi ye-formic kunye ne-1 μg ye-peptides zahlaziywa kusetyenziswa i-Orbitrap Fusion Lumos Tribrid mass spectrometer exhotyiswe nge-nano ESI umthombo ovela kwi-Tune kunye ne-Xcalibur kwi-software yomthengisi 4.3.Iisampulu zahlulwa kwi-75 µm × 15 cm epakishwe ngaphakathi kwikholamu ye-capillary ene-3 µm C18 impahla (ReproSil-pur, #r13.b9.) kwaye iqhagamshelwe kwi-EASY-nLC 1200 UHPLC system (Thermo).I-peptides yahlulwe ngomgca we-95 imizuzu ye-gradient chromatography ukusuka kwi-8% ye-solvent B ukuya kwi-50% ye-solvent B (A = 0.1% ye-asidi ye-formic emanzini, B = 0.1% ye-asidi ye-formic kwi-80% ye-acetonitrile), emva koko yanda ngokulandelelana ukuya kwi-98% B min. kwi-6 min kwinqanaba lokuhamba kwe-300 nl / min.I-Orbitrap Fusion I-Lumos iqokelela idatha ngokutshintshana phakathi kwe-MS scan epheleleyo kunye ne-MS2 scan ngokuxhomekeke kwidatha.I-voltage ye-sputtering yamiselwa kwi-2.1 kV kwaye ubushushu be-ion transport capillary yayiyi-320 ° C.I-MS spectra (350-2000 m / z) iqokelelwe kunye nesisombululo se-120,000, i-AGC 4 × 105, kunye nexesha elide lokufaka i-150 ms.Iiprecursors ezili-10 eziqhelekileyo eziphindwe kabini kwiskena esipheleleyo zahlulwa kusetyenziswa i-HCD ene-normalized collision energy ye-30%, i-quadrupole isolation window ye-1.6 m/z, kunye nokumiselwa kwesisombululo se-30,000.Ithagethi ye-AGC ye-tandem mass spectrometry isebenzisa i-5×104 kunye nexesha legalelo eliphezulu yi-150 ms.Ukukhutshwa okuguquguqukayo kumiselwe kwimizuzwana engama-30. Ii-ion ezingabelwanga okanye abo banentlawulo ye-1 + kunye ne-> 7+ banqatshelwe i-MS / MS. Ii-ion ezingabelwanga okanye abo banentlawulo ye-1 + kunye ne-> 7+ banqatshelwe i-MS / MS. Неназначенные ионы или ионы с зарядом 1+ kunye >7+ были отклонены для МС/МС. Iiyoni ezingabelwanga okanye iiyoni ezinentlawulo ye-1+ kunye> ne-7+ zaliwe kwi-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+ kunye >7+ были отклонены для МС/МС. Iiyoni ezingachazwanga okanye iiyoni ezineentlawulo ze-1+ kunye> 7+ zanqatshwa kwi-MS/MS.
Idatha ekrwada icutshungulwa kusetyenziswa iqonga lecomputing yeFragPipe esekwe kwiMSFragger.I-Mass biases kunye ne-amino acids ehambelanayo inqunywe ngokusebenzisa i-algorithm yokukhangela evulekileyo kunye ne-precursor mass tolerance of -150 ukuya kwi-500 Da.Iipeptide ezilungisiweyo zaye zachongwa kusetyenziswa ukuguqulwa kwe-histidine kunye neenzuzo ezininzi ze-229.0964 kunye + 247.1069 Da kwiPD (Proteome Discoverer 2.5, Thermo).
