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Enzymatic proximity labeling nzira dzakabva pane activated esters kana phenoxy radicals dzinoshandiswa zvakanyanya kumepu subcellular proteomes uye mapuroteni anodyidzana mumaseru mhenyu.Nekudaro, ma esters akabatidzwa haaite zvishoma, zvichikonzera radius yakafararira, uye phenoxy radicals inogadzirwa neperoxide kurapwa inogona kukanganisa redox nzira.Pano isu tinoshuma nzira yepedyo yekunyora dependent photoactivation (PDPL) yakagadzirwa nemajini inobatanidza iyo miniSOG photosensitizer protein kune protein inofarira.Yakakonzerwa nechiedza chebhuruu uye inodzorwa nenguva yekuratidzwa, singlet okisijeni inogadzirwa uye ipapo spatiotemporally yakagadziriswa kunyorwa kwezvisaririra zve histidine ne aniline probe inowanikwa.Isu tinoratidza kuvimbika kwayo kwepamusoro kuburikidza ne organelle-specific proteome mepu.Kuenzanisa-ne-parutivi kuenzaniswa kwePDPL neTurboID kunoratidza yakanyatsojeka uye yakazara proteomic kufukidzwa kwePDPL.Tevere, takaisa PDPL kune chirwere-chakabatana transcriptional coactivator BRD4 uye E3 Parkin ligase uye takawana yaimbozivikanwa yekudyidzana.Nekuwedzeredza kutarisisa, maviri asingazivikanwe substrates, Ssu72 uye SNW1, akaonekwa kune Parkin, iyo kuderedzwa kwayo kunopindirana neubiquitination-proteasome nzira.
Kunyatsoita hunhu hweprotein network kunotsigira akawanda akakosha maserura maitiro.Naizvozvo, yakanyatsojeka spatiotemporal mepu yekudyidzana kweprotein ichapa hwaro hwemamorekuru hwekududzira nzira dzebhaibheri, chirwere chechirwere, uye kukanganisa kusangana uku kwezvinangwa zvekurapa.Kuti izvi zviitike, nzira dzinokwanisa kuona kudyidzana kwechinguvana mumasero mapenyu kana matishu dzinodiwa zvikuru.Affinity Purification Mass Spectrometry (AP-MS) yagara ichishandiswa kuona vanosunga vanobatana navo mapuroteni ekufarira (POIs).Nekuvandudzwa kwehuwandu hwemaitiro eproteomics, Bioplex3.0 yakagadzirwa, iyo yakakura dhatabhesi yeprotein network yakavakirwa paAP-MS.Kunyangwe AP-MS ine simba kwazvo, iyo sero lysis uye dilution nhanho mukufambiswa kwebasa dzakarerekera kune isina kusimba uye yechinguvana inosunga kudyidzana uye kuunza post-lysis artifacts akadai seyekunyepedzera yekudyidzana mapeya anoshaya compartmentalization isati yaitwa lysis.
Kugadzirisa nyaya idzi, maamino acids (UAA) ane maamino acids (UAA) ane mapoka anobatanidza uye enzymatic ari pedyo ekunyora mazita (PL) mapuratifomu (eg APEX neBioID)5 akagadzirwa.Kunyangwe iyo nzira yeUAA yakashandiswa zvinobudirira mune dzakawanda mamiriro uye inopa ruzivo pane yakananga mapuroteni anonamira, optimization yeiyo UAA yekuisa saiti ichiri kudikanwa.Zvinotonyanya kukosha, inzira yekunyora stoichiometric inoshaya kudzoreredzwa kwezviitiko zvekunyora.Mukupesana, enzymatic PL nzira, dzakadai seBioID nzira, fuse the engineered biotin ligase kuPOI7, iyo inozoita activate biotin kuumba reactive biotinyl-AMP ester yepakati.Iyo enzyme nokudaro inokonzeresa uye inoburitsa yakabatidzwa biotin "gore" iyo inonyora proximal lysine zvisaririra.Zvisinei, BioID inoda maawa anopfuura gumi nemaviri kuti iwane chiratidzo chakakwana chakanyorwa, chinodzivisa kushandiswa kwayo nekugadzirisa kwenguva.Ichishandisa shanduko yakanangana nekuratidzwa kwembiriso, TurboID yakagadzirwa yakavakirwa paBioID kuti inyatsoita, ichibvumira kunyoreswa kwakanaka nebiotin mukati memaminitsi gumi, zvichibvumira maitiro ane simba kuti adzidzwe.Nekuti TurboID inoshanda zvakanyanya uye endogenous biotin mazinga akakwana kune yakaderera-lebel yekunyorwa, yekumashure inove dambudziko ringangove dambudziko kana yakakwenenzverwa uye yakarongeka manyorerwo achidikanwa nekuwedzera exogenous biotin.Mukuwedzera, activated esters haina kunyatsogadzirisa (t1/2 ~ 5 min), iyo inogona kutungamirira kune yakakura label radius, kunyanya mushure mekuzara kwemapuroteni akavakidzana ane biotin 5. Mune imwe nzira, genetic fusion ye engineered ascorbate peroxidase (kureva biotin- phenol radicals uye inobvumira mapuroteni kunyora mukati meminiti imwe 9, 10. APEX inoshandiswa zvakanyanya kuziva subcellular proteomes, membrane protein complexes, uye cytosolic signaling protein complexes11, 12. Zvisinei, kudiwa kwehuwandu hweperoxides kunogona kukanganisa redox mapuroteni kana nzira, kukanganisa. cellular process.
Nokudaro, nzira itsva inokwanisa kugadzira mamwe maitiro akanyorwa-radius kudzvinyirira marudzi ane hupamhi hwepamusoro uye hwemazuva pasina kukanganisa zvakanyanya nzira dzemaserura ichave yakakosha kuwedzera kune nzira dziripo. Pakati pezvipenyu zvinogadzirisa, singlet oxygen yakamutsa pfungwa dzedu nekuda kwehupenyu hwayo hupfupi uye shoma yekupararira radius (t1/2 <0.6 µs mumasero)13. Pakati pezvipenyu zvinogadzirisa, singlet oxygen yakamutsa pfungwa dzedu nekuda kwehupenyu hwayo hupfupi uye shoma yekupararira radius (t1/2 <0.6 µs mumasero)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии 3,1/3 (t. Pakati pemaitiro anoshanda, singlet okisijeni yakakwezva kutarisa kwedu nekuda kwehupenyu hwayo hupfupi uye mashoma kupararira radius (t1/2 <0.6 µs mumasero)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）而引起了我們的時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 наше внимание привлекает синглетный кислород из-за его короткого времени наше внимание привлекает синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диф16/кси . Pakati pemafomu anoshanda, singlet okisijeni inokwezva kutarisa kwedu nekuda kwehupenyu hwayo hupfupi uye shoma yekupararira radius (t1/2 <0.6 μs mumasero).Okisijeni imwechete yakanzi iite oxidize methionine, tyrosine, histidine uye tryptophan, zvichiita kuti ive polar 14,15 yekunamatira kune amine kana thiol based probes16,17.Kunyangwe singlet okisijeni yakashandiswa kunyora subcellular compartment RNA, mazano ekugadzirisa endo native POI yepedyo mamaki anoramba asina kuongororwa.Pano, isu tinopa chikuva chinonzi photoactivation-dependent proximity labeling (PDPL), kwatinoshandisa mwenje webhuruu kuvhenekera maPOI akasanganiswa neminiSOG photosensitizer uye inokonzeresa singlet oxygen chizvarwa kuti iite oxidize zvakasara zvakasara, zvichiteverwa neamine-ine gadziriso kuti oxidize kemikari probes kupinda. masero mapenyu epakati..Isu takaedza boka remakemikari probes kuti tiwedzere tag chaiyo uye kuona nzvimbo dzekushandura tichishandisa yakavhurika yeproteomics workflow.Kuenzanisa-ne-parutivi kuenzaniswa kwePDPL neTurboID kunoratidza yakanyatsojeka uye yakazara proteomic kufukidzwa kwePDPL.Isu takashandisa nzira iyi kune organelle-chaiwo mamaki eiyo subcellular proteome uye general proteome kuzivikanwa kwevanosunga vanobatana nekenza-inosanganisirwa epigenetic regulatory protein BRD4 uye chirwere cheParkinson-chakabatana E3 ligase Parkin, iyo yakasimbisa zvese zvinozivikanwa uye zvisingazivikanwe network yeprotein. kudyidzana..Kugona kwePDPL kuziva E3 substrates muhombe maprotein complexes inomiririra mamiriro ezvinhu apo kuzivikanwa kwevasina kunanga binders kunodiwa.Maviri asingazivikanwe parkin substrates akapindirana neubiquitination-proteasome akasimbiswa mune situ.
Photodynamic therapy (PDT) 19 uye chromophore-assisted laser inactivation (CALI) 20, umo chiedza chechiedza chine photosensitizers chinobudisa singlet oxygen, inogona kumisa mapuroteni anonangwa kana kukonzera kufa kwesero.Sezvo singlet okisijeni iri chinhu chinoshanda zvakanyanya chine theoretical diffusion chinhambwe chingangoita 70 nm, nzvimbo yakaganhurwa oxidation yakatenderedza photosensitizer inogona kudzorwa.Zvichibva pane iyi pfungwa, takasarudza kushandisa singlet okisijeni kuti tiwane padhuze kunyora maprotein complexes mumasero mapenyu.Takagadzira PDPL chemoproteomic nzira yekuzadzisa mabasa mana: (1) kugadzirisa chizvarwa chekushanda singlet oxygen yakafanana nePL enzymatic approach;(2) kupa manyorero akagadziriswa nguva pakutanga kwechiedza;(3) nekuchinja (4) Dzivisa kushandisa endogenous cofactors (senge biotin) kuderedza kumashure, kana kushandisa zvakanyanya kuvhiringidza exogenous reagents (senge peroxides) kuderedza kuratidzwa kwesero kushushikana kwezvakatipoteredza.
