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Izindlela zokulebula ze-Enzymatic proximity ezisuselwe kuma-ester acushiwe noma ama-phenoxy radicals zisetshenziswa kakhulu ukwenza imephu yama-subcellular proteinors nama-protein interactors kumaseli aphilayo.Kodwa-ke, ama-ester acushiwe awasabeli kahle, okuholela ebaleni lokulebula elibanzi, futhi ama-phenoxy radicals akhiqizwa ukwelashwa kwe-peroxide angaphazamisa izindlela ze-redox.Lapha sibika indlela ye-proximity labeling dependent photoactivation (PDPL) ethuthukiswe ngokuxhumanisa ngokofuzo iphrotheni ye-miniSOG ye-photosensitizer kuphrotheni ethandwayo.Icutshungulwa ukukhanya okuluhlaza okwesibhakabhaka futhi ilawulwa isikhathi sokuchayeka, umoya-mpilo we-singlet uyakhiqizwa bese ukulebula okuxazululwe nge-spatiotemporally kwezinsalela ze-histidine nge-aniline probe kufinyelelwa.Sibonisa ukwethembeka kwayo okuphezulu ngokusebenzisa imephu ye-organelle-specific proteome.Ukuqhathaniswa kwehlangothi nehlangothi kwe-PDPL ne-TurboID kukhombisa ukumbozwa kwe-proteomic okucace kakhudlwana nokuphelele kwe-PDPL.Okulandelayo, sisebenzise i-PDPL ku-coactivator ye-transcriptional ehlobene nesifo i-BRD4 ne-E3 Parkin ligase futhi sathola abahlanganyeli ababengaziwa ngaphambili.Ngokuhlolwa kwe-overexpression, ama-substrates amabili angaziwa, i-Ssu72 kanye ne-SNW1, akhonjwe i-Parkin, okuwohloka kwayo kulamula indlela ye-ubiquitination-proteasome.
Ukubonakaliswa okunembile kwamanethiwekhi amaprotheni kuyisisekelo sezinqubo eziningi ezibalulekile zamaselula.Ngakho-ke, imephu ye-spatiotemporal enembe kakhulu yokusebenzisana kwamaprotheni izohlinzeka ngesisekelo samangqamuzana sokucacisa izindlela zebhayoloji, ukugula kwezifo, nokuphazamisa lokhu kusebenzisana ngezinjongo zokwelapha.Kuze kube manje, izindlela ezikwazi ukubona ukusebenzisana kwesikhashana kumaseli aphilayo noma izicubu zifiseleka kakhulu.I-Affinity Purification Mass Spectrometry (AP-MS) ibisetshenziswa ngokomlando ukukhomba ozakwethu ababophayo bamaprotheni of interest (POIs).Ngokuthuthukiswa kwezindlela ze-quantitative proteomics, i-Bioplex3.0 yadalwa, isizindalwazi esikhulu kunazo zonke samanethiwekhi amaprotheni asekelwe ku-AP-MS.Nakuba i-AP-MS inamandla kakhulu, izinyathelo ze-cell lysis kanye ne-dilution ekuhambeni komsebenzi zichemile ekusebenzisaneni okubophezelayo okubuthakathaka futhi okwesikhashana futhi zethula izinto zobuciko zangemuva kokuhlaziya njengamapheya okusebenzelana angamanga antula ukuhlanganisa ngaphambi kwe-lysis.
Ukuze kubhekwane nalezi zinkinga, ama-amino acid angewona awemvelo (UAA) anamaqembu axhumanisayo kanye nezinkundla zokulebula eziseduze ze-enzymatic (PL) (isb. i-APEX ne-BioID)5.Nakuba indlela ye-UAA isetshenziswe ngempumelelo ezimweni eziningi futhi inikeza ulwazi mayelana nokunamathela okuqondile kwamaprotheni, ukuthuthukiswa kwendawo yokufaka i-UAA kusadingeka.Okubaluleke nakakhulu, kuyindlela yokulebula ye-stoichiometric engenakho ukuguqulwa okunamandla kwemicimbi yokulebula.Ngokuphambene, izindlela ze-enzymatic PL, ezifana nendlela ye-BioID, zixubanisa i-biotin ligase eyakhiwe ibe yi-POI7, kamuva eyenza i-biotin isebenze ukuze yakhe indawo emaphakathi ye-biotinyl-AMP ester.Ngakho-ke i-enzyme ivuselela futhi ikhiphe "ifu" le-biotin elicushiwe elilebula izinsalela ze-lysine eziseduze.Nokho, i-BioID idinga amahora angaphezu kwangu-12 ukuze ithole isignali enelebula eyanele, evimbela ukusetshenziswa kwayo ngokulungiswa kwesikhashana.Isebenzisa ukuziphendukela kwemvelo okuqondisiwe okusekelwe ekuboniseni imvubelo, i-TurboID yaklanywa ngokusekelwe ku-BioID ukuze isebenze kahle, ivumele ukulebula okuphumelelayo nge-biotin phakathi nemizuzu eyi-10, okuvumela izinqubo ezinamandla kakhudlwana ukuba zifundwe.Ngenxa yokuthi i-TurboID iyasebenza kakhulu futhi amazinga e-biotin e-endogenous anele ukulebula kwezinga eliphansi, ukulebula kwangemuva kuba inkinga engaba khona lapho ukulebula okuthuthukisiwe kakhulu futhi okunesikhathi kudingwa ngokwengezwa kwe-biotin yangaphandle.Ukwengeza, ama-ester acushiwe awasebenzi kahle (t1/2 ~5 min), okungaholela endaweni enkulu yokulebula, ikakhulukazi ngemva kokugcwala kwamaphrotheni angomakhelwane nge-biotin 5. Ngenye indlela, ukuhlanganiswa kofuzo kwe-ascorbate peroxidase (okungukuthi i-biotin- i-phenol radicals futhi ivumela ukulebula amaprotheni phakathi nomzuzu owodwa9, 10. I-APEX isetshenziselwa kabanzi ukukhomba ama-proteome angaphansi kweselula, i-membrane protein complexes, kanye ne-cytosolic signaling protein complexes11, 12. Nokho, isidingo sokugxila okuphezulu kwama-peroxides singathinta amaprotheni e-redox noma izindlela, ukuphazamisa izinqubo zamaselula.
Ngakho-ke, indlela entsha ekwazi ukukhiqiza izinhlobo zokucindezela zerediyasi ezisebenzayo ngokunemba okuphezulu kwendawo kanye nesikhashana ngaphandle kokuphazamisa kakhulu izindlela zamaselula kuzoba isengezo esibalulekile ezindleleni ezikhona. Phakathi kwezinhlobo ezisebenzayo, umoya-mpilo we-singlet wavusa ukunaka kwethu ngenxa yokuphila kwayo okufushane kanye ne-radius elinganiselwe yokusabalalisa (t1/2 <0.6 µs kumaseli)13. Phakathi kwezinhlobo ezisebenzayo, umoya-mpilo we-singlet wavusa ukunaka kwethu ngenxa yokuphila kwayo okufushane kanye ne-radius elinganiselwe yokusabalalisa (t1/2 <0.6 µs kumaseli)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузий 3, 3/3 (t. Phakathi kwamafomu asebenzayo, umoya-mpilo we-singlet udonse ukunaka kwethu ngenxa yokuphila kwayo okufushane kanye nerediyasi yokusabalalisa elinganiselwe (t1/2 <0.6 µs kumaseli)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）而引起了我們的注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 дифф16/кси, как радиуса диф16/зим2. Phakathi kwamafomu asebenzayo, i-oksijeni ye-singlet idonsa ukunaka kwethu ngenxa yesikhathi sayo esifushane sokuphila kanye nerediyasi elinganiselwe yokusabalalisa (t1/2 <0.6 μs kumaseli).I-oksijeni ye-Singlet kubikwe ukuthi i-oxidize ngokungahleliwe i-methionine, i-tyrosine, i-histidine ne-tryptophan, okwenza kube yi-polar 14,15 yokunamathisela kuma-amine noma ama-thiol based probes16,17.Nakuba umoya-mpilo we-singlet usetshenziselwe ukulebula i-subcellular compartment RNA, amasu okuphinda asebenzise izimpawu eziseduze ze-POI ahlala engahloliwe.Lapha, sethula inkundla ebizwa ngokuthi i-photoactivation-dependent proximity labeling (PDPL), lapho sisebenzisa khona ukukhanya okuluhlaza okwesibhakabhaka ukuze sikhanyise ama-POI ahlanganiswe ne-miniSOG photosensitizer futhi sicuphe isizukulwane somoya-mpilo we-singlet ukuze oxidize izinsalela eziseduze, okulandelwa izinguquko eziqukethe i-amine ukuze kufakwe ama-chemical probes. amangqamuzana aphilayo aphakathi..Sihlole iqembu lama-chemical probes ukuze sikhulise ukucaciswa komaka futhi sahlonza amasayithi okulungisa sisebenzisa ukuhamba komsebenzi okuvulekile kwe-proteomics.Ukuqhathaniswa kwehlangothi nehlangothi kwe-PDPL ne-TurboID kukhombisa ukumbozwa kwe-proteomic okucace kakhudlwana nokuphelele kwe-PDPL.Sisebenzise le ndlela kumaka akhethekile we-organelle we-subcellular proteome kanye nokuhlonza okujwayelekile kwe-proteome yabalingani ababophayo beprotheni elawula i-epigenetic ehlotshaniswa nomdlavuza BRD4 kanye ne-Parkinson's ehlotshaniswa nesifo i-E3 ligase Parkin, okuqinisekise kokubili inethiwekhi eyaziwayo nengaziwa yamaprotheni. ukusebenzisana..Ikhono le-PDPL lokubona ama-substrates e-E3 kuma-protein complexes amakhulu limelela isimo lapho ukuqashelwa kokuhlanganisa okungaqondile kuyadingeka.Ama-parkin substrates amabili angaziwa alamula i-ubiquitination-proteasome aqinisekisiwe endaweni.
I-Photodynamic therapy (PDT)19 kanye ne-chromophore-assisted laser inactivation (CALI)20, lapho ukukhanya okukhanyayo okunama-photosensitizer kukhiqiza umoya-mpilo we-singlet, kungenza amaprotheni aqondiwe noma kubangele ukufa kweseli.Njengoba umoya-mpilo we-singlet uyinto esebenza kakhulu enebanga lokusabalalisa elingokomqondo elingu-70 nm, i-oxidation elinganiselwe yendawo ezungeze i-photosensitizer ingalawulwa.Ngokusekelwe kulo mqondo, sinqume ukusebenzisa umoya-mpilo we-singlet ukuze sifinyelele ukulebula okuseduze kwama-protein complexes kumaseli aphilayo.Senze indlela ye-PDPL ye-chemoproteomic yokufeza imisebenzi emine: (1) ukwenza ukukhiqizwa kwe-oksijeni ye-singlet esebenzayo efana nendlela ye-PL enzymatic;(2) ukuhlinzeka ngokulebula okuxazululwe isikhathi ekuqalisweni kokukhanya;(3) ngokushintsha (4) Gwema ukusebenzisa ama-cofactor angapheli (njenge-biotin) ukuze unciphise ingemuva, noma usebenzise ama-reagents angaphandle aphazamisa kakhulu (njengama-peroxides) ukuze unciphise ukuchayeka kweseli ekucindezelekeni kwendawo ezungezile.
Ama-photosensitizer angahlukaniswa abe izigaba ezimbili okuhlanganisa nesisindo samangqamuzana amancane e-fluorophore (isb. i-rose bengal, i-methylene blue)22 kanye namaprotheni amancane afakwe ngokofuzo (isb. i-miniSOG, i-KillerRed)23.Ukuze sizuze umklamo oyimojuli, sithuthukise inkundla yesizukulwane sokuqala se-PDPL ngokwengeza amaprotheni e-photosensitizer (PS) ku-POI24,25 (Umfanekiso 1a).Uma ikhanyiswe ngokukhanya okuluhlaza okwesibhakabhaka, i-singlett oksijeni i-oxidizes proximal nucleophilic amino acid residues, okuholela ekubeni i-umpolung polarity eyi-electrophilic futhi ingasabela ngokuqhubekayo nge-amine probe nucleophiles16,17.I-probe yakhelwe ngesibambo se-alkyne ukuvumela ikhemistri yokuchofoza futhi idonsele phansi ukuze uthole uhlamvu lwe-LC/MS/MS.