Iiseli ezibonisa ngokuzinzileyo ijini ye-miniSOG edityanisiweyo zafakwa kwizitya eziyi-6 cm.Ekufikeleleni kwi-~ 80% ye-confluence, iiseli zahlanjwa kanye nge-HBSS (Gibco, #14025092), emva koko zifakwe kwiikhemikhali ze-probes kwi-HBSS ngeyure ye-1 kwi-37 ° C kwaye zikhanyiswe ngokukhanya okwesibhakabhaka.I-10W LED yemizuzu engama-20 kwiqondo lokushisa.Ukufumanisa ukuba luphi uhlobo lweentlobo ze-oksijeni ezisebenzayo ezibandakanyekayo kwi-PDPL, i-0.5 mM i-vitamin C (MCE, #HY-B0166), i-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 yongezwa kwiiseli njengezongezelelo.Emva kokuhlamba nge-PBS ebandayo, iiseli zikhutshiwe, ziqokelelwe kwiityhubhu ze-1.5 ml ze-centrifuge, kunye ne-sonicated nge-tip ye-1 min kwi-200 μl ye-PBS kunye ne-1x protease inhibitor ngaphandle kwe-EDTA (i-1 s kunye ne-1 s ngaphandle, i-amplitude 35%).Umxube obangelwayo wawuyi-centrifuged kwi-15,871 × g ye-10 min kwi-4 ° C kwaye i-concentration ye-supernatant yahlengahlengiswa kwi-1 mg / mL usebenzisa i-BCA protein assay kit.Ngokumalunga ne-50 µl ye-lysate engentla yafukanywa nge-0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, kunye ne-1 mM CuSO4 ngeyure e-1 kwiqondo lobushushu begumbi kunye nojikelezo ukusuka ezantsi ukuya phezulu.Emva kokucofa ukusabela, imvula kunye ne-acetone iqhutywe ngokudibanisa i-250 μl ye-acetone epholileyo ngaphambili kwiisampuli, i-incubating kwi -20 ° C nge-20 min kunye ne-centrifuging kwi-6010 × g ye-10 min kwi-4 ° C.Qokelela i-pellet kwaye ubilise nge-50 µl ye-1x ye-buffer ye-Laemmli i-10 min kwi-95 °C.Iisampulu zaye zahlalutywa kwi-SDS-PAGE iigels ezinde kwaye zabonwa kusetyenziswa i-Bio-rad ChemiDoc MP Touch imaging system kunye ne-Image Lab Touch software.
Ukubonakaliswa kunye nokuhlanjululwa kwe-miniSOG-6xIprotheni ye-recombinant yenziwa njengoko kuchaziwe ngaphambili.Ngokufutshane, iiseli ze-E. coli BL21(DE3) (TransGen, #CD701-02) zatshintshwa nge-pET21a-miniSOG-6xHis kunye nokubonakaliswa kweprotheyini ye-0.5 mM IPTG (Sangon, #A600168).Emva kwe-cell lysis, iiprotheni zahlanjululwa kusetyenziswa ubuhlalu be-Ni-NTA agarose (MCE, no. 70666), i-dialysed against PBS, kwaye igcinwe kwi-80 ° C.
Kwi-antibody-based in vitro label proximity assay, xuba i-100 μM i-miniSOG ecocekileyo, i-1 mM probe 3, kunye ne-1 μg ye-anti-label mouse monoclonal antibody (TransGen, #HT501-01) kwi-PBS ukuya kumthamo opheleleyo wokuphendula we-50 μl..Umxube wokuphendula uhlaziywe ngokukhanya kwe-LED eluhlaza okwesibhakabhaka kwi-0, 2, 5, 10, kunye ne-20 min kwiqondo lokushisa.Umxube waqanjwa nge-0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, kunye ne-1 mM CuSO4 ngeyure e-1 kwiqondo lobushushu begumbi kwishaker eshukumayo enyukayo.Emva kokusabela ngokukhawuleza, yongeza i-4x ye-Laemmli ye-buffer ngqo kumxube kwaye ubilise kwi-95 ° C nge-10 min.Iisampulu zahlalutywa kwiigels ze-SDS-PAGE kwaye zahlalutywa ngokuchithwa kwentshona kunye ne-streptavidin-HRP (1: 1000, Solarbio, #SE068).