Photosensitizers inogona kukamurwa kuita mapoka maviri anosanganisira madiki mamorekuru huremu hwefluorophores (eg rose bengal, methylene blue)22 uye mapuroteni madiki ane genetically encoded (semuenzaniso miniSOG, KillerRed)23.Kuti tiwane modular dhizaini, takagadzira chizvarwa chekutanga PDPL chikuva nekuwedzera photosensitizer (PS) mapuroteni kuPOI24,25 (Mufananidzo 1a).Kana yakasvibiswa nechiedza chebhuruu, singlet oxygen oxidizes proximal nucleophilic amino acid residues, zvichiita kuti umpolung polarity iyo inonzi electrophilic uye inogona kuenderera mberi neamine probe nucleophiles16,17.Iyo probe yakagadzirirwa ine alkyne mubato kubvumidza kudzvanya chemistry uye kudonhedza pasi kune LC/MS/MS maitiro.
Schematic mufananidzo wekunyorwa kweprotein complexes inopindirana ne miniSOG.Kana yakafumurwa kuchiedza chebhuruu, masero anoratidza miniSOG-POI anoburitsa okisijeni imwe chete, inoshandura mapuroteni anodyidzana asi asiri mapuroteni asingasunge.Zvigadzirwa zvepakati zvephotooxidation zvinobatwa nerelay labels yeamine chemical probe kugadzira covalent adducts.Boka re alkynyl pa chemistry probe rinobvumira kudzvanya chemistry yekufumisa nekudhonza-pasi ichiteverwa neLC-MS/MS quantitation.b Kemikari chimiro cheamine probes 1-4.c Representative fluorescent gel kuongororwa kwemitochondrial localized miniSOG-mediated proteomic markers vachishandisa probes 1-4 uye huwandu hwehuwandu hunobva pane gel densitometry.Chiratidzo-kumashure-reshiyo yemakemikari probes yakaongororwa uchishandisa maitiro asina kunaka ekudzora asingasanganisi mwenje webhuruu kana kushandisa HEK293T maseru pasina miniSOG kutaura.n = 2 biologically yakazvimirira samples.Dot rega rega rinomiririra biological replica.d Kuonekwa kwemumiriri uye kuwanda kwePDPL uchishandisa optimized probe 3 pamberi kana kusavapo kwezvikamu zvakaratidzwa zvePDPL se c.n = 3 biologically yakazvimirira samples.Dot rega rega rinomiririra biological replica.Mitsetse yepakati nendebvu zvinomiririra kureva uye ± kutsauka kwemaitiro.CBB: Coomassie Brilliant Blue.e Confocal imaging ye singlet oxygen ine kure-tsvuku Si-DMA tsvina.Scale bar: 10 µm.Gel imaging uye confocal miedzo yakadzokororwa yakadzokororwa kanenge kaviri nemhedzisiro yakafanana.
Takatanga kuedza kukwanisa kweakura photosensitizers miniSOG26 uye KillerRed23, yakanyatsoratidzwa muHEK293T, kuyananisa propargylamine kunyora kweproteome semakemikari probe (Supplementary Fig. 1a).Gel fluorescence ongororo yakaratidza kuti yakazara proteome kunyora yakawanikwa uchishandisa miniSOG uye yebhuruu mwenje irradiation, nepo pasina chinooneka chigadzirwa chakaonekwa neKillerRed.Kuvandudza chiratidzo-kumashure reshiyo, takazoedza seti yemakemikari probes ane aniline (1 uye 3), propylamine (2), kana benzylamine (4).Takacherechedza kuti HEK293T masero pachawo aiva nechiratidzo chepamusoro chepamusoro kana chichienzaniswa nechiedza chebhuruu, zvichida nekuda kweiyo endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27. Aniline-based chemical probes 1 uye 3 yakapa humbowo huri nani, iine HEK293T ichiratidza zvakadzikama miniSOG mumitochondria ichiratidzira >8-kupeta kuwedzera kwechiratidzo che probe 3, nepo probe 2 ichishandiswa muRNA-labeling nzira CAP-seq ichingoratidza ~2.5- peta chiratidzo chekuwedzera, zvichida nekuda kwezvakasiyana reactivity zvido pakati peRNA neprotein (Fig. 1b, c). Aniline-based chemical probes 1 uye 3 yakapa humbowo huri nani, iine HEK293T ichiratidza zvakadzikama miniSOG mumitochondria ichiratidzira >8-kupeta kuwedzera kwechiratidzo che probe 3, nepo probe 2 ichishandiswa muRNA-labeling nzira CAP-seq ichingoratidza ~2.5- peta chiratidzo chekuwedzera, zvichida nekuda kwezvakasiyana reactivity zvido pakati peRNA neprotein (Fig. 1b, c).Aniline-based chemical probes 1 uye 3 yakaratidza kunyatsojeka: HEK293T, iyo inonyatsoratidza miniSOG mumitochondria, inoratidza zvinopfuura 8-kupeta kuwedzera kwechiratidzo che probe 3, nepo probe 2, inoshandiswa muCAP-seq RNA nzira yekunyora, chete. inoratidza ~ 2.5-fold signal kuwedzera, zvichida nekuda kwezvakasiyana reactivity zvido pakati peRNA neprotein (Fig. 1b, c).基于 苯胺 的 化学 化学 1 和 3 具有 更 好 的, Hek293t 在 线 粒体 中 稳定 表达 Minisig, 探针 3 的, 而 用 于 Rna 标记 方法 Cap-Seq 的 探针 2 仅 显示 ~ 2.5-倍信号增加，可能是由于RNA 和蛋白赎之间的不同反应偏好（图1b，c）.基于 苯胺 的 化学 化学 化学 1 和 3 具有 更 的, Hek293t 在 粒体 中 稳定 表达 Minisig, 探针 3 倍 信号 增加 增加> 8 倍 而 于】 Rna -倍信号增加，可能是由于RNAAniline-based chemical probes 1 uye 3 yakanga iine hunhu huri nani, HEK293T yakaratidza zvakadzikama miniSOG mumitochondria, uye probe 3 yaive ne8-yakapetwa kuwedzera kwechiratidzo, nepo probe 2 yeCAP-seq RNA yekunyora nzira yaingoratidza ~ 2.5 -peta kuwedzera.muchiratidzo, zvichida nekuda kwekusiyana kwekuita zvido pakati peRNA neprotein (Fig. 1b, c).Mukuwedzera, probe 3 isomers uye hydrazine probes (probes 5, 6, 7) yakaedzwa, ichisimbisa kugadziriswa kweprobe 3 (Supplementary Fig. 1b, c).Saizvozvo, mu-gel fluorescence ongororo yakaratidza mamwe akakwenenzverwa ekuedza paramita: irradiation wavelength (460 nm), kemikari probe concentration (1 mM), uye irradiation nguva (20 min) (Supplementary Fig. 2a-c).Kusiya chero chikamu kana danho muPDPL protocol kwakaguma nekukosha kwechiratidzo chekudzoka kumashure (Fig. 1d).Zvinonzwisisika, protein labeling yakaderedzwa zvakanyanya pamberi pe sodium azide kana trolox, iyo inozivikanwa kudzima singlet oxygen.Kuvapo kweD2O, iyo inozivikanwa kudzikamisa singlet oxygen, inosimudzira chiratidzo chekunyora.Kuti tiongorore mupiro wemamwe marudzi eokisijeni anoshanda pakuisa mazita, mannitol uye vhitamini C zvakawedzerwa kumisikidza hydroxyl uye superoxide radical scavengers, zvichiteerana, 18, 29, asi havana kuwanikwa kuti vaderedze mazita.Kuwedzerwa kweH2O2, asi kwete kuvhenekera, hakuna kuguma nekunyora (Supplementary Fig. 3a).Fluorescence singlet oxygen imaging ine Si-DMA probes yakasimbisa kuvapo kweiyo singlet oxygen muHEK293T-miniSOG waya, asi kwete mune yekutanga HEK293T waya.Mukuwedzera, mitoSOX Tsvuku yaisagona kuona kugadzirwa kwe superoxide mushure mekuvhenekera (Fig. 1e uye Supplementary Fig. 3b) 30. Iyi data inonyatsoratidza kuti singlet oxygen ndiyo inonyanya kushandiswa kweokisijeni yemhando inotarisirwa kune inotevera proteomic labeling.Cytotoxicity yePDPL yakaongororwa kusanganisira blue light irradiation uye makemikari probes, uye hapana cytotoxicity inokosha yakaonekwa (Supplementary Fig. 4a).
Kuti tidzidze maitiro ekunyora uye kugonesa proteomic kuzivikanwa kweprotein complexes uchishandisa LC-MS/MS, isu chekutanga tinoda kuona kuti ndeapi amino acids akagadziridzwa uye delta misa yemaprobe label.Methionine, histidine, tryptophan uye tyrosine zvakanzi zvakashandurwa ne singlet oxygen14,15.Isu tinobatanidza iyo TOP-ABPP31 kufambiswa kwebasa pamwe nekusarerekera kuvhurika kutsvaga kwakapihwa neFragPipe komputa chikuva chakavakirwa paMSFragger32.Mushure me singlet oxygen modification uye chemical probe labeling, click chemistry yakaitwa pachishandiswa biotin reduction label ine cleavable linker, inoteverwa neutravidin stretching uye trypsin digestion.Iyo peptide yakagadziridzwa, ichiri kusungwa kune resin, yaive photocleaved yeLC-MS / MS kuongororwa (Mufananidzo 2a uye Supplementary Data 1).Nhamba huru yekugadziridza yakaitika mukati meproteome ine inopfuura 50 peptide mepu (PSM) machisi akanyorwa (Fig. 2b).Sezvineiwo, isu takangoona kuchinjika kwe histidine, pamwe nekuda kwepamusoro reactivity ye oxidized histidine kune aniline probes kupfuura mamwe amino acids.Zvinoenderana neyakaburitswa nzira ye histidine oxidation ne singlet okisijeni, 21,33 iyo yakarongwa delta-mass chimiro che +229 Da inoenderana neiyo adduct ye probe 3 ine 2-oxo-histidine mushure mekuviri oxidation, nepo +247 Da ndiyo hydrolysis chigadzirwa. ye +229 Da (Supplementary Fig. 5).Kuongororwa kweiyo MS2 spectrum kwakaratidza kuvimbika kwakanyanya kwekuzivikanwa kweakawanda ey uye b ions, kusanganisira kuzivikanwa kweakagadziridzwa fragment ions (y uye b) (Fig. 2c).Kuongororwa kwemamiriro ezvinhu emunharaunda yePDPL-modified histidines yakaratidza chimiro chepakati chemaitiro kune zviduku zviduku zvehydrophobic pa ± 1 nzvimbo (Supplementary Fig. 4b).Paavhareji, 1.4 histidines yakaonekwa paprotein, uye nzvimbo dzezvicherechedzo izvi zvakagadziriswa ne solvent accessible surface area (SASA) uye hukama hwekugadzirisa kuwanikwa (RSA) kuongorora (Supplementary Fig. 4c, d).