Umdwebo ohleliwe wokulebula ama-protein complexes mediated by miniSOG.Uma evezwe ekukhanyeni okuluhlaza okwesibhakabhaka, amaseli aveza i-miniSOG-POI akhiqiza umoya-mpilo we-singlet, oshintsha amaphrotheni asebenzisanayo kodwa hhayi amaprotheni angabophi.Imikhiqizo emaphakathi ye-photooxidation ibanjwa ngamalebula adluliselwe we-amine chemical probe ukuze kwakheke ama-covalent adducts.Iqembu le-alkynyl ku-chemistry probe livumela ikhemistri yokuchofoza ukuze inothiswe ngokudonsela phansi kulandele ubuningi be-LC-MS/MS.b Isakhiwo samakhemikhali se-amine probes 1-4.c Ukuhlaziywa kwejeli ye-fluorescent emele izinkomba ze-mitochondrial zasendaweni ze-miniSOG-mediated proteomic kusetshenziswa ama-probe 1-4 kanye nokulinganisa okuhlobene okusekelwe ku-densitometry yejeli.Isilinganiso sesignali nengemuva sama-probe amakhemikhali sihlolwe kusetshenziswa izivivinyo zokulawula okungekuhle okungabandakanyi ukukhanya okuluhlaza okwesibhakabhaka noma kusetshenziswa amaseli e-HEK293T ngaphandle kwesisho se-miniSOG.n = 2 amasampula azimele ngokwebhayoloji.Ichashazi ngalinye limelela ikhophi yebhayoloji.d Ukutholwa kommeleli kanye nomthamo we-PDPL kusetshenziswa i-probe 3 ethuthukisiwe lapho kukhona noma zingekho izingxenye ezibonisiwe ze-PDPL ezifana c.n = 3 amasampula azimele ngokwebhayoloji.Ichashazi ngalinye limelela ikhophi yebhayoloji.Imigqa emaphakathi kanye nentshebe imele isilinganiso kanye ± ukuchezuka okujwayelekile.I-CBB: I-Coomassie Brilliant Blue.e I-confocal imaging ye-singlett oxygen enebala elibomvu kakhulu le-Si-DMA.Ibha yesikali: 10 µm.I-imaging ye-gel kanye nokuhlolwa kwe-confocal kwaphindwa ngokuzimela okungenani kabili ngemiphumela efanayo.
Siqale sahlola ikhono lama-photosensitizers avuthiwe miniSOG26 kanye ne-KillerRed23, evezwe ngokuqinile ku-HEK293T, ukulamula ukulebula kwe-propargylamine kwe-proteome njengophenyo lwamakhemikhali (I-Supplementary Fig. 1a).Ukuhlaziywa kwe-Gel fluorescence kubonise ukuthi ukulebula okuphelele kwe-proteome kwafinyelelwa kusetshenziswa i-miniSOG kanye nemisebe yokukhanya eluhlaza okwesibhakabhaka, kuyilapho kungekho mkhiqizo olebulayo obonakalayo obonwe nge-KillerRed.Ukuze sithuthukise isilinganiso sesignali-kuya-ngemuva, sibe sesihlola isethi yama-chemical probe aqukethe i-aniline (1 no-3), i-propylamine (2), noma i-benzylamine (4).Siqaphele ukuthi amaseli e-HEK293T ngokwawo anesignali ephakeme yangemuva uma kuqhathaniswa nokungabikho kokukhanya okuluhlaza okwesibhakabhaka, okungenzeka ngenxa ye-photosensitizer ye-riboflavin engapheli, i-flavin mononucleotide (FMN) 27. Ama-probe amakhemikhali asekelwe ku-aniline 1 kanye no-3 anikeze ukucaciswa okungcono kangcono, i-HEK293T iveza ngokuzinzile i-miniSOG ku-mitochondria ibonisa ukukhuphuka kwesignali >8 ngokuphindwe ka-8 ku-probe 3, kuyilapho i-probe 2 isetshenziswa endleleni yokulebula ye-RNA CAP-seq ibonisa kuphela ~2.5- ukunyuswa kwesignali yokugoqa, cishe ngenxa yezintandokazi ezihlukile zokuphinda zisebenze phakathi kwe-RNA namaprotheni (Umfanekiso we-1b, c). Ama-probe amakhemikhali asekelwe ku-aniline 1 kanye no-3 anikeze ukucaciswa okungcono kangcono, i-HEK293T iveza ngokuzinzile i-miniSOG ku-mitochondria ibonisa ukukhuphuka kwesignali >8 ngokuphindwe ka-8 ku-probe 3, kuyilapho i-probe 2 isetshenziswa endleleni yokulebula ye-RNA CAP-seq ibonisa kuphela ~2.5- ukunyuswa kwesignali yokugoqa, cishe ngenxa yezintandokazi ezihlukile zokuphinda zisebenze phakathi kwe-RNA namaprotheni (Umfanekiso we-1b, c).Ama-probe amakhemikhali asekelwe ku-aniline 1 kanye no-3 abonise ukucaciswa okungcono kakhulu: I-HEK293T, eveza ngokuzinzile i-miniSOG ku-mitochondria, ikhombisa ukukhuphuka okuphindwe kayisi-8 kwesignali ye-probe 3, kuyilapho i-probe 2, isetshenziswa ngendlela yokulebula ye-CAP-seq RNA, kuphela ibonisa i- ~ 2.5-fold fold signal ukwanda, mhlawumbe ngenxa yezintandokazi ezihlukile zokuphinda zisebenze phakathi kwe-RNA namaprotheni (Fig. 1b, c).基于 苯胺 的 化学 探针 1 和 3 具有具有 好的, Hek293t 在在 中线 稳定线 稳定 稳定2.5-倍信号增加，可能是由于RNA 和蛋白质之间的不同反应偏好（图1b，c）.基于 苯胺 的 化学 探针 1 和 3 具有具有 的 的, Hek2993t 在在-倍信号增加，可能是由于RNAAma-probe amakhemikhali asekelwe ku-Aniline 1 kanye no-3 ayenokucaciswa okungcono kangcono, i-HEK293T izwakalise ngokuzinzile i-miniSOG ku-mitochondria, futhi i-probe 3 ibe nokukhuphuka kwesignali okuphindwe izikhathi ezingu-8, kuyilapho i-probe 2 yendlela yokulebula ye-CAP-seq RNA ibonise kuphela ukunyuka okungu-~2.5 -fold.esignali, mhlawumbe ngenxa yokukhetha okuhlukile kokusabela phakathi kwe-RNA namaprotheni (Fig. 1b, c).Ngaphezu kwalokho, i-probe 3 isomers kanye ne-hydrazine probes (i-probes 5, 6, 7) ihlolwe, iqinisekisa ukuthuthukiswa kwe-probe 3 (I-Supplementary Fig. 1b, c).Ngokufanayo, ukuhlaziywa kwe-in-gel fluorescence kwembula amanye amapharamitha wokuhlola athuthukisiwe: i-irradiation wavelength (460 nm), i-chemical probe concentration (1 mM), kanye nesikhathi sokukhanyisa (20 min) (Supplementary Fig. 2a–c).Ukweqa noma iyiphi ingxenye noma isinyathelo kuphrothokholi ye-PDPL kubangele ukuguqulwa kwesignali ebalulekile ngemuva (Umfanekiso 1d).Ngokuphawulekayo, ukulebula kwamaprotheni kwehliswe kakhulu phambi kwe-azide ye-sodium noma i-trolox, eyaziwa ngokucisha umoya-mpilo owodwa.Ukuba khona kwe-D2O, eyaziwa ngokuzinzisa umoya-mpilo we-singlet, kuthuthukisa isignali yokulebula.Ukuze kuphenywe umnikelo wezinye izinhlobo ze-oxygen esebenzayo ekulebeni, i-mannitol ne-vitamin C yengezwe ukuze kusungulwe i-hydroxyl ne-superoxide radical scavengers, ngokulandelana, i-18, i-29, kodwa ayizange itholakale ukunciphisa ukulebula.Ukwengezwa kwe-H2O2, kodwa hhayi ukukhanya, akuzange kubangele ukulebula (I-Supplementary Fig. 3a).I-Fluorescence singlet imaging oxygen ene-Si-DMA probes iqinisekise ukuba khona komoya-mpilo we-singlet kuntambo ye-HEK293T-miniSOG, kodwa hhayi ocingweni lokuqala lwe-HEK293T.Ukwengeza, i-mitoSOX Ebomvu ayikwazanga ukubona ukukhiqizwa kwe-superoxide ngemva kokukhanyisa (I-Fig. 1e kanye ne-Supplementary Fig. 3b) 30. Le datha iphakamisa ngokuqinile ukuthi i-oksijeni ye-singlet iyinhlobo eyinhloko ye-oksijeni esebenzayo ebhekene nokulebula okulandelayo kwe-proteomic.I-Cytotoxicity ye-PDPL yahlolwa kuhlanganise ne-irradiation yokukhanya okuluhlaza okwesibhakabhaka kanye nama-probes amakhemikhali, futhi akukho cytotoxicity ebalulekile eyabonwa (I-Supplementary Fig. 4a).
Ukuze sifunde indlela yokulebula futhi sikwazi ukuhlonza i-proteomic yezakhiwo zamaprotheni kusetshenziswa i-LC-MS/MS, sidinga kuqala ukunquma ukuthi yimaphi ama-amino acid ashintshiwe kanye ne-delta mass yamalebula okuhlola.I-Methionine, i-histidine, i-tryptophan ne-tyrosine kuye kwabikwa ukuthi ishintshwe yi-singlett oxygen14,15.Sihlanganisa ukuhamba komsebenzi kwe-TOP-ABPP31 nosesho oluvulekile olungachemile olunikezwa inkundla yekhompyutha ye-FragPipe esekelwe ku-MSFragger32.Ngemva kokuguqulwa kwe-singlett komoya-mpilo kanye nokulebula kokuhlolwa kwamakhemikhali, ikhemistri yokuchofoza yenziwa kusetshenziswa ilebula yokunciphisa i-biotin equkethe isixhumi esivulekayo, okulandelwa ukwelula kwe-neutravidin kanye nokugaya kwe-trypsin.I-peptide eguquliwe, isaboshelwe ku-resin, yafakwa isithombe ukuze kuhlaziywe i-LC-MS/MS (Umfanekiso 2a kanye Nedatha Eyengeziwe 1).Inani elikhulu lokuguqulwa lenzekile kuyo yonke i-proteome ngokufana kwemephu ye-peptide (PSM) engaphezu kuka-50 esohlwini (Fig. 2b).Ngokumangazayo, sabona ukuguqulwa kwe-histidine kuphela, mhlawumbe ngenxa yokusebenza kabusha okuphezulu kwe-histidine ene-oxidized kuma-aniline probes kunamanye ama-amino acid.Ngokwendlela eshicilelwe ye-histidine oxidation nge-singlett oxygen, i-21,33 isakhiwo se-delta-mass esihlongozwayo se-+229 Da sihambisana ne-adduct ye-probe 3 ne-2-oxo-histidine ngemva kwe-oxidation emibili, kuyilapho i-+247 Da ingumkhiqizo we-hydrolysis. kwe-+229 Da (I-Supplementary Fig. 5).Ukuhlolwa kwe-spectrum ye-MS2 kubonise ukwethembeka okuphezulu kokuhlonza iningi lama-y kanye ne-b ions, okuhlanganisa ukuhlonza ama-ion aguquliwe we-fragment (y no-b) (Fig. 2c).Ukuhlaziywa kokuqukethwe kokulandelana kwendawo kwe-PDPL-modified histidines kwembula okuthandwayo kwe-motif okumaphakathi kwezinsalela ze-hydrophobic ezincane ezikhundleni ze-±1 (I-Supplementary Fig. 4b).Ngokwesilinganiso, i-1.4 histidines ikhonjwe ngeprotheni ngayinye, futhi iziza zalezi zomaka zinqunywe indawo etholakalayo e-solvent (SASA) kanye nokuhlaziywa kokutholakala kwe-solvent (RSA) (I-Supplementary Fig. 4c, d).