Ipeptide yokwenziwa ene-histidine ene-C-terminal amidation (LHDALDAK-CONH2) yayisetyenziselwa ukuhlalutya i-peptide-based based in vitro labeling.Kulo vavanyo, i-100 μM ihlanjululwe i-miniSOG, i-10 mM probe 3 kunye ne-2 μg / ml i-peptide yokwenziwa yaxutywa kwi-PBS kwi-volume reaction total ye-50 μl.Umxube wokusabela ufakwe i-radiated nge-blue LED ukukhanya kweyure ye-1 kwiqondo lokushisa.Enye i-microliter yesampuli yahlalutywa kusetyenziswa inkqubo ye-LC-MS (Amanzi, i-SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer kunye ne-software ye-MassLynx yohlalutyo lwe-spectrum).
Iiseli ze-HEK293T ezibonisa ngokuzinzileyo i-miniSOG gene fusion yahlwayelwa kwii-10 cm izitya zemigca ene-organelle eyahlukeneyo yendawo (i-Mito, i-ER, i-Nucleus) kunye ne-15 cm izitya ze-Parkin-miniSOG kunye nemigca ye-BRD4-miniSOG.Ekufikeleleni kwi-~ 90% ye-confluence, iiseli zihlanjwe kanye nge-HBSS, emva koko zifakwe kwi-probe 3 kwi-HBSS ngeyure eli-1 kwi-37 ° C kwaye zikhanyiswe nge-10 W blue LED kwiqondo lokushisa.Ngokungaqhagamshelwanga kwelebhile ye-Parkin, i-10 µM ye-proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) ene-probe 3 kwi-HBSS yongezwa ngeyure e-1 kwi-37°C.I-cell lysis, nqakraza i-chemistry, ukunciphisa kunye namanyathelo e-alkylation ayefana njengoko kuchazwe ngasentla, ngaphandle kokuba i-2 mg ye-lysate yongezwa kunye ne-biotin PEG3 azide isetyenziswe kwimpendulo yokuchofoza endaweni ye-photodegradable biotin azide.Emva kokutyebisa, ubuhlalu buhlanjwe ngamaxesha e-3 kunye ne-5 ml ye-PBS equkethe i-0.2% ye-SDS, amaxesha e-3 kunye ne-5 ml ye-PBS equkethe i-1 M urea, kunye namaxesha e-3 kunye ne-5 ml ye-PBS.Emva koko, i-2 µg trypsin yongezwa kwi-300 µl 25 mM ABC equlethe i-1 M urea ukuze kucandwe iprotein ngobusuku obungama-37°C.Ukusabela kwamiswa ngokongeza i-asidi ye-formic de kube i-pH ye-2-3 ifikelelwe.Emva kwe-trypsinization kumaso, isisombululo sepeptide saye sasuswa ityuwa kusetyenziswa ikholamu ye-SOLAµ HRP (Thermo, #60209-001) kwaye yomiswa kwisigxininisi se-Speedvac vacuum.I-Peptides yachithwa kwakhona kwi-0.1% ye-asidi ye-asidi kunye ne-500 ng ye-peptides yahlalutywa kusetyenziswa i-Orbitrap Fusion Lumos Tribrid mass spectrometer exhotywe ngomthombo we-nano-ESI ochazwe ngasentla.I-Peptides yahlulwe kwi-precolumns ye-RP-HPLC yorhwebo (i-75 μm x 2 cm) (i-Thermo, no. 164946) kunye neekholamu ze-RP-HPLC zohlalutyo (75 μm x 25 cm) (Thermo, no. 164941), zombini zizaliswe nge-2 μm.i-gradient