Kufambiswa kwekusarerekera kwekudzidza yakasara yekusarudza uchishandisa FragPipe komputa chikuva chinofambiswa neMSFragger.Cleavable linkers anoshandiswa muKudzvanya chemistry kubvumira photocleavage yemodified peptides kubva kustreptavidin resin.Tsvagiridzo yakavhurika yakatangwa kuti ione zvakawanda zvakagadziridzwa, pamwe chete nemasara akakodzera.b Ipa huwandu hwekugadziriswa kunoitika mukati meproteome.Peptide mepu yePSM.c MS2 spectral annotation ye histidine nzvimbo dzakagadziriswa ne probe 3. Semumiririri wemuenzaniso, covalent reaction ne probe 3 yakawedzera +229.0938 Da kune yakagadziridzwa amino acid.d Mutation assay inoshandiswa kuyedza mamaki ePDPL.PRDX3 (H155A, H225A) uye PRDX1 (H10A, H81A, H169A) yakashandurwa nemaplasmids emhando dzesango kuti aonekwe neAnti-Mureza.e Iyo peptide yekugadzira yakaitwa neyakacheneswa miniSOG pamberi peprobe 3 uye zvigadzirwa zvinoenderana neΔm +247 uye +229 zvakaonekwa muLC-MS spectrum.f In vitro protein-to-protein interactions modeled with miniSOG-6xHis-tag uye anti-6xHis antibody.Antibiotin (streptavidin-HRP) uye anti-mouse Western blot analysis ye miniSOG-6xHis/anti-6xMa antibody complexes ake akanyorwa neprobe 3, zvichienderana nenguva yekuratidzwa kuchiedza.Mazita emapuroteni ega anoratidzwa muhuremu hwemamorekuru hunoenderana: LC antibody light chain, HC antibody heavy chain.Izvi zviedzo zvakadzokororwa zvakazvimiririra kanenge kaviri nemhedzisiro yakafanana.
Nezve biochemical verification yenzvimbo yekunyora, PRDX3 uye PRDX1 yakaonekwa nehukuru spectrometry yakashandurwa kubva kuhistidine kuenda kualanine uye ichienzaniswa nemhando yemusango mukuyedza kutapurirana.Mhedzisiro yePDPL yakaratidza kuti kushanduka kwakaderedza zvakanyanya kunyora (Fig. 2d).Zvichakadaro, kutevedzana kwepeptide kwakaonekwa mukutsvaga kwakavhurika kwakagadzirwa uye kuita mu vitro ine yakacheneswa miniSOG pamberi peprobe 3 uye bhuruu mwenje, inoburitsa zvigadzirwa zvine huwandu hwekuchinja kwe +247 uye +229 Da payakaonekwa neLC-MS (Fig. .2e).)Kuongorora kana kupindirana kweproximal mapuroteni kunogona kunyorwa mu vitro mukupindura miniSOG photoactivation, isu takagadzira yekunyepedzera yepedyo yekuongorora nekudyidzana pakati pe miniSOG-6xHis protein uye anti-His monoclonal antibody in vitro (Mufananidzo 2f).Muchiyedzo ichi, isu taitarisira proximal kunyorwa kweanorema uye akareruka cheni ne miniSOG.Kutaura zvazviri, anti-mouse (ichiziva maketani anorema uye akareruka e-anti-6xHis-labeled antibody) uye streptavidin Western blots yakaratidza yakasimba biotinylation yemaketani anorema uye akareruka.Zvikuru, takaona miniSOG autobiotinylation nekuda kweiyo 6xHis tag uye muchinjiko-zvinongedzo pakati pemwenje uye inorema cheni, iyo inogona kunge ine hukama neyakatsanangurwa gap pakati pe lysine uye 2-oxo-histidine proximal mhinduro.Mukupedzisa, tinogumisa kuti PDPL inogadzirisa histidine nenzira inotsamira.
Chinangwa chedu chinotevera chaive chekuratidza subcellular proteome kuyedza iyo chaiyo in situ labeling.Nokudaro, isu takanyatsoratidza miniSOG mu nucleus, mitochondrial matrix, kana kunze ER membrane yeHEK293T masero (Fig. 3a).Gel fluorescence analysis yakaratidza mabhandi akawanda akanyorwa panzvimbo nhatu dze subcellular pamwe chete nemaitiro akasiyana ekunyora (Fig. 3b).Fluorescence imaging analysis yakaratidza yakakwirira chaiyo yePDPL (Fig. 3c).The PDPL workflow yakateverwa nekudzvanya maitiro ane rhodamine dhayi kudonhedza subcellular proteomes uchishandisa fluorescence microscopy, uye PDPL masaini akaiswa colocalized neDAPI, mitochondrial trackers, kana ER trackers, zvichisimbisa kuvimbika kwakanyanya kwePDPL.Kune iyo matatu organelle nzvimbo, kudivi-ne-kurutivi kuenzanisa kwePDPL neTurboID vachishandisa avidin western blot yakaratidza kuti PDPL yakanyorwa zvakanyanya kana ichienzaniswa neyavo kutonga.Pasi pemamiriro ePDPL, mamwe mabhandi akanyorwa akaonekwa, achiratidza mamwe mapuroteni akanyorwa nePDPL (Supplementary Fig. 6a-d).
a Schematic inomiririra ye miniSOG-mediated organelle-specific proteome labeling.miniSOG inotarisa mitochondrial matrix kuburikidza nefusion kune N-terminal 23 amino acids yevanhu COX4 (mito-miniSOG), nucleus kuburikidza ne fusion kuH2B (nucleus-miniSOG), uye Sec61β kuburikidza ne cytoplasmic side yeER membrane (ER-miniSOG). )Zviratidzo zvinosanganisira imaging yegel, confocal imaging, uye mass spectrometry.b Representative gel mifananidzo yematatu organelle-yakananga PDPL profiles.CBB Coomassie Brilliant Blue.c Inomiririra confocal mifananidzo yeHEK293T masero anoratidza zvakadzikama miniSOG ine akasiyana subcellular localizations akaonekwa nemasoja akanyorwa V5 (tsvuku).Subcellular mamakisi anoshandiswa mitochondria uye ER (girinhi).Iyo PDPL kufambiswa kwebasa kunosanganisira kuonekwa kwe miniSOG (yellow) yakanyorwa subcellular proteoms uchishandisa Cy3-azide click chemistry.Scale bar: 10 µm.d Volcanic plots dzePDPL-tagged proteomes mune akasiyana organelles akaverengerwa neasina kunyorwa quantification (n = 3 yakazvimirira biological experiments).T-bvunzo yeMudzidzi ine miswe miviri yakashandiswa panzvimbo dzegomo rinoputika.HEK293T mhando yemusango yakashandiswa seyakaipa kutonga. Mapuroteni akashandurwa zvakanyanya anojekeswa mutsvuku (p <0.05 uye> 2-fold ion intensity musiyano). Mapuroteni akashandurwa zvakanyanya anojekeswa mutsvuku (p <0.05 uye> 2-fold ion intensity musiyano). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Mapuroteni akashandurwa zvakanyanya akaiswa mutsvuku (p <0.05 uye> 2-peta musiyano muion 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). Mapuroteni akashandurwa zvakanyanya akaiswa mutsvuku (p <0.05 uye> 2-peta musiyano musimba reionic).Mapuroteni ane hukama akakosha kuHEK293T-miniSOG asi asina kukosha kuHEK293T anoratidzwa mugirini.e Ongororo yezvakajeka zveproteomic datasets kubva mukuyedza d.Huwandu hwehuwandu hwehuwandu hwemapuroteni akakosha mune yega organelle (tsvuku uye girinhi madotsi) inotaridzwa kumusoro.Histograms inoratidza mapuroteni anowanikwa mune organelles anobva paMitoCarta 3.0, GO ongororo uye A. Ting et al.vanhu.Akapatsanura dhatabheti emitochondria, nuclei, uye ER.Izvi zviedzo zvakadzokororwa zvakazvimiririra kanenge kaviri nemhedzisiro yakafanana.Raw data inopiwa nenzira yemafaira e data.