Ukugeleza komsebenzi okungachemile kokutadisha ukukhetha kwensalela kusetshenziswa inkundla yekhompyutha ye-FragPipe enikwa amandla yi-MSFragger.Izixhumanisi ezivulekayo zisetshenziswa ku-Click chemistry ukuvumela ukuchithwa kwe-photocleavage yama-peptide aguquliwe kusuka ku-streptavidin resin.Ukusesha okuvulekile kwaqaliswa ukuze kuhlonzwe izinguquko eziningi, kanye nezinsalela ezifanele.b Nikeza inqwaba yokuguqulwa okwenzeka kuyo yonke i-proteome.Imephu ye-Peptide ye-PSM.c MS2 spectral annotation of histidine sites modified with probe 3. Njengesibonelo esimele, ukusabela okuhlangene okune-probe 3 kwengeze +229.0938 Da ku-amino acid eguquliwe.d Ukuhlolwa kokuguquguquka okusetshenziselwa ukuhlola omaka be-PDPL.I-PRDX3 (H155A, H225A) kanye ne-PRDX1 (H10A, H81A, H169A) zadluliselwa ngama-plasmids ohlobo lwasendle ukuze kutholwe i-anti-Flag.e I-peptide yokwenziwa yaphendulwa nge-miniSOG ehlanzekile phambi kwe-probe 3 kanye nemikhiqizo ehambisanayo ne-Δm +247 kanye ne-+229 yaphawulwa ku-spectrum ye-LC-MS.f Ukusebenzisana kwe-in vitro amaprotheni kuya kumaphrotheni okumodela nge-miniSOG-6xHis-tag kanye ne-anti-6xHis antibody.I-Antibiotin (streptavidin-HRP) kanye nokuhlaziywa kwe-anti-mouse blot yaseNtshonalanga ye-miniSOG-6xHis/anti-6xAmasosha omzimba akhe ayinkimbinkimbi alebulwe nge-probe 3, kuye ngesikhathi sokuchayeka ekukhanyeni.Amalebula amaphrotheni ngamanye avezwa ngesisindo samangqamuzana ahambisanayo: Iketango lokukhanya le-LC antibody, iketango elisindayo le-HC antibody.Lezi zivivinyo ziphindwe ngokuzimela okungenani kabili ngemiphumela efanayo.
Ukuze kuqinisekiswe i-biochemical site yokulebula, i-PRDX3 ne-PRDX1 ekhonjwe nge-mass spectrometry yashintshwa isuka ku-histidine yaba i-alanine futhi yaqhathaniswa nohlobo lwasendle ekuhlolweni kokudluliselwa kwegciwane.Imiphumela ye-PDPL ibonise ukuthi ukuguqulwa kunciphise kakhulu ukulebula (Fig. 2d).Ngaleso sikhathi, ukulandelana kwe-peptide okukhonjwe ekusesheni okuvulekile kwahlanganiswa futhi kwaphendulwa ku-vitro nge-miniSOG ehlanzekile phambi kwe-probe 3 nokukhanya okuluhlaza okwesibhakabhaka, imikhiqizo ekhiqizayo ngokushintshwa okukhulu kwe-+247 kanye +229 Da lapho itholwa yi-LC-MS (Fig. ngo. 2e).).Ukuhlola ukuthi ingabe amaprotheni asondelene asebenzisanayo angase alebulwe nge-vitro ngokuphendula ukwenziwa kwesithombe kwe-miniSOG, sidizayine ukuhlolwa kokusondela kokwenziwa ngokusebenzisana phakathi kwe-miniSOG-6xHis protein kanye ne-anti-His monoclonal antibody in vitro (Umfanekiso 2f).Kulesi sivivinyo, besilindele ukulebula okusondelene kwamaketanga asindayo nalula amasosha omzimba nge-miniSOG.Eqinisweni, i-anti-mouse (eqaphela amaketanga asindayo futhi alula we-anti-6xHis-labelele antibody) kanye nama-streptavidin Western blots abonise i-biotinylation eqinile yamaketanga asindayo futhi alula.Ngokuphawulekayo, siqaphele i-miniSOG autobiotinylation ngenxa yethegi engu-6xHis kanye nezixhumanisi phakathi kwamaketanga alula nasindayo, okungenzeka ahlobane negebe elichazwe ngaphambilini phakathi kwe-lysine kanye ne-2-oxo-histidine impendulo eseduze.Sengiphetha, siphetha ngokuthi i-PDPL ilungisa i-histidine ngendlela encike ekusondeleni.
Umgomo wethu olandelayo kwakuwukuphawula i-subcellular proteome ukuhlola ukucaciswa kokulebula kwe-in situ.Ngakho-ke, siveze ngokuzinzile i-miniSOG ku-nucleus, i-mitochondrial matrix, noma ulwelwesi lwangaphandle lwe-ER lwamaseli we-HEK293T (Fig. 3a).Ukuhlaziywa kwe-gel fluorescence kwembule amabhande anamalebula amaningi ezindaweni ezintathu zamaselula kanye namaphethini okulebula ahlukene (Umfanekiso 3b).Ukuhlaziywa kwe-imaging ye-Fluorescence kubonise ukucaciswa okuphezulu kwe-PDPL (Fig. 3c).Ukugeleza komsebenzi we-PDPL kwalandelwa ukusabela ngokuchofoza ngodayi be-rhodamine ukuze kucaciswe ama-proteome angaphansi kweselula esebenzisa i-fluorescence microscopy, futhi amasiginali e-PDPL ahlanganiswa ne-DAPI, ama-trackers e-mitochondrial, noma ama-ER trackers, okuqinisekisa ukwethembeka okuphezulu kwe-PDPL.Ezindaweni ezintathu ze-organelle, ukuqhathaniswa okuhlangene kwe-PDPL ne-TurboID kusetshenziswa i-avidin western blot kubonise ukuthi i-PDPL yayibhalwe ngokucacile uma iqhathaniswa nezilawuli zayo.Ngaphansi kwezimo ze-PDPL, amabhendi anamalebula amaningi avele, abonisa amaprotheni amaningi anelebula le-PDPL (I-Supplementary Fig. 6a-d).
ukumelwa kwe-Schematic kokulebula kwe-miniSOG-mediated organelle-specific proteome.i-miniSOG iqondise i-matrix ye-mitochondrial ngokuhlanganisa ku-N-terminal 23 amino acids yomuntu we-COX4 (mito-miniSOG), i-nucleus nge-fusion ku-H2B (nucleus-miniSOG), kanye ne-Sec61β ngohlangothi lwe-cytoplasmic lwe-ER membrane (ER-miniSOG ).Izinkomba zihlanganisa i-gel imaging, imaging confocal, kanye ne-mass spectrometry.b Izithombe zejeli ezimele amaphrofayili e-PDPL amathathu aqondene ne-organelle.I-CBB Coomassie Brilliant Blue.c Izithombe ezihlangene ezimele amaseli e-HEK293T aveza kahle i-miniSOG enokuhlukahluka kwe-subcellular localizations okutholwe i-antibody ebhalwe ukuthi V5 (bomvu).Omaka be-subcellular basetshenziselwa i-mitochondria ne-ER (eluhlaza).Ukugeleza komsebenzi kwe-PDPL kufaka phakathi ukutholwa kwe-miniSOG (yellow) enelebuli yama-subcellular proteomes kusetshenziswa ikhemistri yokuchofoza i-Cy3-azide.Ibha yesikali: 10 µm.d Iziqephu zentaba-mlilo zama-proteome amakiwe e-PDPL kuma-organelle ahlukahlukene alinganiswa ngobuningi obungalebuli (n = 3 ukuhlola okuzimele kwebhayoloji).Ukuhlolwa kuka-t koMfundi onemisila emibili kusetshenziswe ezindaweni zentaba-mlilo.Uhlobo lwasendle lwe-HEK293T lusetshenziswe njengokulawula okungekuhle. Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswa ngokubomvu (p <0.05 kanye>>2-fold ion intensity umehluko). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswa ngokubomvu (p <0.05 kanye>>2-fold ion intensity umehluko). Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswe ngokubomvu (p <0.05 kanye> umehluko we-2-fold intensity ion).显着变化的蛋白质以红色突出显示（p <0.05 和> 2 倍离子强度差异）。显着变化的蛋白质以红色突出显示（p <0.05和> 2 Значительно измененные белки выделены красным цветом (p < 0,05 и > 2-кратная разница в ионной силе). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswe ngokubomvu (p <0.05 kanye> umehluko we-2-fold in ionic amandla).Amaprotheni ahlobene abalulekile ku-HEK293T-miniSOG kodwa angabalulekile ku-HEK293T aboniswa ngokuluhlaza.e Ukuhlaziywa kokucaciswa kwedathasethi ye-proteomic evela ekuhlolweni d.Isamba senani lamaprotheni abalulekile ngokwezibalo ku-organelle ngayinye (amachashazi abomvu naluhlaza) amakwe phezulu.Ama-histograms abonisa amaprotheni enziwe endaweni kuma-organelles asekelwe ku-MitoCarta 3.0, ukuhlaziywa kwe-GO kanye no-A. Ting et al.abantu.Hlukanisa amasethi edatha we-mitochondria, nuclei, ne-ER.Lezi zivivinyo ziphindwe ngokuzimela okungenani kabili ngemiphumela efanayo.Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Ngokukhuthazwa ijeli nemiphumela yezithombe, ukulinganisa okungenalebula kwasetshenziswa ukuze kulinganise i-proteome ekhonjiwe ku-organelle ngayinye (Idatha Eyengeziwe 2).I-HEK293T engatheleleki isetshenziswe njengesilawuli esinegethivu ukuze kukhishwe omaka bangemuva. Ukuhlaziywa kwesakhiwo sentaba-mlilo kuboniswe amaprotheni anothisiwe kakhulu (p <0.05 kanye > no-2-fold fold ion intensity) kanye namaprotheni e-singleton akhona kuphela emigqeni eveza i-miniSOG (Fig. 3d amachashazi abomvu naluhlaza). Ukuhlaziywa kwesakhiwo sentaba-mlilo kuboniswe amaprotheni anothisiwe kakhulu (p <0.05 kanye > no-2-fold fold ion intensity) kanye namaprotheni e-singleton akhona kuphela emigqeni eveza i-miniSOG (Fig. 3d amachashazi abomvu naluhlaza). 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). Ukuhlaziywa kwesakhiwo sentaba-mlilo kubonise amaprotheni anothisiwe kakhulu (p<0.05 kanye>>2-fold fold ion intensity) kanye namaprotheni angawodwa akhona kuphela emigqeni ye-miniSOG-expressing (Fig. 3d, amachashazi 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）以及仅存在纎中存在纎MINISOG 一亲边存在纎miniSOG 艾红诚）火山图 分析 显示 出 显着 的 蛋白质 (p <0.05 和> 2 倍 离子 强度) 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). Ukuhlaziywa kwesakhiwo sentabamlilo kwembule amaprotheni anothisiwe kakhulu (p<0.05 kanye>>2x amandla e-ionic) kanye namaprotheni angawodwa akhona kulayini wokubonisa we-miniSOG (amachashazi abomvu naluhlaza ku-Fig. 3d).Ngokuhlanganisa le datha, sihlonze i-1364, 461, kanye ne-911 amaprotheni abalulekile ngokwezibalo enuclear, mitochondrial, kanye ne-ER yangaphandle ye-membrane, ngokulandelanayo.Ukuhlaziya ukunemba kwe-organelle-localized PDPL, sisebenzise i-MitoCarta 3.0, i-Gene Ontology (GO) analysis, kanye no-A. Ting et al.isethi yedatha8 yasetshenziselwa i-mitochondria, i-nucleus, ne-ER ukuhlola ukucaciswa kwe-organelle yamaprotheni atholiwe, okuhambisana nokunemba kwe-73.4, 78.5, ne-73.0% (Fig. 3e).Ukucaciswa kwe-PDPL kuqinisekisa ukuthi i-PDPL iyithuluzi elikahle lokuhlonza ama-proteome aqondene ne-organelle.Ngokuphawulekayo, ukuhlaziywa kwe-subochondrial kwamaprotheni e-mitochondrial ahlonziwe kubonise ukuthi i-proteome eboshiwe yasatshalaliswa kakhulu ku-matrix kanye nolwelwesi lwangaphakathi (226 kanye ne-106, ngokulandelana), ibalwa ku-91.7% (362) yenani eliphelele lamaphrotheni e-mitochondrial akhonjiwe.izinga eliphezulu le-PDPL laqinisekiswa ngaphezu kwalokho (I-Supplementary Fig. 7a).Ngokufanayo, ukuhlaziywa kwe-subnuclear kubonise ukuthi i-proteome ethathwe ngokuyinhloko yasakazwa ku-nucleus, nucleoplasm, ne-nucleolus (I-Supplementary Fig. 7b).Ukuhlaziywa kwe-nuclear proteomic nge-peptide yesignali yendawo ye-nuclear (3xNLS) kubonise ukunemba okufanayo nokwakhiwa kwe-H2B (I-Supplementary Fig. 7c–h).Ukuze kutholwe ukucaciswa komaka we-PDPL, i-nuclear laminin A yakhethwa njengesicupho se-POI7 esibekwe ngokucace kakhulu.I-PDPL ikhombe amaprotheni angu-36 anothiswe kakhulu, lapho amaprotheni angu-12 (30.0% kuhlanganise ne-lamin A) ayengamaprotheni asebenzayo e-lamin A achazwe yisizindalwazi se-String, anephesenti eliphezulu kunendlela ye-BioID (amaprotheni angu-122) angu-28 kwangu-28. , 22.9 %) 7. Indlela yethu ihlonze amaprotheni ambalwa, okungenzeka ngenxa yezindawo zokulebula ezilinganiselwe, ezenziwe zaba nokwenzeka ngokusebenza okwengeziwe komoya-mpilo we-singlett.Ukuhlaziywa kwe-GO kubonise ukuthi amaprotheni akhonjiwe ikakhulukazi atholakala ku-nucleoplasm (26), ulwelwesi lwenuzi (10), ulwelwesi lwenuzi (9), nezimbotshana zenuzi (5).Ngokuhlangene, lawa maphrotheni asendaweni yenuzi abalelwa ku-80% wamaprotheni athuthukisiwe, aphinde abonise ukucaciswa kwe-PDPL (I-Supplementary Fig. 8a-d).