ukusuka kwi-8% ukuya kwi-35% ye-ACN kwimizuzu engama-60, emva koko inyuke ngokulandelelana ukuya kwi-98% B kwimizuzu emi-6 kwinqanaba lokuhamba kwe-300 Nl / min.I-MS spectra (350-1500 m / z) iqokelelwe ngesisombululo se-60,000, i-AGC 4 × 105, kunye nexesha elide lokufaka i-50 ms.Ii-ion ezikhethiweyo zahlulwa ngokulandelelanayo yi-HCD kwimijikelo ye-3 kunye namandla okudibana okuqhelekileyo angama-30%, ifestile ye-quadrupole yodwa ye-1.6 m / z, kunye nesisombululo se-15000. I-5 × 104 tandem ye-spectrometer yobunzima be-AGC ekujoliswe kuyo kunye nexesha elininzi lokutofa ye 22 ms yasetyenziswa.Ukukhutshwa okuguquguqukayo kumiselwe kwimizuzwana engama-45. Ii-ion ezingabelwanga okanye abo banentlawulo ye-1 + kunye ne-> 7+ banqatshelwe i-MS / MS. Ii-ion ezingabelwanga okanye abo banentlawulo ye-1 + kunye ne-> 7+ banqatshelwe i-MS / MS. Неназначенные ионы или ионы с зарядом 1+ kunye >7+ были отклонены для МС/МС. Iiyoni ezingabelwanga okanye iiyoni ezinentlawulo ye-1+ kunye> ne-7+ zaliwe kwi-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+ kunye >7+ были отклонены для МС/МС. Iiyoni ezingachazwanga okanye iiyoni ezineentlawulo ze-1+ kunye> 7+ zanqatshwa kwi-MS/MS.
Amanyathelo okulungiselela isampula ukuya ekuphuculeni ubuhlalu be-NeutrAvidin ayefana nohlalutyo lwe-LC-MS / MS oluchazwe ngasentla.Ngokumalunga ne-50 μg ye-lysate isetyenziswe njengegalelo lokulawula ukulayisha kwaye i-2 mg ye-lysate yayisetyenziselwa ukuphendula ngokuchofoza.Emva kokutyebisa kunye nokuhlamba nge-neutravidin, iiprotheni eziboshiweyo zahluthwa ngokudibanisa i-50 μl ye-buffer ye-Laemmli kwi-agarose resin beads kwaye ibiliswe kwi-95 ° C. imizuzu emi-5.Igalelo lomthwalo wokulawula kunye neesampulu eziphuculweyo zebead zahlalutywa yi-SDS-PAGE kwaye zagqithiselwa kwiimbrane ze-PVDF (i-Millipore, #ISEQ00010) ngeendlela eziqhelekileyo zokuchithwa kwe-Western.Iimbumba zavalwa nge-5% yobisi lwe-skim (Sangon, #A600669) kwi-TBS equlethe i-0.1% phakathi kwe-20 (TBST) kwaye ifakwe ngokulandelelana kunye ne-antibodies yokuqala kunye neyesibini.Izilwa-buhlungu eziphambili zixutywe nge-1:1000 kwi-5% yobisi lwe-skim kwi-TBST kwaye ziqatywe ngobusuku kwi-4°C.I-antibodies yesibini isetyenziswe kumlinganiselo we-1: 5000 kwaye ifakwe kwi-1 iyure kwindawo yokushisa.Iimbumba zabonwa nge-chemiluminescence kusetyenziswa inkqubo yokucinga ye-Chemidoc MP.Zonke izikena ezinganqunyulwanga zamablothi kunye neegel kumfanekiso ziboniswa njengedatha ekrwada.