Kukurudzirwa nejeri uye mhedzisiro yekufungidzira, label-isina quantification yakashandiswa kuenzanisa iyo yakaonekwa proteome mune yega organelle (Supplementary Data 2).Untransfected HEK293T yakashandiswa seyakaipa kudzora kubvisa kumashure mamaki. Volcano plot analysis yakaratidza zvakapfumisa mapuroteni (p <0.05 uye> 2-fold ion intensity) pamwe chete neprotein ye singleton inongowanikwa mumitsetse ye miniSOG-inobudisa (Fig. 3d red and green dots). Volcano plot analysis yakaratidza zvakapfumisa mapuroteni (p <0.05 uye> 2-fold ion intensity) pamwe chete neprotein ye singleton inongowanikwa mumitsetse ye miniSOG-inobudisa (Fig. 3d red and green dots). 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). Volcano plot analysis yakaratidza kupfumisa zvakanyanya mapuroteni (p <0.05 uye> 2-fold ion intensity) pamwe chete neprotein imwe chete inongowanikwa mumitsetse ye miniSOG-inoratidza (Fig. 3d, red and green dots).火山图分析显示出显着富集的蛋白质（p <0.05 和>2 倍离子强度）以及仅存在纎mini存在纎miniSOG 一亚存在纎miniSOG 一火山图 分析 显示 出 显着 的 (P <0 0.05 和> 2 倍 离子 强度) 仅 在于 在于 在于 在于 (的 3 3 3d 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). Volcano plot analysis yakaratidza zvakanyanya kupfumisa mapuroteni (p <0.05 uye> 2x ionic simba) pamwe chete neprotein imwe chete inowanikwa mumutsara we miniSOG wekutaura (mavara matsvuku uye matsvuku muFig. 3d).Tichibatanidza idzi data, takaona 1364, 461, uye 911 zvine nhamba yakakosha mapuroteni enyukireya, mitochondrial, uye ER ekunze membrane, zvichiteerana.Kuti tiongorore kurongeka kwe organelle-localized PDPL, takashandisa MitoCarta 3.0, Gene Ontology (GO) kuongorora, uye A. Ting et al.data set8 yakashandiswa kune mitochondria, nucleus, uye ER kuedza iyo organelle chaiyo yemapuroteni akaonekwa, anoenderana nekururama kwe73.4, 78.5, uye 73.0% (Fig. 3e).Iyo chaiyo yePDPL inosimbisa kuti PDPL chishandiso chakanakira kuziva organelle-chaiwo maproteomes.Zvinonzwisisika, ongororo yesubochondrial yemaprotein emitochondrial akaonekwa yakaratidza kuti proteome yakabatwa yainyanya kugoverwa mumatrix uye mukati me membrane (226 ne106, zvichiteerana), ichiverengera 91.7% (362) yehuwandu hwehuwandu hwemapuroteni emitochondrial.chiyero chepamusoro chePDPL chakawedzerwa kusimbiswa (Supplementary Fig. 7a).Saizvozvowo, subnuclear analysis yakaratidza kuti proteome yakatorwa yainyanya kugoverwa mu nucleus, nucleoplasm, uye nucleolus (Supplementary Fig. 7b).Nuclear proteomic analysis nenuclear localization signal peptide (3xNLS) yakaratidza kufanana kwakafanana neH2B kuvaka (Supplementary Fig. 7c-h).Kuti uone kujeka kwechiratidzo chePDPL, nyukireya laminin A yakasarudzwa semusungo wakasarudzika wePOI7.PDPL yakaratidza 36 zvakanyanya kupfumisa mapuroteni, ayo 12 mapuroteni (30.0% anosanganisira lamin A) ainyatsozivikanwa lamin A inopindirana mapuroteni anotsanangurwa neString database, ane chikamu chepamusoro pane BioID nzira (122 mapuroteni) 28 ye28. , 22.9 %) 7. Nzira yedu yakaratidza mapuroteni mashoma, zvichida nekuda kwenzvimbo dzisina kunyorwa, izvo zvakagoneka nekuwedzera kushanda kwe singlet oxygen.GO ongororo yakaratidza kuti mapuroteni akaonekwa ainyanya kuwanikwa munucleoplasm (26), nuclear membrane (10), nuclear membrane (9), uye nuclear pores (5).Pamwe chete, mapuroteni aya ane nyukliya-nzvimbo akaverengera 80% yemapuroteni akafumiswa, achienderera mberi achiratidza kujeka kwePDPL (Supplementary Fig. 8a-d).
Mushure mekutangisa kugona kwePDPL kuita zvekumaka padhuze muma organelles, takazoedza kana PDPL inogona kushandiswa kuongorora POI inosunga vanobatana.Kunyanya, takatsvaga kutsanangura PDPL kuongororwa kwecytosolic mapuroteni, ayo anoonekwa seakaomesesa tariswa pane avo membrane-anogara munzvimbo nekuda kwehunhu hwavo hwakasimba.Iyo bromodomain uye extraterminal (BET) protein BRD4 yakakwezva pfungwa dzedu nokuda kwebasa rayo rinokosha muzvirwere zvakasiyana-siyana 35, 36.Iyo yakaoma yakaumbwa neBRD4 ndeye transcriptal coactivator uye yakakosha yekurapa chinangwa.Nekugadzirisa kutaura kwec-myc uye Wnt5a zvinyorwa zvinyorwa, BRD4 inofungidzirwa kuti ndiyo inonyanya kukosha yeacute myeloid leukemia (AML), multiple myeloma, Burkitt's lymphoma, kenza yekoloni uye zvirwere zvinoputika37,38.Pamusoro pezvo, mamwe mavhairasi anonangidzira BRD4 kudzora hutachiona uye cellular transcript, senge papillomavirus, HIV, uye SARS-CoV-236,39.
Kumepu kusangana kweBRD4 uchishandisa PDPL, takabatanidza miniSOG nepfupi N- kana C-terminal isoform yeBRD4.Zvigumisiro zveproteomic zvakaratidza huwandu hwepamusoro hwekupindirana pakati pezvivakwa zviviri (Supplementary Fig. 9a).Nuclear proteome inowanikwa ne miniSOG-H2B inovhara 77.6% yemapuroteni anobatana neBRD4 (Supplementary Fig. 9b).Zvadaro, nguva dzakasiyana-siyana dzekuvhenekera (2, 5, 10, 20 min) dzakashandiswa kugadzirisa chiratidzo chechiratidzo (Fig. 4a uye data yekuwedzera 3).Isu tinogumisa kuti pamapfupi mafotoperiods, PDPL ichanyanya kunyora vakananga vanosunga vanobatana, nepo nguva refu ichisanganisira mapuroteni akaonekwa munguva pfupi yekutora mafoto pamwe chete nezvinangwa zvisina kunangana mumalebel complexes.Muchokwadi, takawana kupindirana kwakasimba pakati penguva dzenguva (84.6% ye2 uye 5 min; 87.7% ye5 uye 10 min; 98.7% ye10 uye 20 min) (Fig. 4b uye Supplementary Fig. 9c).Mumapoka ese ekuyedza, hatina kuwana chete BRD4 yekuzvinyora, asi akati wandei anozivikanwa tarisiro seMED1, CHD8, BICRA, NIPBL, SMC1A, uye HMGB1 yakatsanangurwa mudhatabhesi yetambo.Ionic simba rezvinangwa izvi zvakaenzana nenguva yekutarisana (Fig. 4c uye Supplementary Fig. 9d).GO kuongororwa kwemapuroteni akaonekwa muboka remaminiti e2 kwakaratidza kuti mapuroteni akaonekwa akaiswa munharaunda uye akabatanidzwa mukugadzirisa chromatin uye RNA polymerase basa.Iyo molecular function yeprotein yakagadziriswa muchromatin binding kana transcriptal coactivation, inopindirana neBRD4 basa (Fig. 4d).String database-enabled protein interaction analysis yakaratidza danho rekutanga rekusangana kusinganangana pakati peBRD4 neHDAC mhuri dzinodyidzana dzakaita seSIN3A, NCOR2, BCOR, uye SAP130 (Fig. 4e uye Supplementary Fig. 9e), inopindirana neBRD4 uye HDAC inosunga acetylated histones ..Mukuwedzera, zvinangwa zvemumiririri zvakaonekwa neLC-MS / MS, kusanganisira Sin3A, NSUN2, Fus, uye SFPQ, zvakasimbiswa neWestern blotting (Fig. 4f).Munguva pfupi yapfuura, iyo pfupi isoform yeBRD4 inonzi inogadzira nuclei ine liquid-liquid phase separation (LLPS) zvivakwa.Iyo RNA inosunga mapuroteni Fus uye SFPQ inopindirana iyo LLPS yeakasiyana masero maitiro uye akaonekwa pano seasina kunyorwa BRD4 anosunga mapuroteni.Kudyidzana pakati peBRD4 neSFPQ kwakasimbiswa ne-co-immunoprecipitation (co-IP) zviedzo (Mufananidzo 4g), zvichiratidza imwe nzira yeBRD4-mediated liquid-liquid phase separation inokodzera kumwe kuongororwa.Zvikatorwa pamwechete, mhedzisiro iyi inoratidza kuti PDPL ipuratifomu yakanakira yekuona inozivikanwa BRD4 inodyidzana pamwe nemapuroteni asingazivikanwe anosunga.
a Schematic inomiririra yeminiSOG-mediated BRD4 padyo yekumaka, nguva yekuratidzwa: 2, 5, 10, uye 20 min.b Kupindirana kwemapuroteni akaonekwa panguva dzakasiyana dzekuvhenekera.Kuwedzera kweprotein kunoonekwa muHEK293T-miniSOG-BRD4 kwakakosha zvakanyanya kana kuchienzaniswa nemhando yemusango HEK293T.c Ion intensity pakuyera mumiriri asina kunyorwa anozivikanwa BRD4-binding mapuroteni panguva yakatarwa.n = 3 biologically yakazvimirira samples.Dhata dzinounzwa sezvinoreva ± zvakajairwa kutsauka.d Gene ontological analysis (GO) yemapuroteni akaonekwa muboka remaminitsi maviri.Matemu gumi ekutanga eGO akanyorwa.Mabhuru ane mavara zvichienderana neGO term chikamu, uye saizi yebubble inoenderana nehuwandu hwemapuroteni anowanikwa mutemu yega yega.e Kuongororwa kwetambo yemapuroteni anosangana neBRD4.Denderedzwa reyero iglue yakananga uye grey denderedzwa ndiro rekutanga reglue isina kunanga.Mitsara mitsvuku inomiririra kudyidzana kwakatemwa uye mitsetse yebhuruu inomiririra kudyidzana kwakafanotaurwa.f Mumiriri weBRD4 anosunga zvinangwa zvakaonekwa muLC-MS/MS zvakasimbiswa neWestern blotting.g Co-immunoprecipitation miedzo inosimbisa kuwirirana pakati peSFPQ neBRD4.Izvi zviedzo zvakadzokororwa zvakazvimiririra kanenge kaviri nemhedzisiro yakafanana.Raw data inopiwa nenzira yemafaira e data.