Ngemva kokuthola ikhono le-PDPL lokwenza ukumaka okuseduze kuma-organelles, sibe sesihlola ukuthi ingabe i-PDPL ingasetshenziswa yini ukuhlaziya ozakwethu ababophayo be-POI.Ikakhulukazi, siye safuna ukuchaza ukuhlaziywa kwe-PDPL kwamaphrotheni e-cytosolic, abhekwa njengezinhloso ezinzima kakhulu kunozakwabo basendaweni be-membrane ngenxa yemvelo yabo enamandla kakhulu.Iphrotheni ye-bromodomain ne-extraterminal (BET) i-BRD4 idonse ukunaka kwethu ngendima yayo ebalulekile ezifweni ezihlukahlukene 35, 36.I-complex eyakhiwe yi-BRD4 iyi-coactivator ebhaliwe futhi iyithagethi ebalulekile yokwelapha.Ngokulawula ukuvezwa kwezici ze-c-myc kanye ne-Wnt5a yokubhala, i-BRD4 kucatshangwa ukuthi iyisinqumo esiyinhloko se-acute myeloid leukemia (AML), i-myeloma eminingi, i-Burkitt's lymphoma, umdlavuza wekoloni kanye nezifo ezivuthayo37,38.Ngaphezu kwalokho, amanye amagciwane aqondise i-BRD4 ukuze ilawule ukubhaliswa kwegciwane kanye neselula, njenge-papillomavirus, i-HIV, ne-SARS-CoV-236,39.
Ukuze wenze imephu ukusebenzisana kwe-BRD4 kusetshenziswa i-PDPL, sihlanganise i-miniSOG ne-N- noma i-C-terminal isoform emfushane ye-BRD4.Imiphumela ye-proteomic yembula izinga eliphezulu lokugqagqana phakathi kwalezi zakhiwo ezimbili (I-Supplementary Fig. 9a).I-nuclear proteome ehlonzwe nge-miniSOG-H2B ihlanganisa u-77.6% wamaprotheni asebenzisana ne-BRD4 (I-Supplementary Fig. 9b).Khona-ke, izikhathi ezihlukene zokukhanya (2, 5, 10, 20 min) zasetshenziswa ukulungisa irediyasi yomaka (Umfanekiso 4a kanye nedatha eyengeziwe yesi-3).Siphetha ngokuthi ngezikhathi ezimfushane zezithombe, i-PDPL ngokuyinhloko izolebula ozakwethu ababophezela ngokuqondile, kuyilapho izikhathi ezinde zizohlanganisa amaphrotheni akhonjwe ngezikhathi ezimfushane zokuthatha izithombe kanye nokuhlosiwe okungaqondile kuzakhiwo zokulebula.Eqinisweni, sithole ukunqwabelana okuqinile phakathi kwamaphoyinti esikhathi aseduze (84.6% ye-2 ne-5 min; 87.7% ye-5 kanye ne-10 min; 98.7% ye-10 ne-20 min) (Fig. 4b kanye ne-Supplementary Fig. 9c).Kuwo wonke amaqembu okuhlola, asitholanga kuphela ukuzilebula kwe-BRD4, kodwa izinjongo ezimbalwa ezaziwayo ezifana ne-MED1, CHD8, BICRA, NIPBL, SMC1A, kanye ne-HMGB1 echazwe kusizindalwazi seyunithi yezinhlamvu.Amandla e-ionic alezi zinhloso alingana nesikhathi sokuchayeka (Fig. 4c kanye ne-Supplementary Fig. 9d).Ukuhlaziywa kwe-GO kwamaprotheni ahlonzwe eqenjini lemizuzu engu-2 kubonise ukuthi amaprotheni akhonjiwe atholakala ku-nucleus futhi abandakanyeka ekulungiseni kabusha i-chromatin kanye nomsebenzi we-RNA polymerase.Umsebenzi wamangqamuzana weprotheni wawunothisiwe ekubopheni i-chromatin noma ukuhlanganiswa kwe-transcriptal, okuhambisana nomsebenzi we-BRD4 (Fig. 4d).Ukuhlaziywa kokusetshenziswa kweprotein okunikwe amandla esizindalwazi sezintambo kwembula izinga lokuqala lokusebenzelana okungaqondile phakathi kwe-BRD4 nomndeni we-HDAC wezakhiwo ezisebenzisanayo ezifana ne-SIN3A, NCOR2, BCOR, ne-SAP130 (Fig. 4e kanye ne-Supplementary Fig. 9e), ngokuhambisana ne-BRD4 kanye ne-HDAC ebopha ama-histones ane-acetylated ..Ukwengeza, izinhloso ezimele ezihlonzwe yi-LC-MS/MS, okuhlanganisa i-Sin3A, i-NSUN2, i-Fus, ne-SFPQ, yaqinisekiswa yi-Western blotting (Fig. 4f).Muva nje, i-isoform emfushane ye-BRD4 kubikwe ukuthi yenza i-nuclei enezakhiwo zokuhlukaniswa kwesigaba se-liquid (LLPS).Amaprotheni abopha i-RNA i-Fus ne-SFPQ alamula i-LLPS yezinqubo ezihlukahlukene zamaselula futhi akhonjwe lapha njengamaprotheni abophayo e-BRD4 angarekhodiwe.Ukusebenzisana phakathi kwe-BRD4 ne-SFPQ kuqinisekiswe ukuhlolwa kwe-co-immunoprecipitation (co-IP) (Umfanekiso 4g), okuphakamisa enye indlela yokuhlukaniswa kwesigaba soketshezi noketshezi lwe-BRD4 okufanele kuphenywe kabanzi.Ihlanganiswe ndawonye, le miphumela iphakamisa ukuthi i-PDPL iyinkundla ekahle yokuhlonza ukuxhumana kwe-BRD4 eyaziwayo kanye namaprotheni abophayo angaziwa.
ukumelwa kweSchematic kokumaka i-miniSOG-mediated BRD4 proximity marking, izikhathi zokuchayeka: 2, 5, 10, kanye ne-20 min.b Ukunqwabelana kwamaphrotheni akhonjwe ngezikhathi zokukhanya ezahlukene.Ukunothiswa kwamaprotheni okuhlonzwe ku-HEK293T-miniSOG-BRD4 kwakubaluleke ngokwezibalo uma kuqhathaniswa nohlobo lwasendle lwe-HEK293T.c Amandla e-ion lapho kubalwa ummeleli ongenalebuli owaziwayo wamaprotheni abophezelayo e-BRD4 ngesikhathi esibekiwe sokuchayeka.n = 3 amasampula azimele ngokwebhayoloji.Idatha yethulwa njengokuchezuka okujwayelekile ± okujwayelekile.d Ukuhlaziywa kwe-Gene ontological (GO) kwamaprotheni akhonjwe eqenjini lemizuzu emi-2.Amagama okuqala ayishumi e-GO asohlwini.Amabhamuza anemibala ngokuya ngesigaba sethemu le-GO, futhi usayizi webhamuza ulingana nenani lamaphrotheni atholakala kuthemu ngayinye.e Ukuhlaziywa kwentambo yamaprotheni asebenzisana ne-BRD4.Imibuthano ephuzi iyiglue eqondile futhi imibuthano empunga iyingqimba yokuqala yeglue engaqondile.Imigqa ebomvu imele ukusebenzisana okunqunywe ukuhlola futhi imigqa eluhlaza okwesibhakabhaka imele ukusebenzisana okubikezelwe.f Izinhloso ezibophezelayo ezimele i-BRD4 ezihlonzwe ku-LC-MS/MS zaqinisekiswa yi-Western blotting.g Ukuhlolwa kwe-Co-immunoprecipitation kuqinisekisa ukusebenzisana phakathi kwe-SFPQ ne-BRD4.Lezi zivivinyo ziphindwe ngokuzimela okungenani kabili ngemiphumela efanayo.Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Ngaphezu kokuhlonza okuhlosiwe okungabhalisiwe okuhlotshaniswa ne-POI, silinganisa ukuthi i-PDPL izofaneleka ukuhlonza ama-substrates ama-enzyme, okungadinga ukucaciswa kwamaprotheni abophayo angaqondile kumakhompleksi amakhulu ukuze achazele ama-substrates angabhalisiwe.I-Parkin (efakwe ikhodi yi-PARK2) iyi-E3 ligase futhi ukuguqulwa kwezakhi ku-parkin kwaziwa ngokubangela isifo se-autosomal recessive juvenile Parkinson (AR-JP)42.Ngaphezu kwalokho, i-parkin iye yachazwa njengebalulekile ku-mitophagy (i-mitochondrial autophagy) kanye nokususwa kwezinhlobo ze-oxygen esebenzayo.Kodwa-ke, nakuba ama-substrates amaningana e-parkin ekhonjiwe, indima ye-parkin kulesi sifo ayikacaci.Ukuze kuchazwe ama-substrates ayo angenasici, i-PDPL ihlolwe ngokwengeza i-miniSOG ku-N- noma i-C-terminus ye-parkin.Amaseli aphathwe nge-carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) ukuze avule i-parkin ngendlela ye-PINK1-Parkin.Uma kuqhathaniswa nemiphumela yethu ye-BRD4 PDPL, i-parkin N-terminus fusion yembule isethi enkulu yamaprotheni aqondiwe, nakuba ihlanganise ingxenye enkulu ye-C-terminus (177 out of 210) (Figure 5a,b and Supplementary Data 4).umphumela uhambisana nemibiko yokuthi amathegi we-N-terminal angakwazi ukusebenzisa i-Parkin44 ngokungafanele.Ngokumangazayo, bekunamaphrotheni ayi-18 kuphela agqagqene kudatha yethu enemiphumela eshicilelwe ye-AP-MS ye-Parkin43, okungenzeka ngenxa yomehluko phakathi kwemigqa yamaseli nokugeleza komsebenzi we-proteomics.Ngaphandle kwamaprotheni amane aziwayo (i-ARDM1, i-HSPA8, i-PSMD14, ne-PSMC3) ekhonjwe ngezindlela ezimbili (Fig. 5c)43.Ukuze kuqhutshekwe kuqinisekiswe imiphumela ye-LC-MS/MS, ukwelashwa kwe-PDPL kanye nokucinywa kwaseNtshonalanga okwalandela kwasetshenziswa ukuze kuqhathaniswe imiphumela yokuhlolwa kweseli yomzali ye-HEK293T kanye nomugqa ozinzile we-N-terminal parkin.Okuhlosiwe okungaziwa ngaphambilini i-CDK2, i-DUT, i-CTBP1, ne-PSMC4 ihlolwe nge-binder eyaziwayo, i-DNAJB1 (Fig. 5d).