Ii-antibodies eziphambili ezisetyenzisiweyo kolu phononongo zibandakanya umvundla we-anti-SFPQ monoclonal antibody (CST, no. 71992), umvundla we-anti-FUS monoclonal antibody (CST, no. 67840), umvundla u-anti-NSUN2 i-polyclonal antibody (Proteintech, no. 20854-1-- I-AP), i-rabbit anti-mSin3A i-polyclonal antibody (Abcam, #ab3479), i-mouse anti-tag monoclonal antibody (TransGen, #HT201-02), i-mouse anti-β-actin monoclonal antibody (TransGen, #HC201-01), i-rabbit antibody -CDK2 monoclonal antibody (ABclonal, #A0094), umvundla monoclonal antibody ukuya CTBP1 (ABclonal, #A11600), rabbit polyclonal antibody to DUT (ABclonal, #A2901), rabbit polyclonal antibody to PSMC4 (ABclonal anti-5), #Abclonal-5), #Abclonal I-DNAJB1 i-polyclonal antibody (ABclonal, # A5504).Ezi zilwa-buhlungu zisetyenziswe kwi-1:1000 dilution kwi-5% yobisi lwe-skim kwi-TBST.Ii-antibodies zesibini ezisetyenzisiweyo kolu phononongo zibandakanya i-anti-rabbit IgG (TransGen, #HS101-01), i-anti-mouse IgG (TransGen, #HS201-01) kwi-1: 5000 dilution.
Ukuqhubela phambili uphando malunga nokuba i-BRD4 isebenzisana ne-SFPQ, i-HEK293T ezinzileyo kunye ne-BRD4-miniSOG iiseli ezigqithise i-HEK293T zifakwe kwii-10 cm izitya.Iiseli zahlanjwa nge-PBS ebandayo kwaye zafakwa kwi-1 ml ye-Pierce IP lysis buffer (Thermo Fisher, #87787) kunye ne-EDTA-free protease inhibitor imizuzu engama-30 kwi-4 ° C.Emva koko, i-lysates yaqokelelwa kwii-tubes ze-1.5 ml ze-centrifuge kunye ne-centrifuged kwi-15,871 xg ye-10 min kwi-4 ° C.I-supernatant yavunwa yaza yaqalwa nge-5 µg ye-anti-V5 ebhalwe imouse monoclonal antibody (CST, #80076) ngobusuku kwi-4°C.Hlamba malunga ne-50 µl yeprotheyini A/G amaso kazibuthe (MCE, #HY-K0202) kabini nge-PBS equlethe i-0.5% Tween-20.Emva koko i-cell lysates ifakwe ngamaso amagnetic kwiiyure ezi-4 kwi-4 ° C kunye nokujikeleza ukusuka ezantsi ukuya phezulu.Emva koko ubuhlalu buhlanjwe ngamaxesha amane nge-1 ml ye-PBST buffer kwaye ibilisiwe kwi-95 ° C kwi-5 min.Iisampulu zahlalutywa kwiijeli ze-SDS-PAGE kwaye zagqithiselwa kwiimbrane ze-PVDF kusetyenziswa iindlela eziqhelekileyo ze-Western blot.Iinwebu zavalwa kwi-5% ye-skim milk kwi-TBST kwaye zafukanywa ngokulandelelana kunye nezilwa-buhlungu zaseprayimari nezesekondari.I-Primary Antibody Rabbit anti-SFPQ i-monoclonal antibody (CST, #71992) isetyenziswe kumlinganiselo we-1: 1000 kwi-5% yobisi lwe-skim kwi-TBST kwaye incubated ngobusuku kwi-4 ° C.I-Anti-Rabbit IgG isetyenziswe kwisilinganiselo se-1: 5000 kwaye ifakwe kwi-1 iyure kwindawo yokushisa.Iimbumba zabonwa nge-chemiluminescence kusetyenziswa inkqubo yokucinga ye-Chemidoc MP.
Zonke izakhiwo ezisetyenziselwa uhlalutyo lweNdawo yoMphezulu oFikelelekayo kwiSolvent (SASA) zifunyenwe kwiProtein Data Bank (PDB)52 okanye kwiAlphaFold Protein Structure Database53.I-Absolute SASA ibalwe kwintsalela nganye kusetyenziswa inkqubo yeFreeSASA.Kuphela yidatha ye-SASA epheleleyo nengacacanga ebhalwe i-histidine kunye nabamelwane bayo esetyenzisiweyo ukufumana umlinganiselo we-SASA kulwakhiwo ngalunye.Ufikelelo lwe-solvent ehambelanayo (RSA) ye-histidine nganye ibalwe ngokwahlula ixabiso elipheleleyo le-SASA ngowona mmandla uphezulu wentsalela okhoyo ofumanekayo kwisinyibilikisi.Zonke i-histidines zaye zahlelwa njengezifihliweyo ukuba i-RSA ephakathi yayingaphantsi kwe-20%, kungenjalo ibonakaliswe56.