Pamusoro pekuziva zvisina kunyoreswa zvinosanganisirwa nePOI, isu tinofungidzira kuti PDPL ichave yakakodzera kuzivisa ma substrates ema enzymes, izvo zvingada hunhu hwemapuroteni anosunga asina kunanga mumacomplexes makuru kuti atsanangure ma substrates asina kunyoreswa.Parkin (yakavharidzirwa nePARK2) iE3 ligase uye kuchinja muparkin kunozivikanwa kukonzera autosomal recessive juvenile Parkinson's disease (AR-JP)42.Mukuwedzera, parkin yakatsanangurwa seyakakosha kune mitophagy (mitochondrial autophagy) uye kubviswa kweiyo reactive oxygen marudzi.Nekudaro, kunyangwe akati wandei parkin substrates akaonekwa, basa re parkin muchirwere ichi harina kujeka.Kuzivisa ma substrates ayo asina kurongeka, PDPL yakaedzwa nekuwedzera miniSOG kune N- kana C-terminus yeparkin.Masero akabatwa necarbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) kuti ashandise parkin kuburikidza nePINK1-Parkin nzira.Kuenzaniswa neyedu BRD4 PDPL zvabuda, parkin N-terminus fusion yakaratidza yakakura seti yemapuroteni akatarwa, kunyangwe akafukidza chikamu chikuru cheC-terminus (177 kunze kwe210) (Mufananidzo 5a,b uye Supplementary Data 4).mhedzisiro yacho inoenderana nemishumo yekuti N-terminal tags inogona kuita aberrantly activate Parkin44.Sezvineiwo, kwaingova negumi nemasere mapuroteni aipindirana mune yedu data neyakaburitswa AP-MS mhedzisiro yeParkin43, zvichida nekuda kwekusiyana pakati pemitsara yesero uye maproteomics workflows.Mukuwedzera kune mapuroteni mana anozivikanwa (ARDM1, HSPA8, PSMD14, uye PSMC3) anozivikanwa nenzira mbiri (Fig. 5c) 43.Kuti uwedzere kusimbisa mhedzisiro yeLC-MS/MS, kurapwa kwePDPL uye kuvharika kwekuMadokero kwakatevera kwakashandiswa kuenzanisa mhedzisiro yeHEK293T yevabereki cell assay uye yakagadzikana N-terminal parkin line.Zvinangwa zvisati zvazivikanwa CDK2, DUT, CTBP1, uye PSMC4 zvakaedzwa nebhainda rinozivikanwa, DNAJB1 (Fig. 5d).
Volcano chirongwa cheparkin-chinopindirana mapuroteni muHEK293T maseru ane akanyatso kuratidzwa miniSOG akasanganiswa kune N- kana C-terminus yeparkin (n = 3 yakazvimirira biological kuyedza).T-bvunzo yeMudzidzi ine miswe miviri yakashandiswa panzvimbo dzegomo rinoputika.HEK293T yakashandiswa sekutonga kusina kunaka. Mapuroteni akashandurwa zvakanyanya anojekeswa mutsvuku (p <0.05 uye> 2-fold ion intensity musiyano). Mapuroteni akashandurwa zvakanyanya anojekeswa mutsvuku (p <0.05 uye> 2-fold ion intensity musiyano). Значительно измененные белки выделены красным цветом (p <0,05 и >2-кратная разница в интенсивности ионов). Mapuroteni akashandurwa zvakanyanya akaiswa mutsvuku (p <0.05 uye> 2-peta musiyano muion 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). Mapuroteni akashandurwa zvakanyanya akaiswa mutsvuku (p <0.05 uye> 2-peta musiyano musimba reionic).Mapuroteni ane hukama akakosha kuHEK293T-miniSOG asi asina kukosha kuHEK293T anoratidzwa mugirini.b Venn dhayagiramu inoratidza anopindirana mapuroteni pakati peN-terminal uye C-terminal inovaka.N-terminal ma tag anogona kuita aberrantly activate parkin uye zvichikonzera mamwe mapuroteni anozivikanwa.c Venn dhayagiramu inoratidza anopindirana mapuroteni pakati pePDPL neAP-MS.Vanozivikanwa vanobatana vakanyorwa, kusanganisira 4 ye18 inopindirana mapuroteni uye 11 ye159 mapuroteni akanyatso zivikanwa muPDPL.d Zvinangwa zvemumiriri zvakaonekwa neLC-MS/MS zvakasimbiswa neWestern blotting.e Ssu72 uye SNW1 vakaonekwa sevasina kunyoreswa parkin substrates.Aya maFLAG-tagged protein plasmids akachinjirwa muHEK293T uye HEK293T-Parkin-miniSOG achiteverwa neCCCP kurapwa panguva dzakasiyana.Kusvibiswa kwacho kwainyanya kutaurwa muParkin overexpression line.f Kushandisa proteasome inhibitor MG132, yakasimbiswa kuti kusvibiswa kweSsu72 uye SNW1 kunopindirana neproteasome-ubiquitination.Izvi zviedzo zvakadzokororwa zvakazvimiririra kanenge kaviri nemhedzisiro yakafanana.Raw data inopiwa nenzira yemafaira e data.
Zvinonyanya kukosha, mapuroteni akaonekwa nePDPL anofanira kusanganisira parkin-binding proteins uye substrates dzavo.Kuti tione parkin substrates dzisina kunyoreswa, takasarudza mapuroteni manomwe akaonekwa (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 uye SNW1) uye maplasmids akatapurwa kuti aburitse majini aya kune akajairwa HEK293T uye anyatso kuratidza miniSOG-Parkin's HEK293T kurapwa.Mazinga eSsu72 uye SNW1 mapuroteni akaderedzwa zvakanyanya mumutsara wakagadzikana miniSOG-Parkin (Fig. 5e).Kurapa neCCCP kwemaawa gumi nemaviri kwakaguma nekuderera kwakanyanya kwezvose zviri zviviri substrates.Kuti uongorore kana kusvibiswa kweSsu72 uye SNW1 kunotungamirirwa neproteasome-ubiquitination, proteasome inhibitor MG132 yakawedzerwa kuti inhibit basa reproteasome, uye chaizvoizvo takaona kuti kusvibiswa kwavo kwakadziviswa (Fig. 5f).Zvimwe zvisiri-substrate zvinangwa zvakasimbiswa seParkin interactors vachishandisa Western blotting (Supplementary Fig. 10), iyo yakaratidza migumisiro inowirirana neLC-MS / MS.Mukupedzisa, kubatanidzwa kwePDPL workflow nechinangwa cheprotein transfection verification inobvumira kuzivikanwa kweE3 ligase substrates dzisina kunyoreswa.
Isu takagadzira yakajairwa yepedyo yekumaka chikuva iyo inokutendera iwe kuti uone munzvimbo uye nguva inopindirana maPOI.Iyi puratifomu yakavakirwa pane miniSOG photosensitizer protein, inongosvika gumi nemaviri kDa, isingasviki hafu yehukuru hweiyo APEX2 enzyme (27 kDa) uye chikamu chimwe muzvitatu saizi yeTurboID (35 kDa).Iyo diki saizi inofanirwa kuwedzera zvakanyanya huwandu hwemaapplication ekudzidza madiki maprotein interactomes.Kuwedzera kuongororwa kwemamwe mafotosensitizers, angave mapuroteni ane genetically encoded kana mamorekuru madiki, anodiwa kuwedzera huwandu hwegoho re singlet oxygen uye kuwedzera kunzwisiswa kweiyi nzira.Kune iyo yazvino vhezheni yeminiSOG, yakakwirira yenguva resolution inogona kuwanikwa uchishandisa bhuruu kuvhenekera kumisa mamaki epedyo.Pamusoro pezvo, nguva yakareba yekuratidzwa yakaburitsa hombe "gore" re singlet oxygen, zvichikonzera kugadziridzwa kweakawanda distal histidine residues, yakawedzera labeling radius, uye kugona kugadzirisa iyo PDPL spatial resolution.Isu takaedzawo zvinomwe makemikari probes kuti tiwedzere chiratidzo-kune-kumashure reshiyo uye tikaongorora iyo molecular mashandiro kuseri kweiyi nzira.Iyo TOP-ABPP workflow yakabatanidzwa nekutsvaga kusingafungidzirwe yakavhurika yakasimbisa kuti kugadziridzwa kwakaitika chete mu histidines uye hapana inopindirana microenvironment yakacherechedzwa yekuwedzera histidine modifications, kunze kwekunge kuri pakati pekuda kwe histidines munharaunda ye loop.
PDPL yakashandiswawo kuratidza subcellular proteomes ine proteome chaiyo uye kufukidzwa ingangoenzaniswa nedzimwe nzira dzepadhuze dzekunyora uye organelle-chaiyo makemikari probe nzira.Zvikwangwani zvepedyo zvakashandiswawo zvakabudirira kuratidza pamusoro, lysosomal, uye secretome-inosanganiswa maproteom46,47.Isu tinotenda kuti PDPL ichaenderana neaya subcellular organelles.Pamusoro pezvo, takadenha PDPL nekuona zvinangwa zve cytosolic protein binding iyo yakaoma kunzwisisa kupfuura membrane bound mapuroteni nekuda kwesimba rawo uye kubatanidzwa mukudyidzana kwakawanda kwenguva.PDPL yakashandiswa kune mapuroteni maviri, iyo transcriptional coactivator BRD4 uye chirwere-chakabatana ligase E3 Parkin.Aya mapuroteni maviri akasarudzwa kwete chete nekuda kwemabasa avo akakosha ebhayoloji, asiwo nekuda kwehutano hwavo hwekiriniki uye kugona kwekurapa.Kune aya maPOI maviri, vanozivikanwa vanosunga vanobatana pamwe chete nezvinangwa zvisina kunyoreswa zvakaonekwa.Zvinonzwisisika, chikamu chekuparadzanisa-chakabatana neprotein SFPQ yakasimbiswa ne-co-IP, iyo inogona kuratidza nzira itsva iyo BRD4 (pfupi isoform) inogadzirisa LLPS.Panguva imwecheteyo, isu tinotenda kuti kuzivikanwa kweParkin substrates ndiyo mamiriro ekuti kuzivikanwa kweasina kunanga adhesives kunodiwa.Takaona maviri asingazivikanwe parkin substrates uye takasimbisa kuderedzwa kwavo munzira yeubiquitination-proteasome.Munguva pfupi yapfuura, nzira-yakavakirwa pakuteya zano rakagadzirwa kuti rione hydrolase substrates nekuvateya nema enzymes.Kunyangwe iyi iri nzira ine simba kwazvo, haina kukodzera kuongororwa kwe substrates inobatanidzwa mukuumbwa kwehukuru hwakakura uye inoda kuumbwa kwe covalent zvisungo pakati pe enzyme ne substrate.Tinotarisira kuti PDPL inogona kuwedzerwa kuti idzidze mamwe maprotein complexes uye mhuri dze enzyme, dzakadai se deubiquitinase uye metalloprotease mhuri.