Isakhiwo sentaba-mlilo samaphrotheni asebenzisana ne-parkin kumaseli e-HEK293T ane-miniSOG evezwe kahle ehlanganiswe ne-N- noma i-C-terminus ye-parkin (n = 3 uhlolo oluzimele lwebhayoloji).Ukuhlolwa kuka-t koMfundi onemisila emibili kusetshenziswe ezindaweni zentaba-mlilo.I-HEK293T isetshenziswe njengesilawuli esinegethivu. Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswa ngokubomvu (p <0.05 kanye>>2-fold ion intensity umehluko). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswa ngokubomvu (p <0.05 kanye>>2-fold ion intensity umehluko). Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswe ngokubomvu (p <0.05 kanye> umehluko we-2-fold intensity ion).显着变化的蛋白质以红色突出显示（p <0.05 和> 2 倍离子强度差异）。显着变化的蛋白质以红色突出显示（p <0.05和> 2 Значительно измененные белки выделены красным цветом (p < 0,05 и > 2-кратная разница в ионной силе). Amaprotheni ashintshiwe ngokuphawulekayo agqanyiswe ngokubomvu (p <0.05 kanye> umehluko we-2-fold in ionic amandla).Amaprotheni ahlobene abalulekile ku-HEK293T-miniSOG kodwa angabalulekile ku-HEK293T aboniswa ngokuluhlaza.b Umdwebo we-Venn obonisa amaprotheni agqagqene phakathi kwe-N-terminal kanye ne-C-terminal constructs.Omaka be-N-terminal bangenza i-parkin isebenze ngokungafanele futhi baphumele kumaphrotheni abonakala kakhudlwana.c Umdwebo we-Venn obonisa amaprotheni agqagqene phakathi kwe-PDPL ne-AP-MS.Abasebenzisanayo abaziwayo bafakwe ohlwini, okuhlanganisa amaphrotheni angu-4 kwangu-18 agqagqene kanye namaprotheni angu-11 kwangu-159 ahlonzwe ngokuqondile ku-PDPL.d Okuhloswe ngabameleli okuhlonzwe yi-LC-MS/MS kwaqinisekiswa yi-Western blotting.I-e Ssu72 ne-SNW1 zihlonzwe njengama-parkin substrates angabhalisiwe.Lawa ma-plasmids amaprotheni ane-FLAG adluliselwe ku-HEK293T kanye ne-HEK293T-Parkin-miniSOG elandelwa ukwelashwa kwe-CCCP ngezikhathi ezihlukahlukene.Ukucekelwa phansi kwagqama kakhulu kulayini weParkin overexpression.f Ngokusebenzisa i-proteasome inhibitor MG132, kwaqinisekiswa ukuthi inqubo yokuwohloka kwe-Ssu72 ne-SNW1 ixhunyaniswa ne-proteasome-ubiquitination.Lezi zivivinyo ziphindwe ngokuzimela okungenani kabili ngemiphumela efanayo.Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Ngokuphawulekayo, amaprotheni akhonjwe yi-PDPL kumele ahlanganise amaprotheni abopha ama-parkin kanye nama-substrates awo.Ukuze sithole ama-parkin substrates angabhalisiwe, sikhethe amaprotheni ayisikhombisa ahlonziwe (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 kanye ne-SNW1) kanye nama-plasmid ashintshiwe ukuze adalule lezi zakhi zofuzo ku-HEK293T evamile futhi siveze ngokuzinzile ukwelashwa kwe-miniSOG-Parkin nge-HEK293T ye-HEK293T.Amazinga we-Ssu72 kanye ne-SNW1 amaprotheni ancishiswe kakhulu emgqeni ozinzile we-miniSOG-Parkin (Fig. 5e).Ukwelashwa nge-CCCP amahora angu-12 kubangele ukuwohloka okukhulu kwawo womabili ama-substrates.Ukuze uphenye ukuthi ingabe ukucekelwa phansi kwe-Ssu72 ne-SNW1 kulawulwa yi-proteasome-ubiquitination, i-proteasome inhibitor MG132 yengezwe ukuze kuvinjwe umsebenzi we-proteasome, futhi eqinisweni sithole ukuthi inqubo yabo yokwehlisa ivinjiwe (Fig. 5f).Okuhlosiwe okungeziwe okungewona ama-substrate kwaqinisekiswa njengabahlanganyeli be-Parkin kusetshenziswa i-Western blotting (I-Supplementary Fig. 10), ebonise imiphumela engaguquki nge-LC-MS/MS.Ekuphetheni, ukuhlanganiswa kokuhamba komsebenzi kwe-PDPL nokuqinisekiswa kokudluliselwa kwamaprotheni okuhlosiwe kuvumela ukuhlonza ama-substrates e-E3 ligase angabhalisiwe.
Senze iplathifomu evamile yokumaka ekuvumela ukuthi ukhombe esikhaleni kanye nesikhathi esisebenzisanayo se-POI.Inkundla isuselwe kuphrotheni ye-photosensitizer miniSOG, ecishe ibe ngu-12 kDa kuphela, ngaphansi kwesigamu sosayizi we-enzyme evuthiwe ye-APEX2 (27 kDa) kanye nosayizi owodwa kokuthathu we-TurboID (35 kDa).Usayizi omncane kufanele unwebe kakhulu ububanzi bezinhlelo zokusebenza zokufunda ama-protein interactives amancane.Ukuhlolwa okwengeziwe kwama-photosensitizer engeziwe, kungakhathaliseki ukuthi amaprotheni afakwe izakhi zofuzo noma ama-molecule amancane, kuyadingeka ukuze kwandiswe isivuno se-quantum ye-singlett oxygen futhi kwandiswe ukuzwela kwale ndlela.Kunguqulo yamanje ye-miniSOG, ukulungiswa kwesikhashana okuphezulu kungafinyelelwa kusetshenziswa ukukhanya okuluhlaza okwesibhakabhaka ukuze kusebenze omaka bokusondela.Ukwengeza, isikhathi eside sokuchayeka sikhiphe "ifu" elikhudlwana le-singlett oxygen, okuholela ekuguqulweni kwezinsalela ezikude ze-histidine, i-radius yokulebula eyandisiwe, kanye nekhono lokulungisa kahle ukulungiswa kwendawo ye-PDPL.Siphinde sahlola ama-chemical probes ayisikhombisa ukuze sandise isilinganiso sesignali-kuya-ingemuva futhi sahlola indlela yamangqamuzana angemuva kwale ndlela.Ukugeleza komsebenzi we-TOP-ABPP kuhlanganiswe nokusesha okuvulekile okungachemile kuqinisekisile ukuthi ukuguqulwa kwenzeke kuphela kuma-histidines futhi akukho mvelo engaguquki engaguquki eyabonwa ukuze kuthuthukiswe ukuguqulwa kwe-histidine, ngaphandle kokuthandwa okulinganiselwe kwe-histidines esifundeni se-loop.
I-PDPL iphinde yasetshenziselwa ukuveza ama-subcellular proteome anemininingwane ye-proteome kanye nokufakwa okungenani okuqhathaniswa nokulebula okuseduze nezindlela zokuhlola amakhemikhali ezithize ze-organelle.Omaka be-Proximity nabo basetshenziswe ngempumelelo ukukhombisa indawo engaphezulu, i-lysosomal, nama-proteoms ahlobene ne-secretome46,47.Sikholelwa ukuthi i-PDPL izosebenzisana nalawa ma-subcellular organelles.Ngaphezu kwalokho, sabekela inselele i-PDPL ngokuhlonza okuhlosiwe kokubophezela kwamaprotheni e-cytosolic ayinkimbinkimbi kakhulu kunamaprotheni aboshwe ulwelwesi ngenxa yezakhiwo zawo eziguquguqukayo kanye nokubandakanyeka ekusebenzelaneni okwengeziwe kwesikhathi.I-PDPL isetshenziswe kumaprotheni amabili, i-transcriptional coactivator BRD4 kanye ne-ligase E3 Parkin ehlobene nesifo.Lawa maprotheni amabili awakhethwanga kuphela ngenxa yemisebenzi yawo eyisisekelo yezinto eziphilayo, kodwa futhi ngenxa yokufaneleka kwawo emtholampilo namandla okwelapha.Kulawa ma-POI amabili, abalingani ababophezelayo abaziwayo kanye nokuhlosiwe okungabhalisiwe kukhonjwe.Ngokuphawulekayo, i-protein ye-SFPQ ehlobene nokuhlukaniswa kwesigaba yaqinisekiswa yi-co-IP, engabonisa indlela entsha i-BRD4 (i-isoform efushane) elawula ngayo i-LLPS.Ngesikhathi esifanayo, sikholelwa ukuthi ukuhlonza ama-Parkin substrates kuyisimo lapho kudingeka khona ukuhlonza okunamathelayo okungaqondile.Sihlonze ama-parkin substrates amabili angaziwa futhi saqinisekisa ukuwohloka kwawo endleleni ye-ubiquitination-proteasome.Muva nje, isu lokucupha elisuselwa kumshini liye lasungulwa ukuze kutholwe ama-substrates e-hydrolase ngokuwacupha ngama-enzyme.Nakuba lena kuyindlela enamandla kakhulu, ayifanele ukuhlaziya ama-substrates ahilelekile ekwakhiweni kwezinkimbinkimbi ezinkulu futhi idinga ukwakheka kwezibopho ezihlangene phakathi kwe-enzyme ne-substrate.Silindele ukuthi i-PDPL inganwetshwa ukuze itadishe ezinye izakhiwo zamaprotheni nemindeni yama-enzyme, njengemindeni ye-deubiquitinase ne-metalloprotease.
Uhlobo olusha lwe-miniSOG, olubizwa nge-SOPP3, lwenziwe ngokukhiqizwa okuthuthukisiwe komoya-mpilo we-singlet.Siqhathanise i-miniSOG ne-SOPP3 futhi sathola ukusebenza kokumaka okuthuthukisiwe, nakuba isilinganiso sesignali-kuya-noise sahlala singashintshiwe (I-Supplementary Fig. 11).Sicabange ukuthi ukulungiselelwa kwe-SOPP3 (isb, ngokuziphendukela kwemvelo okuqondisiwe) kuzoholela kumaprotheni e-photosensitizer asebenza kahle kakhulu adinga izikhathi ezimfishane zokukhanya futhi ngaleyo ndlela kuvumele izinqubo zamaselula ezinamandla kakhulu ukuthi zithwetshulwe.Ngokuphawulekayo, inguqulo yamanje ye-PDPL ilinganiselwe endaweni yeselula njengoba idinga ukukhanya okuluhlaza okwesibhakabhaka futhi ayikwazi ukungena ezicutshini ezijulile.Lesi sici sivimbela ukusetshenziswa kwaso ezifundweni zemodeli yezilwane.Nokho, inhlanganisela ye-optogenetics ne-PDPL inganikeza ithuba lokucwaninga ngezilwane, ikakhulukazi ebuchosheni.Ngaphezu kwalokho, amanye ama-photosensitizer enziwe ngobunjiniyela be-infrared nawo asusa lo mkhawulo.Kumanje ucwaningo luyaqhubeka kule ndawo.
Ulayini weseli we-HEK293T utholwe kwa-ATCC (CRL-3216).Ulayini weseli uhlolwe ukuthi awunayo ukutheleleka kwe-mycoplasma futhi wakhuliswa ku-DMEM (Thermo, #C11995500BT) yengezwe ngo-10% we-fetus bovine serum (FBS, Vistech, #SE100-B) kanye no-1% penicillin/streptomycin (Hyclone, #SV30010).ukhule ku.
I-3-Aminophenylene (isampula 3) kanye (4-ethynylphenyl)methanamine (isampula 4) ithengwe kwa-Bidepharm.I-Propylamine (i-probe 2) ithengwe kumakhemikhali Amandla.I-N-(2-Aminophenyl)pent-4-ynamide (probe 1) yahlanganiswa ngokwezindlela ezishicilelwe.
Ithebula Lokwengeza 1 libala izakhi zofuzo ezisetshenziswe kulolu cwaningo.Ukulandelana kwe-miniSOG ne-KillerRed kwenziwe ku-plasmid yesipho evela ku-P. Zou (Peking University).Ukulandelana kokukhomba kwe-mitochondrial matrix kwathathwa kuma-amino acid angama-23 we-N-terminal we-COX4 futhi kwenziwa ama-vectors abonisiwe kusetshenziswa ukuhlanganiswa kwe-Gibson (Beyotime, #D7010S).Ukuze uqondise ulwelwesi kanye ne-nucleus ye-endoplasmic reticulum, i-SEC61B i-DNA yomuntu (NM_006808.3) (NEB, #M0491L) ekhuliswe yi-PCR kusukela kumtapo wezincwadi we-cDNA wamaseli e-HEK293T, kanye ne-H2B DNA (enikelwe u-D. Lin, iLabhorethri yaseShenzhen Bay) futhi yenziwe , njengoba kushiwo ngenhla.Ngaphandle kwalapho kuboniswe ngenye indlela, ezinye izakhi zofuzo zamaphrotheni ezisetshenziselwa ukudlulisela kanye nokwakhiwa kwemigqa yamaseli ezinzile zakhuliswa nge-PCR kusuka kulabhulali ye-HEK293T yeseli cDNA.I-G3S (GGGS) kanye ne-G4S (GGGGS) zisetshenziswe njengezixhumi phakathi kweprotein ye-bait ne-miniSOG.I-V5 epitope tag (GKPIPNPLLGLDST) yengezwe kulokhu kuhlanganiswa kwezakhi.Ukuze kuvezwe izilwane ezincelisayo kanye nokusungula ulayini weseli ozinzile, ukwakhiwa kwe-miniSOG fusion kwafakwa ngaphansi kwe-pLX304 lentiviral vector.Mayelana nokuvezwa kwebhaktheriya, i-miniSOG yenziwe yaba yivekhtha ye-pET21a ebhalwe ukuthi 6xHis ku-C-terminus.