Iifayile ezikrwada ezifunyenwe kwimowudi ye-DDA zakhangelwa kusetyenziswa i-Proteome Discoverer (v2.5) okanye i-MSfragger (i-Fragpipe v15.0) kwindawo efanelekileyo yesiseko seprotheyini eqinisekisiweyo ye-SwissProt equlethe ungcoliseko oluqhelekileyo.Iipeptide zazifuna i-trypsin epheleleyo eneendawo ezimbini ezilahlekileyo zokuqhekeka, i-carbamoyl methylation njengolungiso olusisigxina kunye ne-methionine oxidation njengokuguqulwa okuguquguqukayo.I-Precursor kunye ne-fragment weight tolerances yamiselwa kwi-10 ppm kunye ne-0.02 Da (MS2 Orbitrap), ngokulandelanayo. Ukubetha okungcolileyo kwasuswa, kwaye iiprotheni zahluzwa ukuze zifumane izinga lokufumanisa ubuxoki <1%. Ukubetha okungcolileyo kwasuswa, kwaye iiprotheni zahluzwa ukuze zifumane izinga lokufumanisa ubuxoki <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэффициент ложного обнаружения <1%. Ukubetha okungcolileyo kwasuswa kwaye iiprotheni zahluzwa ukunika izinga lokufumanisa ubuxoki <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%. Ukubetha okungcolileyo kwasuswa kwaye iiprotheyini zahluzwa ukuze kuphunyezwe izinga elifanelekileyo lobuxoki <1%.Ukuhlalutya ubungakanani ngaphandle kokusetyenziswa kweelebhile, umxholo weprotheyini oqhelekileyo ovela kwizinto ezintathu eziphindaphindiweyo zebhayoloji zisetyenzisiwe.Uhlalutyo lwe-protein subcellular localization lwenziwa ngokusetyenziswa kwe-Gene Ontology (GO) uhlalutyo oluvela kwi-DAVID Bioinformatics Resources, i-MitoCarta 3.0 kunye nedatha eqokelelweyo kwaye ipapashwe liqela le-Alice Ting.Imephu yentaba-mlilo yafunyanwa ePerseus (v1.6.15.0). Iiprotheyini zobuninzi beenguqu ezitshintshileyo zavavanywa ngokubaluleka kwezibalo zisebenzisa i-t-test-side, kunye neeprotheyini ezithintekayo zichongiwe ngotshintsho oluninzi> i-2 (ngaphandle kokuba kuchazwe ngenye indlela) kunye nexabiso le-p <0.05. Iiprotheyini zobuninzi beenguqu ezitshintshileyo zavavanywa ngokubaluleka kwezibalo zisebenzisa i-t-test-side, kunye neeprotheyini ezithintekayo zichongiwe ngotshintsho oluninzi> i-2 (ngaphandle kokuba kuchazwe ngenye indlela) kunye nexabiso le-p <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. Iiprotheyini eziguquguqukayo zeprotheyini zihlolwe ukubaluleka kwezibalo kusetyenziswa i-t-test-tailed two-tailed, kwaye i-protein matchs ichongiwe kunye noshintsho lomxholo> i-2 (ngaphandle kokuba kuphawulwe ngenye indlela) kunye nexabiso le-ap <0.05.1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 星 星 0 智慧 ＄0 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. Ukubaluleka kwamanani okutshintsha komxholo weprotheyini kwavavanywa kusetyenziswa uvavanyo lwe-t-tailed-tailed, kunye neprotheyini ehambelanayo yamiselwa utshintsho lomxholo> 2 (ngaphandle kokuba kuboniswe ngenye indlela) kunye ne-p-values ​​<0.05.Uhlalutyo lokusebenzisana kweeprotheyini lwenziwa kusetyenziswa uhlalutyo lwe-GO kunye ne-database ye-String.