Iyo itsva fomu ye miniSOG, inonzi SOPP3, yakagadziridzwa neyakagadziridzwa singlet oxygen kugadzirwa.Isu takafananidza miniSOG neSOPP3 uye takawana yakagadziridzwa yekumaka kuita, kunyangwe chiratidzo-ku-ruzha chiyero chakaramba chisina kuchinjwa (Supplementary Fig. 11).Isu takafungidzira kuti optimization yeSOPP3 (semuenzaniso, kuburikidza neyakagadziriswa shanduko) yaizotungamira kune akanyatsoshanda photosensitizer mapuroteni anoda nguva pfupi yechiedza uye nekudaro kubvumira maitiro emagetsi emagetsi kuti abatwe.Zvinonzwisisika, iyo yazvino vhezheni yePDPL inogumira kune cellular nharaunda sezvo ichida mwenje webhuruu kuvhenekerwa uye haigone kupinda mukati mematishu akadzika.Ichi chimiro chinodzivisa kushandiswa kwayo muzvidzidzo zvemuenzaniso wemhuka.Zvisinei, kusanganiswa kweoptogenetics nePDPL kunogona kupa mukana wekutsvakurudza mhuka, kunyanya muuropi.Uye zvakare, mamwe mainjiniya infrared photosensitizers anobvisawo ichi chinogumira.Tsvagiridzo iri kuenderera mberi munzvimbo iyi.
Iyo HEK293T cell line yakawanikwa kubva kuATCC (CRL-3216).Mutsara wesero wakaongororwa kuti hauna hutachiona hwe mycoplasma uye wakagadzirwa muDMEM (Thermo, #C11995500BT) yakawedzerwa ne10% fetal bovine serum (FBS, Vistech, #SE100-B) uye 1% penicillin/streptomycin (Hyclone, #SV30010).kukura mukati.
3-Aminophenylene (sample 3) uye (4-ethynylphenyl)methanamine (sample 4) yakatengwa kubva kuBidepharm.Propylamine (probe 2) yakatengwa kubva kuEnergy-chemicals.N-(2-Aminophenyl)pent-4-ynamide (probe 1) yakagadzirwa maererano nenzira dzakadhindwa.
Supplementary Tafura 1 inodonongodza maumbirwo emajini akashandiswa muchidzidzo ichi.Iyo miniSOG uye KillerRed sequences dzakagadzirwa kubva kune chipo plasmid kubva kuP. Zou (Peking University).Iyo mitochondrial matrix yakanangana nenhevedzano yakatorwa kubva ku23 N-terminal amino acids yeCOX4 uye yakaumbwa mumavheji anoratidzwa uchishandisa Gibson assembly (Beyotime, #D7010S).Kutarisa membrane uye nucleus ye endoplasmic reticulum, SEC61B DNA yemunhu (NM_006808.3) (NEB, #M0491L) yakasimudzirwa nePCR kubva muraibhurari ye cDNA yeHEK293T masero, uye H2B DNA (yakapiwa naD. Lin, Shenzhen Bay Laboratory) uye akaumbwa, sezvataurwa pamusoro apa.Kunze kwekunge zvaratidzwa neimwe nzira, mamwe mapuroteni majini anoshandiswa kutapurirana uye kuvaka yakagadzikana maseru mitsara PCR yakakwidziridzwa kubva HEK293T cell cDNA raibhurari.G3S (GGGS) uye G4S (GGGGS) yakashandiswa sekubatanidza pakati peprotein yebheti uye miniSOG.V5 epitope tag (GKPIPNPLLGLDST) yakawedzerwa kune aya ma fusion anovaka.Nekutaura mumhuka dzinoyamwisa uye kumisikidza yakagadzikana sero mutsara, iyo miniSOG fusion yekuvaka yakaiswa mu pLX304 lentiviral vector.Nekutaura kwebhakitiriya, miniSOG yakaumbwa mu pET21a vector yakanyorwa 6xHis paC-terminus.
HEK293T maseru akadyarwa pa2.0 x 105 masero patsime mumatanho matanhatu-tsime uye akatapurwa kwemaawa makumi maviri nemana gare gare aine recombinant lentiviral plasmids (2.4 μg pLX304) uye hutachiona hwekurongedza plasmids (1.5 μg psPAX2 uye 1.2 μg00 MD240 pMD) , #C0533), inenge 80% fusion.Mushure mekutapurirwa kwehusiku, svikiro racho rakachinjwa ndokuiswa kwemamwe maawa makumi maviri nemana.Kuunganidzwa kwehutachiona kwakaitwa mushure memaawa makumi maviri nemana, makumi mana nemasere uye makumi manomwe nemaviri.Pamberi pehutachiona hwemitsara yemasero, hutachiona hwehutachiona hwakanatswa kuburikidza ne 0.8 μm sefa (Merck, #millex-GP) uye polybrene (Solarbio, #H8761) yakawedzerwa kuhuwandu hwe8 μg / ml.Mushure memaawa makumi maviri nemana, maseru akabvumidzwa kupora nekushandura svikiro.Masero akasarudzwa achishandisa 5 μg/ml blasticidin (Solarbio, #3513-03-9) yezvikamu zvitatu zvekutanga sesarudzo yakaderera.Yakabva yashandisa 20 μg/ml sechirongwa chakaomarara pandima nhatu dzinotevera.
Masero akadyarwa mumakamuri gumi nemaviri-mutsime (Ibidi, #81201) pahuwandu hwemaseru angangoita 20,000 patsime.Kuti uvandudze kunamatira kweHEK293T maseru, wedzera 50 µg/ml fibronectin (Corning, #356008) yakadirwa muphosphate buffered saline (PBS, Sangon, #B640435) pa37°C.Iwo machamber aka pretreated for 1 hour ndokuzobviswa nePBS.Mushure memaawa makumi maviri nemana, maseru akagezwa kamwe chete nePBS, akaiswa 1 mM probe 3 mune nyowani Hanks' balanced salt solution (HBSS, Gibco, #14025092) kwe1 h pa37°C, ndokuzoiswa neblue LED (460 nm. )) dzakaitwa neradiation kwemaminetsi gumi patembiricha yekamuri.Mushure maizvozvi, masero akashambidzwa kaviri nePBS uye akagadziriswa ne4% formaldehyde muPBS (Sangon, #E672002) kwemaminitsi gumi nemashanu pakupisa kwekamuri.Yakawandisa formaldehyde yakabviswa kubva mumasero akaiswa nekugeza katatu nePBS.Masero akabva apinzwa ne 0.5% Triton X-100 (Sangon, #A600198) muPBS uye akagezwa katatu nePBS.Wobva wabvisa chamber wowedzera kumuenzaniso wega wega 25 µl wekudzvanya reaction musanganiswa une 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) uye 0.5 mg / ml sodium ascorbate (Aladdin, no. S105024) uye incubated kwemaminitsi makumi matatu pakushambidzika kwekamuri.Mushure mekuita kwekukurumidza, maseru akagezwa katanhatu nePBS ine 0.05% Tween-20 (Sangon, #A600560) (PBST) ndokuzovharwa ne5% BSA (Abcone, #B24726) muPBST kwemaminetsi makumi matatu pakupisa kwekamuri.
Nezve colocalization immunostaining, maseru akaiswa neakirairira masoja ekudzivirira chirwere zvichienderana nemamiriro akaratidzwa: mbeva anti-V5 tag mAb (1: 500, CST, #80076), tsuro anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), tsuro polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) kana tsuro anti-lamin A/C monoclonal antibody (1:500; CST, #2032) pa4 °C husiku.Mushure mekugeza katatu nePBST, maseru akaiswa nemasoja echipiri: mbudzi inorwisa tsuro Alexa Fluor 488 (Thermo, #A11034) yakanyungudutswa 1:1000, mbudzi anti-mbeva Alexa Fluor 594 (CST, #8889) yakaderedzwa 1:1000.dilution Dilute pakamuri tembiricha kwemaminitsi makumi matatu.Masero akazoshambidzwa 3 nguva nePBST uye akapokana neDAPI (Thermo, #D1306) muPBS kwemaminetsi gumi pakupisa kwekamuri.Mushure mekugezwa kwe3 nePBS, maseru akavharwa mu50% glycerol (Sangon, #A600232) muPBS yekufungidzira.Mifananidzo yeImmunofluorescent yakawanikwa pachishandiswa ZEISS LSM 900 Airyscan2 confocal microscope uye ZNE 3.5 software.
Kune singlet oxygen fluorescent imaging, maseru akagezwa kaviri neHanks HEPES buffer asati awedzera 100 nM Si-DMA muHanks HEPES buffer (DJINDO, #MT05).Mushure mekusangana nechiedza, masero akaiswa mu CO2 incubator pa 37 ° C kwemaminitsi makumi mana.Masero akazogezwa kaviri neHanks 'HEPES buffer uye akaverengerwa neHoechst muHanks'HEPES buffer kwemaminetsi gumi patembiricha yekamuri uye akaonekwa achishandisa ZEISS LSM 900 confocal microscope., #M36008) muHBSS buffer ine calcium uye magnesium.Mushure mekusangana nechiedza kana doxorubicin (MCE, #HY-15142A), masero akaiswa muCO2 incubator pa 37 ° C. kwemaminitsi gumi, akashambidzwa kaviri neHBSS buffer, uye akaiswa neHoechst muHBSS bhafa pane tembiricha yekamuri.maminitsi.Doxorubicin yakashandiswa seyakanaka probe control uko maseru aibatwa ne 20 μM doxorubicin muHBSS ine 1% BSA ye30 min.Immunofluorescent mifananidzo yakawanikwa uchishandisa Zeiss LSM 900 confocal microscope.