Amaseli we-HEK293T afakwe ku-2.0 x 105 amaseli emthonjeni ngamunye kumapuleti ayisithupha wemithombo futhi adluliselwa emahoreni angu-24 kamuva nge-recombinant lentiviral plasmids (2.4 μg pLX304) kanye nama-plasmids okupakisha amagciwane (1.5 μg psPAX2 kanye ne-1.2 μg00 ye-MD2) , #C0533), cishe ukuhlanganiswa okungama-80%.Ngemva kokudluliselwa ebusuku, i-medium yashintshwa futhi ifakwe amanye amahora angu-24.Ukuqoqwa kwaleli gciwane kwenziwa ngemuva kwamahora angama-24, 48 kanye nama-72.Ngaphambi kokutheleleka kwemigqa yamaseli okuhlosiwe, i-viral medium yahlungwa ngesihlungi esingu-0.8 μm (Merck, #millex-GP) kanye ne-polybrene (Solarbio, #H8761) yengezwe ekuhlanganiseni kwe-8 μg/ml.Ngemuva kwamahora angama-24, amaseli avunyelwe ukuthi alulame ngokushintsha okuphakathi.Amaseli akhethwe kusetshenziswa i-5 μg/ml blasticidin (Solarbio, #3513-03-9) kumavesi amathathu okuqala njengokukhetha okuqinile okuphansi.Bese kusetshenziswa u-20 μg/ml njengomuthi oqinile ezindimeni ezintathu ezilandelayo.
Amaseli atshalwe emakamelweni anemithombo engu-12 (Ibidi, #81201) ekuminyaneni kwamaseli angaba ngu-20,000 emthonjeni ngamunye.Ukuze uthuthukise ukunamathela kwamaseli e-HEK293T, engeza i-50 µg/ml fibronectin (Corning, #356008) ehlanjululwe ku-phosphate buffered saline (PBS, Sangon, #B640435) ku-37°C.Amakamelo alungiswa ihora elingu-1 abese ekhishwa nge-PBS.Ngemuva kwamahora angu-24, amaseli ayewashwa kanye nge-PBS, efakwe ngo-1 mM probe 3 esixazululweni esisha sikasawoti esilinganiselwe sika-Hanks (HBSS, Gibco, #14025092) ihora elingu-1 ngo-37°C, bese efakwa i-LED eluhlaza okwesibhakabhaka (460 nm. ).) zifakwe imisebe imizuzu eyi-10 endaweni yokushisa yasekamelweni.Ngemva kwalokho, amaseli ahlanzwa kabili nge-PBS futhi alungiswa nge-4% formaldehyde ku-PBS (Sangon, #E672002) imizuzu engu-15 ekamelweni lokushisa.I-formaldehyde eyengeziwe ikhishwe kumaseli angashintshi ngokuwashwa kathathu nge-PBS.Amaseli abe esefakwa uketshezi ngo-0.5% Triton X-100 (Sangon, #A600198) ku-PBS futhi wawashwa izikhathi ezingu-3 nge-PBS.Bese ususa igumbi bese wengeza kusampula ngayinye engu-25 µl yengxube yokusabela ngokuchofoza equkethe i-50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) kanye ne-0.5 mg / ml ye-sodium ascorbate (i-Aladdin, no. S105024) futhi ifakwe imizuzu engu-30 ekamelweni lokushisa.Ngemva kokusabela okuphuthumayo, amaseli aye wawashwa izikhathi eziyisithupha nge-PBS equkethe u-0.05% Tween-20 (Sangon, #A600560) (PBST) futhi avinjwa ngo-5% BSA (Abcone, #B24726) ku-PBST imizuzu engu-30 ekamelweni lokushisa.
Ukuze uthole i-colocalization immunostaining, amaseli afakwe amasosha omzimba ayisisekelo ngokuvumelana nezimo ezibonisiwe: igundane anti-V5 tag mAb (1:500, CST, #80076), i-rabbit anti-Hsp60 mAb (1:1000), ABclonal, #A0564), i-rabbit polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) noma anti-lamin A/C monoclonal antibody onogwaja (1:500; CST, #2032) ngo-4 °C ngobusuku obubodwa.Ngemva kokugeza izikhathi ezingu-3 nge-PBST, amaseli afakwe amasosha omzimba esibili: imbuzi elwa nonogwaja i-Alexa Fluor 488 (Thermo, #A11034) ehlanjululwe ngu-1:1000, imbuzi ephikisana negundane i-Alexa Fluor 594 (CST, #8889) ihlanjululwe 1:1000.dilution Nciphisa ekamelweni lokushisa imizuzu engu-30.Amaseli abe esegezwa izikhathi ezingu-3 nge-PBST futhi aphikisana ne-DAPI (Thermo, #D1306) ku-PBS imizuzu engu-10 ekamelweni lokushisa.Ngemuva kokugeza oku-3 nge-PBS, amaseli avalwa ku-50% glycerol (Sangon, #A600232) ku-PBS ukuze kuthathwe izithombe.Izithombe ze-Immunofluorescent zitholwe kusetshenziswa i-ZEISS LSM 900 Airyscan2 confocal microscope kanye nesofthiwe ye-ZNE 3.5.
Ngomfanekiso we-singlelt oxygen fluorescent, amaseli awashwe kabili ngebhafa ye-Hanks HEPES ngaphambi kokwengeza i-100 nM Si-DMA ku-Hanks HEPES buffer (DAJINDO, #MT05).Ngemva kokuchayeka ekukhanyeni, amaseli afakwa ku-CO2 incubator engu-37°C imizuzu engu-45.Amaseli abe esewashwa kabili ngebhafa ye-Hanks' HEPES futhi aphikiswa nge-Hoechst ku-Hanks' HEPES buffer imizuzu engu-10 ekamelweni lokushisa futhi abonwa ngeso lengqondo kusetshenziswa i-ZEISS LSM 900 confocal microscope., #M36008) kubhafa ye-HBSS equkethe i-calcium ne-magnesium.Ngemva kokuchayeka ekukhanyeni noma e-doxorubicin (MCE, #HY-15142A), amaseli afakwa ku-incubator engu-CO2 engu-37° C. imizuzu engu-10, ahlanzwa kabili nge-HBSS buffer, futhi afakwa i-Hoechst ku-HBSS buffer ekamelweni lokushisa.imizuzu.I-Doxorubicin isetshenziswe njengesilawuli se-probe esihle lapho amaseli aphathwa nge-20 μM doxorubicin ku-HBSS equkethe u-1% BSA imizuzu engama-30.Izithombe ze-Immunofluorescent zitholwe kusetshenziswa i-Zeiss LSM 900 confocal microscope.
Amaseli we-HEK293T aveza i-mito-miniSOG ngokuzinzile afakwe ekumineni okucishe kube ngu-30% ezitsheni ezingu-15 cm.Ngemva kwamahora angu-48, lapho ~ 80% ukuhlangana kufinyelelwa khona, amaseli ayewashwa kanye nge-PBS, efakwe i-1 mM Probe 3 kubhafa ye-HBSS entsha ihora elingu-1 ku-37°C abese ekhanyiswa nge-LED eluhlaza okwesibhakabhaka imizuzu eyi-10 ekamelweni. izinga lokushisa..Ngemva kwalokho, amaseli aye wawashwa kabili nge-PBS, aklwejwa futhi aphinde amiswa kubhafa ye-PBS ebandayo equkethe i-EDTA-free protease inhibitors (MCE, #HY-K0011).Amaseli akhishwa ngokufaka ithiphu iminithi elingu-1 (isekhondi elingu-1 livuliwe kanye nesekhondi elingu-1 ephulwe ku-35% amplitude).Ingxube ewumphumela yafakwa i-centrifuged ku-15,871 xg imizuzu eyi-10 ku-4°C ukuze kukhishwe udoti, futhi ukugxilisa okunamandla amakhulu kwalungiswa kwaba ngu-4 mg/mL kusetshenziswa ikhithi yokuhlola amaprotheni ye-BCA (Beyotime, #P0009).Hlanganisa i-1 ml ye-lysate engenhla ne-0.1 mM ye-biotin azide ebolekayo (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), kanye ne-1 mM ye-CuSO4 ye-incuba engezansi zungeza ihora elingu-1 ekamelweni lokushisa.Ngemva kokusabela ngokushesha, engeza ingxube ekhambineni elixutshwe ngaphambili (MeOH:CHCl3:H2O = 4 ml:1 ml:3 ml) ebhodleleni lengilazi elingu-10 ml.Amasampula axutshwe futhi agxilwe ku-4500 g imizuzu engu-10 ekamelweni lokushisa.Izixazululo eziphansi nezingaphezulu zalahlwa, imvula yahlanzwa kabili nge-1 ml ye-methanol futhi i-centrifuged ku-15871 × g imizuzu engu-5 ku-4 ° C.Engeza u-1 ml we-8 M urea (Aladdin, no. U111902) ku-25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) ukuze uncibilikise imvula.Amasampuli aphinde ahlanganiswa ne-10 mM dithiothreitol (Sangon, #A100281 ku-25 mM ABC) imizuzu engu-40 ku-55°C okulandelwa ukwengezwa kwe-iodoacetamide entsha engu-15 mM (Sangon, #A600539) ekamelweni lokushisa ebumnyameni.Alkylation ngaphakathi kwemizuzu engama-30..I-dithiothreitol engu-5 mM eyengeziwe yengeziwe ukuze kumiswe ukusabela.Lungiselela cishe ubuhlalu be-NeutrAvidin agarose (Thermo, #29202) obungaba ngu-100 µl wesampula ngayinye ngokugeza izikhathi ezi-3 nge-1 ml ye-PBS.Isixazululo esingenhla se-proteome sihlanjululwe ngo-5 ml we-PBS futhi safakwa ngobuhlalu be-agarose e-NeutrAvidin obugezwe ngaphambili amahora angu-4 ekamelweni lokushisa.Ubuhlalu bese bugezwa izikhathi ezi-3 nge-5 ml PBS equkethe u-0.2% SDS (Sangon, #A600485), izikhathi ezingu-3 nge-5 ml PBS equkethe i-urea engu-1M, kanye nezikhathi ezingu-3 nge-5 ml ddH2O.Ubuhlalu bese buvunwa nge-centrifugation futhi buphinde bumiswe ku-200 μl we-25 mM ABC equkethe u-1 M urea, 1 mM CaCl 2 (Macklin, #C805228) kanye no-20 ng/μl trypsin (Promega, #V5280).Zama ubusuku bonke ku-37°C ngokuzungezisa.Ukusabela kwamiswa ngokungeza i-formic acid (i-Thermo, # A117-50) kuze kube yilapho i-pH ifinyelela ku-2-3.Ubuhlalu buhlanjululwe izikhathi ezingu-3 nge-1 ml ye-PBS equkethe i-0.2% SDS, izikhathi ezingu-3 nge-1 ml ye-PBS equkethe i-1 M urea, bese izikhathi ezingu-3 nge-1 ml yamanzi e-distilled.Ama-peptide ashintshiwe akhishwe yi-light lysis (365 nm) imizuzu engama-90 kusetshenziswa u-200 μl we-70% MeOH.Ngemuva kwe-centrifugation, i-supernatant yaqoqwa.Ubuhlalu bese bugezwa kanye ngo-100 μl ka-70% we-MeOH bese ama-supernatant ahlanganiswa.Amasampuli omisiwe ku-concentrator ye-Speedvac vacuum futhi agcinwe ku -20 ° C kuze kube yilapho kuhlaziywa.