Ukuphindaphindwa kwebhayoloji ezintathu zenziwa ngeziphumo ezifanayo.Uhlalutyo lwamanani lwenziwa ngeGraphPad Prism (isoftware yeGraphPad) kunye neendawo zentaba-mlilo zenziwa ngePerseus (v1.6.15.0).Ukuthelekisa la maqela mabini, amaxabiso e-p aye amiselwa kusetyenziswa uvavanyo loMfundi olunemisila emibini.Iiprotheni ze-singleton kuphela ezichongiweyo ubuncinci kabini kwiqela lovavanyo zibandakanyiwe kwiiplani zentaba-mlilo, kwaye amaxabiso alahlekileyo ahambelanayo kwiqela lolawulo athatyathelwa indawo yiPerseus ukusuka kulwabiwo oluqhelekileyo ukuze ixabiso le-p libalwe.Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.Kuhlalutyo lweproteomic kuhlalutyo lwamanani, ubuninzi beeprotheni ezivele ubuncinane kwii-biological replicates ezimbini zagcinwa.Iindlela zezibalo azizange zisetyenziswe ukumisela kwangaphambili ubungakanani besampulu.Imifuniselo ayizenzelwanga.Abaphandi abazange bayimfama kwimisebenzi ngexesha lovavanyo kunye novavanyo lweziphumo.
Ukufumana ulwazi oluthe kratya malunga noyilo lwesifundo, bona iNgxelo yoPhando lweNdalo eqhagamshelwe kweli nqaku.
Idatha ye-mass spectrometry efunyenwe kolu phononongo ifakwe kwi-ProteomeXchange Consortium ngokusebenzisa i-iProX57 iqabane elinguvimba phantsi kwedatha ye-ID PXD034811 (PDPL-MS dataset).Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.Eli nqaku libonelela ngedatha yokuqala.
I-Gingras, AC, Abe, KT & Raught, B. Ukwazi ummelwane: usebenzisa i-biotinylation exhomekeke ekusondeleni ukubonisa iimpawu zeprotheni kunye ne-organelles yemephu. I-Gingras, AC, Abe, KT & Raught, B. Ukwazi ummelwane: usebenzisa i-biotinylation exhomekeke ekusondeleni ukubonisa iimpawu zeprotheni kunye ne-organelles yemephu.I-Gingras, i-AS, i-Abe, i-KT kunye ne-Raut, i-B. Ukuqhelana neendawo ezingqongileyo: usebenzisa i-biotinylation exhomekeke kufuphi ukuze ibonise iiprotheyini zeprotheyini kunye ne-organelles yemephu. 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. Ukuqonda ubumelwane: sebenzisa ukuxhomekeka kobumelwane kubomi bebhayoloji.I-Gingras, i-AS, i-Abe, i-KT kunye ne-Raut, i-B. Ukuqonda ukusondela: ukubonakaliswa kweeprotheyini eziyinkimbinkimbi kunye nemephu ye-organelles usebenzisa i-biotinylation exhomekeke ngokusondeleyo.Okwangoku.Uluvo lwam.Ikhemikhali.ibhayoloji 48, 44-54 (2019).
Geri, JB et al.Ukwenza imephu ye-microenvironment ngokudlulisela amandla e-Dexter kwiiseli zomzimba.Inzululwazi 367, 1091-1097 (2020).
Hertling, EL et al.Uthungelwano lwe-proteome scale scale lubona ukulungiswa kwe-cell-specific of the interactome yabantu.Iiseli 184, 3022-3040.e3028 (2021).