HEK293T masero anoratidza zvakadzikama mito-miniSOG akadyarwa pahuwandu hunosvika 30% mumidziyo ye15 cm.Mushure memaawa makumi mana nemasere, pakasvika ~ 80% confluence, maseru akagezwa kamwe chete nePBS, akaiswa 1 mM Probe 3 muHBSS buffer nyowani kweawa imwe pa37 ° C ndokuvhenekerwa neblue LED kwemaminitsi gumi mukamuri. tembiricha..Mushure maizvozvo, maseru akagezwa kaviri nePBS, akakweshwa uye akamiswazve muchando-inotonhora PBS buffer ine EDTA-isina protease inhibitors (MCE, #HY-K0011).Masero akaiswa lysed ne sonicating the tip for 1 miniti (1 second on uye 1 second off at 35% amplitude).Musanganiswa wakaguma wakaiswa centrifuged pa 15,871 xg ye 10 min pa 4 ° C kuti ibvise marara, uye iyo supernatant concentration yakagadziridzwa ku 4 mg / mL uchishandisa BCA protein assay kit (Beyotime, #P0009).Sanganisa 1 ml ye lysate iri pamusoro ne 0.1 mM ine photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), uye 1 mMtor CuSO4 yepasi kutenderera kumusoro kweawa imwe pane tembiricha yekamuri.Mushure mekuita kwekukurumidza, wedzera musanganiswa kune pre-yakasanganiswa mhinduro (MeOH:CHCl3:H2O = 4 ml: 1 ml: 3 ml) mu 10 ml yegirazi vial.Iwo masampula akavhenganiswa uye akaiswa centrifuged pa4500 g kwemaminetsi gumi pane tembiricha.Mishonga yepasi nepamusoro yakaraswa, mvura inonaya yakashambidzwa kaviri ne 1 ml ye methanol uye centrifuged pa 15871 × g ye 5 min pa 4 ° C.Wedzera 1 ml ye8 M urea (Aladdin, no. U111902) mu 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) kunyungudutsa mvura inonaya.Samples akaumbwa zvakare ne10 mM dithiothreitol (Sangon, #A100281 mu25 mM ABC) kwemaminetsi makumi mana pa55 ° C ichiteverwa nekuwedzera kwe15 mM nyowani iodoacetamide (Sangon, #A600539) pakamuri tembiricha murima.Alkylation mukati memaminitsi makumi matatu..Imwe 5 mM dithiothreitol yakawedzerwa kumisa kuita.Gadzirira zvingangoita 100 µl NeutrAvidin agarose beads (Thermo, #29202) yemuenzaniso wega wega nekugeza katatu ne 1 ml PBS.Mushonga weproteome uri pamusoro wakacheneswa ne5 ml PBS uye wakaiswa mabeads eNeutrAvidin agarose ekare kwemaawa mana patembiricha yekamuri.Mabhero acho akazoshambidzwa katatu ne5 ml PBS ine 0.2% SDS (Sangon, #A600485), 3 nguva ne5 ml PBS ine 1M urea, uye 3 nguva ne5 ml ddH2O.Mabhero acho akazokohwewa ne centrifugation ndokumiswazve mu200 μl ye25 mM ABC ine 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) uye 20 ng/μl trypsin (Promega, #V5280).Trypsinize usiku hwose pa37°C nekutenderera.Kuita kwakamiswa nekuwedzera formic acid (Thermo, # A117-50) kusvika pH yasvika 2-3.Mabhesi akashambidzwa katatu ne 1 ml yePBS ine 0.2% SDS, 3 nguva ne 1 ml yePBS ine 1 M urea, uyezve katatu ne 1 ml yemvura yakasvibiswa.Mapeptides akagadziridzwa akaburitswa nelight lysis (365 nm) ye90 min uchishandisa 200 μl ye70% MeOH.Mushure me centrifugation, iyo supernatant yakaunganidzwa.Mabhero acho akazogezwa kamwe chete ne100 μl ye70% MeOH uye mashura akasanganiswa.Samples dzakaomeswa mu Speedvac vacuum concentrator uye dzakachengetwa pa -20 ° C kusvika kuongororwa.
Kuziva uye kuverengera singlet oxygen yakagadziridzwa peptides, samples dzakanyungudika zvakare mu 0.1% formic acid uye 1 μg yepeptides yakaongororwa pachishandiswa Orbitrap Fusion Lumos Tribrid mass spectrometer ine nano ESI sosi kubva kuTune uye Xcalibur kubva kune mutengesi software 4.3.Samples dzakapatsanurwa pa75 µm × 15 cm mukati yakarongedzwa capillary column ine 3 µm C18 zvinhu (ReproSil-pur, #r13.b9.) uye yakabatana kune EASY-nLC 1200 UHPLC system (Thermo).Mapeptide akapatsanurwa nemutsara 95 maminitsi gradient chromatography kubva pa8% solvent B kusvika 50% solvent B (A = 0.1% formic acid mumvura, B = 0.1% formic acid mu80% acetonitrile), yakawedzera mutsara kusvika 98% B min. mu 6 min pachiyero chekuyerera kwe300 nl / min.Orbitrap Fusion Lumos inounganidza data neimwe nzira pakati peMS scan yakazara uye MS2 scan zvichienderana nedata.Iyo sputtering voltage yakaiswa ku2.1 kV uye tembiricha yeion transport capillary yaive 320°C.MS spectra (350-2000 m/z) yakaunganidzwa ine resolution ye120,000, AGC 4 × 105, uye yakanyanya kupinza nguva ye150 ms.Iwo gumi anonyanyozivikanwa anochajiswa emberi ega ega ega akazara akapatsanurwa achishandisa HCD ine yakajairika kudhumhana simba re30%, quadrupole yekuzviparadzanisa hwindo re1.6 m/z, uye kugadzirisa kwe30,000.Chinangwa cheAGC chetandem mass spectrometry uchishandisa 5 × 104 uye yakanyanya kupinza nguva 150 ms.Iyo inosarudzika yekusarudzika inoiswa kumasekonzi makumi matatu. Ioni dzisina kugoverwa kana avo vane mutero we1+ uye > 7+ vakarambwa nokuda kweMS/MS. Ioni dzisina kugoverwa kana avo vane mutero we1+ uye > 7+ vakarambwa nokuda kweMS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ioni dzisina kugoverwa kana maion ane mutero we1+ uye > 7+ dzakarambwa nokuda kweMS/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. Ioni dzisina kutaurwa kana ions dzine mhosva dze1+ uye> 7+ dzakarambwa nokuda kweMS/MS.
Iyo mbishi data inogadziriswa uchishandisa iyo FragPipe komputa chikuva chakavakirwa paMSFragger.Misa yakarerekera uye inowirirana amino acids yakatemwa kushandisa yakavhurika yekutsvaga algorithm ine precursor mass tolerance ye -150 kusvika 500 Da.Peptides yakagadziridzwa yakabva yaonekwa kushandiswa kwe histidine kuchinjwa nekuwedzera kwe +229.0964 uye +247.1069 Da muPD (Proteome Discoverer 2.5, Thermo).
Masero ainyatso ratidza iyo fused miniSOG gene akaiswa mu6 cm ndiro.Pakusvika ~ 80% confluence, masero akagezwa kamwe chete neHBSS (Gibco, #14025092), ndokuzoiswa nemakemikari probes muHBSS kweawa 1 pa37 ° C uye akavhenekerwa nechiedza chebhuruu.10W LED kwemaminetsi makumi maviri pane tembiricha yekamuri.Kuti uone kuti ndeupi rudzi rwemhando yeokisijeni inoshandiswa muPDPL, 0.5 mM vhitamini 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 yakawedzerwa kumaseru sekuwedzera.Mushure mekugeza nePBS inotonhora, masero akachekwa, akaunganidzwa mu 1.5 ml centrifuge tubes, uye sonicated netip ye 1 min mu 200 μl yePBS ine 1x protease inhibitor pasina EDTA (1 s uye 1 s pasina, amplitude 35%).Musanganiswa wakaguma wakaiswa centrifuged pa 15,871 × g ye 10 min pa 4 ° C uye iyo supernatant concentration yakagadziriswa ku 1 mg / mL uchishandisa BCA protein assay kit.Inenge 50 µl ye lysate iri pamusoro yakabatanidzwa ne 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, uye 1 mM CuSO4 kweawa 1 pakupisa kwekamuri nekutenderera kubva pasi kusvika kumusoro.Mushure mekuita kwekudzvanya, kunaya neacetone kwakaitwa nekuwedzera 250 μl yepre-chilled acetone kumasampuli, ichiisa pa -20°C kwemaminetsi makumi maviri uye centrifuging pa6010×g kwemaminetsi gumi pa4°C.Unganidza pellet uye bika mu50 µl ye1x Laemmli's buffer kwemaminetsi gumi pa95 °C.Samples dzakazoongororwa paSDS-PEJI marefu gels uye akaonekwa achishandisa Bio-rad ChemiDoc MP Kubata imaging system ine Image Lab Touch software.
Kutaura uye kucheneswa kweiyo recombinant miniSOG-6xProtein yake yakaitwa sezvakatsanangurwa kare.Muchidimbu, E. coli BL21(DE3) masero (TransGen, #CD701-02) akashandurwa aine pET21a-miniSOG-6xHis uye mapuroteni ekutaura akaitwa ne 0.5 mM IPTG (Sangon, #A600168).Mushure mesero lysis, mapuroteni akacheneswa achishandisa Ni-NTA agarose beads (MCE, no. 70666), dialyzed against PBS, uye akachengetwa pa -80 ° C.
Kune antibody-based in vitro label proximity assay, sanganisa 100 μM yakacheneswa miniSOG, 1 mM probe 3, uye 1 μg anti-label mouse monoclonal antibody (TransGen, #HT501-01) muPBS kusvika kuhuwandu hwekuita huwandu hwe50 μl..Iyo reaction musanganiswa yakavhenekerwa neblue LED mwenje we0, 2, 5, 10, uye 20 min pakamuri tembiricha.Musanganiswa wacho wakaiswa 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, uye 1 mM CuSO4 kwe1 h pakamuri tembiricha pane inokwidza inofamba shaker.Mushure mekuita kwekukurumidza, wedzera 4x Laemmli's buffer zvakananga kumusanganiswa uye bika pa 95 ° C kwemaminetsi gumi.Mienzaniso yakaongororwa paSDS-PEJI gels uye yakaongororwa nekumadokero blotting ne streptavidin-HRP (1: 1000, Solarbio, #SE068).