Ukuze kuhlonzwe futhi kulinganise ama-peptide ashintshiwe komoya-mpilo e-singlett, amasampula aphinde achithwa ku-0.1% ye-formic acid kanye ne-1 μg yama-peptides ahlaziywa kusetshenziswa i-Orbitrap Fusion Lumos Tribrid mass spectrometer efakwe umthombo we-nano ESI ovela ku-Tune ne-Xcalibur evela kusofthiwe yomthengisi 4.3.Amasampuli ahlukaniswe kukholomu ye-capillary egcwele ngaphakathi engu-75 µm × 15 cm enento engu-3 µm C18 (ReproSil-pur, #r13.b9.) futhi yaxhunywa kusistimu ye-EASY-nLC 1200 UHPLC (Thermo).Ama-peptides ahlukaniswa ngomugqa we-chromatography ye-gradient yamaminithi angu-95 ukusuka ku-8% we-solvent B ukuya ku-50% ye-solvent B (A = 0.1% ye-formic acid emanzini, B = 0.1% ye-formic acid ku-80% acetonitrile), yabe isikhuphukela ku-98% B min. emizuzwini engu-6 ngesilinganiso sokugeleza esingu-300 nl/min.I-Orbitrap Fusion Lumos iqoqa idatha ngokushintshana phakathi kokuskena okugcwele kwe-MS nokuskena kwe-MS2 kuye ngedatha.I-voltage ye-sputtering isethwe ku-2.1 kV futhi izinga lokushisa le-ion transport capillary lalingu-320°C.I-MS spectra (350-2000 m/z) iqoqwe ngokulungiswa okungu-120,000, AGC 4 × 105, kanye nesikhathi sokufaka esiphezulu esingu-150 ms.Izandulela eziyi-10 ezivame ukushajwa ngokuphindwe kabili ekuskeneni ngakunye okugcwele zahlukaniswa kusetshenziswa i-HCD enamandla okushayisana ajwayelekile angu-30%, iwindi lokuhlukaniswa eliyi-quadrupole elingu-1.6 m/z, kanye nesilungiselelo sokuxazulula esingu-30,000.Ithagethi ye-AGC ye-tandem mass spectrometry isebenzisa u-5×104 kanye nesikhathi sokufaka esiphezulu esingu-150 ms.Okuhlukile okuguquguqukayo kusethelwe kumasekhondi angama-30. Ama-ion angabelwe noma lawo anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-MS/MS. Ama-ion angabelwe noma lawo anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ama-ion angabelwe noma ama-ion anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-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. Ama-ion noma ama-ion anezindleko zokungu-1+ kanye > 7+ anqatshiwe ku-MS/MS.
Idatha eluhlaza icutshungulwa kusetshenziswa inkundla yekhompyutha ye-FragPipe esekelwe ku-MSFragger.Ukuchema kwenqwaba kanye nama-amino acid ahambisanayo kwanqunywa kusetshenziswa i-algorithm yokusesha evulekile ene-precursor mass tolerance of -150 kuya ku-500 Da.Ama-peptide ashintshiwe abe esehlonzwa kusetshenziswa ukuguqulwa kwe-histidine ngenzuzo enkulu ye-+229.0964 kanye +247.1069 Da ku-PD (Proteome Discoverer 2.5, Thermo).
Amaseli aveza ngokuqinile isakhi sofuzo se-miniSOG esihlanganisiwe afakwe ezitsheni ezingu-6 cm.Lapho efinyelela ku-~80% confluence, amaseli aye wawashwa kanye nge-HBSS (Gibco, #14025092), wabe esefafazwa ngama-chemical probe ku-HBSS ihora elingu-1 ku-37°C futhi akhanyiswa ngokukhanya okuluhlaza okwesibhakabhaka.I-10W LED imizuzu engu-20 ekamelweni lokushisa.Ukunquma ukuthi yiluphi uhlobo lwezinhlobo ze-oxygen esebenzayo ezihilelekile ku-PDPL, 0.5 mM uvithamini 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 yengezwe kumaseli njengezithasiselo.Ngemva kokugeza nge-PBS ebandayo, amaseli aklwejwa, aqoqwa kumashubhu e-centrifuge angu-1.5 ml, futhi afakwa ithiphu le-1 min ku-200 μl we-PBS nge-1x protease inhibitor ngaphandle kwe-EDTA (1 s kanye ne-1 s ngaphandle, i-amplitude engu-35%).Ingxube ewumphumela yafakwa i-centrifuged ku-15,871 × g imizuzu engu-10 ku-4 °C futhi ukugxilisa okunamandla kakhulu kwalungiselelwa ku-1 mg/mL kusetshenziswa ikhithi yokuhlola amaprotheni ye-BCA.Cishe u-50 µl we-lysate engenhla yafakwa ku-0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, kanye ne-1 mM CuSO4 ihora elingu-1 ekamelweni lokushisa ngokuzungezisa ukusuka phansi ukuya phezulu.Ngemuva kokuphendula ngokuchofoza, imvula ene-acetone yenziwa ngokungeza u-250 μl we-acetone ebanda ngaphambili kumasampula, incubating ku-20°C imizuzu engama-20 kanye ne-centrifuging ku-6010×g imizuzu eyi-10 ku-4°C.Qoqa i-pellet bese ubilise ku-50 µl we-1x ye-Laemmli buffer imizuzu engu-10 ku-95 °C.Amasampula abe esehlaziywa kumajeli amade e-SDS-PAGE futhi abonwa ngeso lengqondo kusetshenziswa isistimu yokuthwebula ye-Bio-rad ChemiDoc MP Touch enesoftware ye-Image Lab Touch.
Ukuvezwa nokuhlanzwa kwe-recombinant miniSOG-6xHis protein kwenziwa njengoba kuchazwe ngaphambilini.Kafushane, amaseli e-E. coli BL21(DE3) (TransGen, #CD701-02) aguqulwa abe ne-pET21a-miniSOG-6xHis kanye ne-protein expression etholwe ngo-0.5 mM IPTG (Sangon, #A600168).Ngemva kwe-cell lysis, amaprotheni ahlanjululwa kusetshenziswa ubuhlalu be-Ni-NTA agarose (MCE, no. 70666), ahlanjululwa ngokumelene ne-PBS, futhi agcinwa ku -80 ° C.
Ukuze uthole i-antibody-based in vitro proximity assay, hlanganisa 100 μM purified miniSOG, 1 mM probe 3, kanye ne-1 μg anti-lebula yegundane le-monoclonal antibody (TransGen, #HT501-01) ku-PBS kuvolumu yokusabela ephelele engu-50 μl..Ingxube yokusabela ifakwe imisebe ye-LED eluhlaza okwesibhakabhaka imizuzu engu-0, 2, 5, 10, kanye nemizuzu engu-20 ekamelweni lokushisa.Ingxube ifakwe ku-0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, kanye ne-1 mM CuSO4 ihora elingu-1 kuthempelesha yegumbi kusiniki-manzi esinyakazayo esikhuphukayo.Ngemva kokusabela ngokushesha, engeza isiphazamiso se-4x Laemmli ngokuqondile engxubeni bese ubilisa ku-95°C imizuzu engu-10.Amasampuli ahlaziywa kumajeli e-SDS-PAGE futhi ahlaziywa ngokucishwa kwentshonalanga nge-streptavidin-HRP (1:1000, Solarbio, #SE068).
I-peptide yokwenziwa equkethe i-histidine ene-C-terminal amidation (LHDALDAK-CONH2) isetshenziselwe ukuhlaziya ilebula eseduze ye-peptide-based in vitro.Kulesi sivivinyo, i-miniSOG ehlanzekile engu-100 μM, i-10 mM probe 3 kanye ne-2 μg/ml peptide yokwenziwa zixutshwe ku-PBS ngevolumu yokusabela ephelele engu-50 μl.Ingxube yokusabela ifakwe ukukhanya kwe-LED eluhlaza okwesibhakabhaka ihora elingu-1 ekamelweni lokushisa.I-microliter eyodwa yesampula yahlaziywa kusetshenziswa uhlelo lwe-LC-MS (Amanzi, i-SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer nge-MassLynx spectrum analysis software).
Amaseli we-HEK293T aveza ngokuzinzile uhlobo lwe-miniSOG lokuhlanganisa afakwe ezitsheni ezingu-10 cm emigqeni ene-organelle ehlukile (i-Mito, i-ER, i-Nucleus) nezitsha ezingu-15 cm zemigqa ye-Parkin-miniSOG kanye ne-BRD4-miniSOG.Lapho efinyelela ku-~90% confluence, amaseli ayewashwa kanye nge-HBSS, abese efakwa nge-probe 3 ku-HBSS ihora elingu-1 ku-37°C futhi akhanyiswa nge-LED engu-10 W eluhlaza okwesibhakabhaka ekamelweni lokushisa.Ngokulebula okungaxhunywana naye kwe-Parkin, i-10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) ene-probe 3 ku-HBSS yengezwe ihora elingu-1 ku-37°C.I-cell lysis, uchofoze ikhemistri, izinyathelo zokunciphisa kanye ne-alkylation ziyefana njengoba kuchazwe ngenhla, ngaphandle kokuthi u-2 mg we-lysate wengeziwe futhi i-biotin PEG3 azide yasetshenziswa ekuphenduleni ngokuchofoza esikhundleni se-biotin azide e-photodegradable.Ngemva kokucetshiswa, ubuhlalu bagezwa izikhathi ezingu-3 nge-5 ml ye-PBS equkethe i-0.2% SDS, izikhathi ezingu-3 nge-5 ml ye-PBS equkethe i-1 M urea, kanye nezikhathi ezingu-3 nge-5 ml ye-PBS.Ngemva kwalokho, i-trypsin engu-2 µg yengezwa ku-300 µl 25 mM ABC equkethe i-urea engu-1 M ukuze ihlukanise amaprotheni ngobusuku obungu-37°C.Ukusabela kwamiswa ngokungeza i-formic acid kuze kufinyelelwe i-pH engu-2-3.Ngemuva kwe-trypsinization kubuhlalu, isixazululo se-peptide sasuswa usawoti kusetshenziswa ikholomu ye-SOLAµ HRP (Thermo, #60209-001) futhi yomiswa kusixhumi se-Speedvac vacuum.Ama-peptide aphinde ahlakazwa ku-0.1% ye-formic acid futhi ama-500 ng ama-peptide ahlaziywa kusetshenziswa i-Orbitrap Fusion Lumos Tribrid mass spectrometer efakwe umthombo we-nano-ESI ochazwe ngenhla.Ama-Peptide ahlukaniswa ngamakholomu angaphambili e-RP-HPLC (75 μm x 2 cm) (Thermo, no. 164946) namakholomu e-RP-HPLC okuhlaziya (75 μm x 25 cm) (Thermo, no. 164941), womabili agcwaliswe ngo-2 μm.i-gradient isuka ku-8% iye ku-35% ACN emaminithini angu-60, bese ikhuphuka ngokomugqa ibe ngu-98% B emizuzwini engu-6 ngesilinganiso sokugeleza esingu-300 Nl/min.I-MS spectra (350-1500 m/z) iqoqwe ngokulungiswa okungu-60,000, AGC 4 × 105, kanye nesikhathi sokufaka esiphezulu esingu-50 ms.Ama-ion akhethiwe ahlukaniswa ngokulandelana yi-HCD emijikelezweni emi-3 enamandla okushayisana ajwayelekile angu-30%, iwindi lokuhlukaniswa kwe-quadrupole elingu-1.6 m/z, nokulungiswa okungu-15000. Ithagethi ye-AGC ye-spectrometer enkulu engu-5 × 104 kanye nesikhathi somjovo esiphezulu. ye-22 ms isetshenzisiwe.Ukukhishwa okuguquguqukayo kusethelwe kumasekhondi angu-45. Ama-ion angabelwe noma lawo anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-MS/MS. Ama-ion angabelwe noma lawo anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ama-ion angabelwe noma ama-ion anenkokhiso engu-1+ kanye > ne-7+ anqatshiwe ku-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. Ama-ion noma ama-ion anezindleko zokungu-1+ kanye > 7+ anqatshiwe ku-MS/MS.
Izinyathelo zokulungiselela isampula kuze kufike ekuthuthukisweni kobuhlalu be-NeutrAvidin zazifana nokuhlaziywa kwe-LC-MS/MS okuchazwe ngenhla.Cishe u-50 μg we-lysate wasetshenziswa njengokufakwayo kokulawula ukulayisha futhi u-2 mg we-lysate wasetshenziselwa ukusabela ngokuchofoza.Ngemva kokunothiswa nokugeza nge-neutravidin, amaprotheni abophekile akhishwa ngokungeza u-50 μl we-buffer ye-Laemmli kubuhlalu be-agarose resin bese ubilisa ku-95 ° C. imizuzu engu-5.Okokufaka kokulayisha kokulawula kanye namasampuli anobuhlalu anobuhlalu ahlaziywa yi-SDS-PAGE futhi adluliselwa kulwelwesi lwe-PVDF (Millipore, #ISEQ00010) ngezindlela ezijwayelekile zokuvala zaseWestern.Ulwelwesi lwaluvinjwe ngo-5% wobisi lwe-skim (Sangon, #A600669) ku-TBS equkethe u-0.1% phakathi no-20 (TBST) futhi lwafakwa ngokulandelana ngamasosha omzimba ayinhloko nawesibili.Amasosha omzimba ayisisekelo ahlanjululwe ngo-1:1000 ku-5% wobisi lwe-skim ku-TBST futhi afakwa ubusuku bonke ku-4°C.Amasosha omzimba esibili asetshenziswa ngesilinganiso esingu-1:5000 futhi afukanyelwa ihora eli-1 ekamelweni lokushisa.Ulwelwesi lwabonwa ngeso lengqondo nge-chemiluminescence kusetshenziswa i-Chemidoc MP imaging system.Konke ukuskena okungasikiwe kwama-blots namajeli esithombeni kuvezwa njengedatha eluhlaza.
Amasosha omzimba ayisisekelo asetshenziswe kulolu cwaningo ahlanganisa i-anti-SFPQ monoclonal antibody (CST, no. 71992), anti-FUS monoclonal antibody (CST, no. 67840), anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1- AP), anti-mSin3A polyclonal antibody (Abcam, #ab3479), anti-tag monoclonal antibody (TransGen, #HT201-02), igundane anti-β-actin monoclonal antibody (TransGen, #HC201-01), anti-rabbit -CDK2 monoclonal antibody (ABclonal, #A0094), antibody monoclonal unogwaja kuya ku-CTBP1 (ABclonal, #A11600), antibody onogwaja we-polyclonal ku-DUT (ABclonal, #A2901), unogwaja we-polyclonal antibody ku-PSMC4 (ABclonal anti-5), #Abclonal-5), I-DNAJB1 i-polyclonal antibody (ABclonal, # A5504).Lawa amasosha omzimba asetshenziswe ekuhlanjululweni okungu-1:1000 ku-5% wobisi lwe-skim ku-TBST.Amasosha omzimba esibili asetshenziswe kulolu cwaningo ahlanganisa i-anti-rabbit IgG (TransGen, #HS101-01), i-anti-mouse IgG (TransGen, #HS201-01) ekuhlanjululweni okungu-1:5000.
Ukuze kuqhutshekwe kuphenywe ukuthi i-BRD4 iyasebenzisana yini ne-SFPQ, i-HEK293T ezinzile kanye ne-BRD4-miniSOG amaseli e-HEK293T agcizelela ngokweqile i-HEK293T afakwe ezitsheni ezingu-10 cm.Amaseli ahlanzwa nge-PBS ebandayo futhi afakwa ku-1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) nge-EDTA-free protease inhibitor imizuzu engu-30 ku-4°C.Ngemva kwalokho, ama-lysates aqoqwe kumashubhu e-centrifuge angu-1.5 ml futhi afakwa i-centrifuge ku-15,871 xg imizuzu engu-10 ku-4 ° C.I-supernatant yavunwa futhi yafakwa ku-5 µg ye-anti-V5 ebhalwe ukuthi igundane le-monoclonal antibody (CST, #80076) ngobusuku obungu-4°C.Geza cishe u-50 µl weprotheni A/G yobuhlalu kazibuthe (MCE, #HY-K0202) kabili nge-PBS equkethe u-0.5% Tween-20.Khona-ke ama-cell lysates afukanyelwa ngobuhlalu kazibuthe amahora angu-4 ku-4 ° C ngokuzungeza kusuka phansi kuya phezulu.Khona-ke ubuhlalu babugezwa izikhathi ezine nge-1 ml ye-PBST buffer futhi ibilisiwe ku-95 ° C imizuzu engu-5.Amasampuli ahlaziywa kumajeli e-SDS-PAGE futhi adluliselwa kulwelwesi lwe-PVDF kusetshenziswa izindlela zokuvala ezijwayelekile zaseNtshonalanga.Ama-membrane ayevinjwe ku-5% wobisi lwe-skim ku-TBST futhi afakwa ngokulandelana ngokulandelana nama-antibodies ayisisekelo kanye nesesibili.I-Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) isetshenziswe ngesilinganiso esingu-1:1000 ku-5% wobisi lwe-skim ku-TBST futhi yafakwa ngobusuku obungu-4°C.I-anti-rabbit IgG isetshenziswe ngesilinganiso se-1: 5000 futhi ifakwe ihora elingu-1 ekamelweni lokushisa.Ulwelwesi lwabonwa ngeso lengqondo nge-chemiluminescence kusetshenziswa i-Chemidoc MP imaging system.
Zonke izakhiwo ezisetshenziselwa ukuhlaziywa kwe-Solvent Accessible Surface Area (SASA) zitholwe ku-Protein Data Bank (PDB)52 noma ku-AlphaFold Protein Structure Database53.I-Absolute SASA ibalwe ngensalela ngayinye kusetshenziswa uhlelo lweFreeSASA.Kwasetshenziswa kuphela idatha ye-SASA ephelele nengacacile yelebula le-histidine kanye nomakhelwane bayo ukuze kutholwe isilinganiso se-SASA sesakhiwo ngasinye.Ukufinyeleleka kwe-solvent ehlobene (RSA) ye-histidine ngayinye kubalwe ngokuhlukanisa inani eliphelele le-SASA ngendawo eyinsalela engase ibe khona etholakala ku-solvent.Wonke ama-histidine abe esehlukaniswa njengafihliwe uma isilinganiso se-RSA sasingaphansi kwama-20%, ngaphandle kwalokho sivezwa56.
Amafayela angahlungiwe atholwe ngemodi ye-DDA aseshwa kusetshenziswa i-Proteome Discoverer (v2.5) noma i-MSfragger (Fragpipe v15.0) kusizindalwazi esifanelekile samaprotheni esiqinisekisiwe se-SwissProt esiqukethe ukungcola okuvamile.Ama-peptide ayedinga i-trypsin ephelele enezindawo ezimbili zokuqhekeka ezilahlekile, i-carbamoyl methylation njengenguquko engaguquki kanye ne-methionine oxidation njengokuguqulwa okuguquguqukayo.Ukubekezelela isisindo se-Precursor ne-fragment kwakusethwe ku-10 ppm naku-0.02 Da (MS2 Orbitrap), ngokulandelanayo. Amahithi angcolile asusiwe, futhi amaprotheni ahlungwa ukuze kutholwe izinga lokutholwa okungamanga elingu-<1%. Amahithi angcolile asusiwe, futhi amaprotheni ahlungwa ukuze kutholwe izinga lokutholwa okungamanga elingu-<1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэффициент ложного обнаружения <1%. Amahithi angcolile asusiwe futhi amaprotheni ahlungwa ukunikeza izinga lokutholwa okungamanga elingu-<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%. Amahithi angcolile asusiwe futhi amaprotheni ahlungwa ukuze kuzuzwe isilinganiso esingamanga esihle esingu-<1%.Ukuze kuhlaziywe ubuningi ngaphandle kokusetshenziswa kwamalebula, okuqukethwe kwamaprotheni okujwayelekile okuvela eziphindaphindweni ezintathu zebhayoloji kusetshenziswe.Ukuhlaziywa kwe-protein subcellular localization kwenziwa kusetshenziswa ukuhlaziywa kwe-Gene Ontology (GO) okuvela ku-DAVID Bioinformatics Resources, i-MitoCarta 3.0 kanye nemininingwane yolwazi ehlanganiswe futhi yanyatheliswa iqembu lika-Alice Ting.Imephu yentaba-mlilo yatholwa ePerseus (v1.6.15.0). Izinguquko zokugoqa kokuchichima kwamaprotheni zahlolelwa ukubaluleka kwezibalo kusetshenziswa i-t-test yezinhlangothi ezimbili, futhi amahithi amaprotheni ahlonzwe ngoshintsho lwenala >2 (ngaphandle kwalapho kuchazwe ngenye indlela) kanye nenani elingu-p <0.05. Izinguquko zokugoqa kokuchichima kwamaprotheni zahlolelwa ukubaluleka kwezibalo kusetshenziswa i-t-test yezinhlangothi ezimbili, futhi amahithi amaprotheni ahlonzwe ngoshintsho lwenala >2 (ngaphandle kwalapho kuchazwe ngenye indlela) kanye nenani elingu-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. Izinguquko zokugoqa kokuqukethwe kwamaprotheni zihlolelwe ukubaluleka kwezibalo kusetshenziswa i-t-test enemisila emibili, futhi amaphrotheni afanayo atholwe nokushintsha kokuqukethwe >2 (ngaphandle kwalapho kuphawulwe ngenye indlela) kanye nenani le-ap engu-<0.05.使用双边t 检验测试蛋白质丰度倍数变化的统计显着性，并确定蛋白质丰度倍数变化的统计显着性，并确定蛋白质丰度倍数变化的统计显着性，并确定蛋白质命中数变化的统计显着性，并确定蛋白质命中数变化。使用 双边 T 检验检验 蛋白质 倍 倍倍 变化变化 统计统计 并并 命中中的 的丰度 的> 2 (另另 说明) 和 P 值 <0.05. Статистическую значимость кратных изменений содержания белка проверяли с использованием двустороннего t-критерия, а совпадения белков определяли для изменений содержания >2 (если не указано иное) и p-значений <0,05. Ukubaluleka kwezibalo zezinguquko ezigoqekayo kokuqukethwe kwamaprotheni kwahlolwa kusetshenziswa i-t-test enemisila emibili, futhi okufanayo kwamaprotheni kwanqunywa izinguquko zokuqukethwe >2 (ngaphandle kwalapho kuboniswe ngenye indlela) kanye namavelu we-p <0.05.Ukuhlaziywa kokusebenzisana kwamaprotheni kwenziwa kusetshenziswa ukuhlaziya kwe-GO kanye nesizindalwazi seString.
Ukuphindaphinda okuthathu kwebhayoloji kwenziwa ngemiphumela efanayo.Ukuhlaziywa kwezibalo kwenziwe nge-GraphPad Prism (isofthiwe ye-GraphPad) futhi iziqephu zentaba-mlilo zenziwe nge-Perseus (v1.6.15.0).Ukuze uqhathanise amaqembu amabili, amanani we-p anqunywa kusetshenziswa ukuhlolwa kuka-t koMfundi okunemisila emibili.Amaprotheni e-singleton kuphela ahlonzwe okungenani kabili eqenjini lokuhlola afakwe eziqintini zentaba-mlilo, futhi amanani angekho ahambisanayo eqenjini elilawulayo athathelwa indawo i-Perseus kusuka ekusabalaliseni okuvamile ukuze kubalwe inani le-p.Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.Ekuhlaziyweni kwe-proteomic ukuze kuhlaziywe izibalo, inala yamaprotheni avele okungenani kuzimpinda ezimbili zebhayoloji agciniwe.Izindlela zezibalo azisetshenziswanga ukuze kunqunywe kusengaphambili usayizi wesampula.Ukuhlolwa akukona okungahleliwe.Abacwaningi bebengaphuphutheki emisebenzini ngesikhathi sokuhlolwa nokuhlolwa kwemiphumela.
Ukuze uthole ulwazi olwengeziwe mayelana nesakhiwo socwaningo, bheka i-Nature Research Report abstract exhunywe kulesi sihloko.
Idatha ye-mass spectrometry etholwe kulolu cwaningo ihanjiswe ku-ProteomeXchange Consortium kusetshenziswa ikhosombe likazakwethu we-iProX57 ngaphansi kwedathasethi ye-ID PXD034811 (PDPL-MS dataset).Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.Lesi sihloko sinikeza idatha yoqobo.
I-Gingras, AC, Abe, KT & Raught, B. Ukwazi indawo yakubo: kusetshenziswa i-biotinylation encike ekusondeleni ukuze kubonakale ama-protein complexes nama-organelles emephu. I-Gingras, AC, Abe, KT & Raught, B. Ukwazi indawo yakubo: kusetshenziswa i-biotinylation encike ekusondeleni ukuze kubonakale ama-protein complexes nama-organelles emephu.I-Gingras, AS, Abe, KT kanye ne-Raut, B. Ukujwayelana nendawo ezungezile: kusetshenziswa i-biotinylation encike ekusondeleni ukuze kubonakale ama-protein complexes nama-organelles emephu. 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。 I-Gingras, AC, Abe, KT & Raught, B. Ukuqonda indawo: sebenzisa ukuncika komakhelwane empilweni yebhayoloji.I-Gingras, i-AS, i-Abe, i-KT ne-Raut, B. Ukuqonda ukusondelana: ukubonakaliswa kwama-protein complexes kanye nemephu ye-organelles kusetshenziswa i-proximity-dependent biotinylation.Okwamanje.Umbono wami.Amakhemikhali.biology 48, 44–54 (2019).
Geri, JB et al.Ukwenza imephu ye-microenvironment ngokudlulisela amandla e-Dexter kumaseli omzimba.Isayensi 367, 1091–1097 (2020).
Hertling, EL et al.Amanethiwekhi esikali se-proteome amabili athola ukulungiswa kabusha kweseli ethize ye-internome yomuntu.Amaseli 184, 3022–3040.e3028 (2021).
Isikhathi sokuthumela: Sep-15-2022