Iyo histidine-ine synthetic peptide ine C-terminal amidation (LHDALDAK-CONH2) yakashandiswa kuongorora peptide iri pedyo-based in vitro labeling.Muchiyedzo ichi, 100 μM yakanatswa miniSOG, 10 mM probe 3 uye 2 μg/ml synthetic peptide yakasanganiswa muPBS muhuwandu hwekuita huwandu hwe50 μl.Iyo reaction musanganiswa yakavhenekerwa neblue LED mwenje kweawa 1 pakudziya kwekamuri.Imwe microliter yemuenzaniso yakaongororwa pachishandiswa LC-MS system (Mvura, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer ine MassLynx spectrum analysis software).
HEK293T maseru anoratidza zvakadzikama miniSOG fusion gene akadyarwa mu10 cm madhishi emitsara ine akasiyana organelle localization (Mito, ER, Nucleus) uye 15 cm ndiro dzeParkin-miniSOG uye BRD4-miniSOG mitsetse.Pakusvika ~ 90% confluence, maseru akagezwa kamwe chete neHBSS, ndokuzoiswa neprobe 3 muHBSS kweawa imwe pa37 ° C uye akavhenekerwa ne10 W yebhuruu LED pakamuri tembiricha.Kune isiri-yekubata mazita eParkin, 10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) ine probe 3 muHBSS yakawedzerwa kweawa imwe pa37°C.Iyo sero lysis, tinya chemistry, kuderedza uye alkylation matanho aive akafanana sezvakatsanangurwa pamusoro, kunze kwekuti 2 mg ye lysate yakawedzerwa uye biotin PEG3 azide yakashandiswa mukudzvanya maitiro pane photodegradable biotin azide.Mushure mekupfumisa, mabhesi akashambidzwa katatu ne5 ml yePBS ine 0.2% SDS, 3 nguva ne5 ml yePBS ine 1 M urea, uye 3 nguva ne5 ml yePBS.Mushure mezvo, 2 µg trypsin yakawedzerwa ku300 µl 25 mM ABC ine 1 M urea kuti itsemure puroteni usiku hwose pa37°C.Kuita kwakamiswa nekuwedzera formic acid kusvika pH ye2-3 yasvika.Mushure me trypsinization pamabead, mhinduro yepeptide yakabviswa munyu uchishandisa SOLAµ HRP column (Thermo, #60209-001) uye yakaomeswa mu Speedvac vacuum concentrator.Mapeptide akanyungudika zvakare mu 0.1% formic acid uye 500 ng yemapeptides akaongororwa pachishandiswa Orbitrap Fusion Lumos Tribrid mass spectrometer yakagadzirirwa nano-ESI sosi inotsanangurwa pamusoro.Peptides akaparadzaniswa pakutengeserana RP-HPLC precolumns (75 μm x 2 cm) (Thermo, no. 164946) uye analytical RP-HPLC mbiru (75 μm x 25 cm) (Thermo, no. 164941), zvose akazadzwa 2 μm.gradient kubva ku8% kusvika ku35% ACN mumaminitsi makumi matanhatu, zvino yakawedzera kusvika ku98% B mumaminitsi matanhatu pachiyero chekuyerera kwe300 Nl / min.MS spectra (350-1500 m/z) yakaunganidzwa ine resolution ye60,000, AGC 4 × 105, uye yakanyanya kupinza nguva ye50 ms.Maion akasarudzwa akatsemurwa zvakatevedzana neHCD muma 3 s cycles ane normalized collision energy ye 30%, quadrupole isolation window ye 1.6 m/z, uye resolution ye 15000. A 5 × 104 tandem mass spectrometer AGC chinangwa uye nguva yakawanda yejekiseni ye22 ms yakashandiswa.Kusarudzika kune simba kwakaiswa kumasekonzi makumi mana nemashanu. Ioni dzisina kugoverwa kana avo vane mutero we1+ uye > 7+ vakarambwa nokuda kweMS/MS. Ioni dzisina kugoverwa kana avo vane mutero we1+ uye > 7+ vakarambwa nokuda kweMS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ioni dzisina kugoverwa kana maion ane mutero we1+ uye > 7+ dzakarambwa nokuda kweMS/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. Ioni dzisina kutaurwa kana ions dzine mhosva dze1+ uye> 7+ dzakarambwa nokuda kweMS/MS.
Maitiro ekugadzirira nhanho kusvika pakupfumiswa kweNeutrAvidin mabheji aive akafanana muLC-MS / MS kuongororwa kwakatsanangurwa pamusoro apa.Inenge 50 μg ye lysate yakashandiswa sechipo chekuisa kudzora uye 2 mg ye lysate yakashandiswa kudzvanya maitiro.Mushure mekugadzirisa uye kugezwa neutravidin, mapuroteni akasungwa akabviswa nekuwedzera 50 μl yeLaemmli's buffer kune agarose resin beads uye kuvira pa 95 ° C. kwemaminitsi mashanu.Kudzora mutoro wekuisa uye bead akafumisa masampuli akaongororwa neSDS-PAGE uye akaendeswa kune PVDF membranes (Millipore, #ISEQ00010) neyakajairwa Western blot nzira.Ma membranes akavharwa ne5% skim mukaka (Sangon, #A600669) muTBS ine 0.1% pakati-20 (TBST) uye incubated zvakatevedzana nekutanga uye yechipiri masoja ekudzivirira chirwere.Masoja ekudzivirira chirwere akadzikiswa 1:1000 mu5% mumukaka weskim muTBST uye akaiswa usiku hwose pa4°C.Masoja ekudzivirira chirwere akashandiswa muchiyero che1:5000 uye akaiswa kweawa imwe patembiricha yekamuri.Iyo membrane yakaonekwa ne chemiluminescence uchishandisa iyo Chemidoc MP imaging system.Ese asina kuchekwa scans emablots uye gels mumufananidzo anounzwa se data mbishi.
Masoja ekudzivirira ekutanga anoshandiswa muchidzidzo ichi aisanganisira tsuro inorwisa SFPQ monoclonal antibody (CST, no. 71992), tsuro inorwisa FUS monoclonal antibody (CST, no. 67840), tsuro inorwisa-NSUN2 polyclonal antibody (Proteintech, no. 20854-1-- AP), tsuro anti-mSin3A polyclonal antibody (Abcam, #ab3479), mouse anti-tag monoclonal antibody (TransGen, #HT201-02), mouse anti-β-actin monoclonal antibody (TransGen, #HC201-01), tsuro inorwisa -CDK2 monoclonal antibody (ABclonal, #A0094), tsuro monoclonal antibody ku CTBP1 (ABclonal, #A11600), tsuro polyclonal antibody kuDUT (ABclonal, #A2901), tsuro polyclonal antibody kuPSMC4 (ABclonal anti-5), #Abclonal-5), DNAJB1 polyclonal antibody (ABclonal, # A5504).Masoja ekudzivirira chirwere aya akashandiswa pa1:1000 dilution muchikamu che5% cheskim mukaka muTBST.Echipiri masoja ekudzivirira chirwere akashandiswa muchidzidzo ichi aisanganisira anti-tsuro IgG (TransGen, #HS101-01), anti-mbeva IgG (TransGen, #HS201-01) pa1:5000 dilution.
Kuenderera mberi nekuongorora kana BRD4 inodyidzana neSFPQ, yakagadzikana HEK293T uye BRD4-miniSOG maseru akawandisa HEK293T akaiswa mu10 cm ndiro.Masero akagezwa nePBS inotonhora uye akaiswa mu1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) neEDTA-free protease inhibitor kwemaminetsi makumi matatu pa4°C.Mushure mezvo, ma lysates akaunganidzwa mu 1.5 ml centrifuge tubes uye centrifuged pa 15,871 xg ye 10 min pa 4 ° C.Iyo supernatant yakakohwewa ikaiswa 5 µg yeanti-V5 yakanyorwa mbeva monoclonal antibody (CST, #80076) husiku hwese pa4°C.Geza ingangoita 50 µl yeprotein A/G magineti bead (MCE, #HY-K0202) kaviri nePBS ine 0.5% Pakati-20.Ipapo sero lysates akafukidzwa nemagineti mabhedhi kwemaawa mana pa4 ° C nekutenderera kubva pasi kuenda kumusoro.Zvadaro mabheji akashambidzwa kana ne 1 ml yePBST buffer uye yakabikwa pa 95 ° C kwemaminitsi mashanu.Samples akaongororwa paSDS-PAGE gels uye akaendeswa kune PVDF membranes vachishandisa yakajairwa Western blot nzira.Mamembrane akavharirwa mu5% skim mukaka muTBST uye akaiswa sequentially ne primary and secondary antibodies.Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) yakashandiswa pachiyero che1:1000 mu5% skim mukaka muTBST uye yakavharirwa usiku hwose pa4°C.Anti-tsuro IgG yakashandiswa pachiyero che1: 5000 uye yakavharirwa kweawa imwe patembiricha yekamuri.Iyo membrane yakaonekwa ne chemiluminescence uchishandisa iyo Chemidoc MP imaging system.
Zvese zvimiro zvakashandiswa pakuongorora kweSolvent Accessible Surface Area (SASA) zvakawanikwa kubva kuProtein Data Bank (PDB)52 kana AlphaFold Protein Structure Database53.Absolute SASA yakaverengerwa kune yega yega yasara uchishandisa chirongwa cheFreeSASA.Chete chakazara uye chisina kujeka data reSASA rakanyorwa histidine nevavakidzani vayo rakashandiswa kuwana iyo inorehwa SASA yechimiro chega chega.The hama sont accessibility (RSA) ye histidine yega yega yakaverengerwa nekugovanisa absolute SASA ukoshi ne empirical maximum inogoneka yakasara yenzvimbo inowanikwa kune iyo solvent.Yese histidines yakazoiswa seyakavanzika kana iyo inorehwa RSA yaive pasi pe20%, neimwe nzira yakafumurwa56.
Raw mafaira akawanikwa muDDA mode akatsvaga achishandisa Proteome Discoverer (v2.5) kana MSfragger (Fragpipe v15.0) muSwissProt verified protein database ine zvinowanzosvibiswa.Iyo peptides yaida trypsin yakakwana ine mbiri yakashaikwa cleavage nzvimbo, carbamoyl methylation seyakagadziriswa gadziriso uye methionine oxidation seshanduko ine simba.Precursor uye chidimbu uremu kushivirira kwakaiswa ku10 ppm uye 0.02 Da (MS2 Orbitrap), zvichiteerana. Yakasvibiswa hits yakabviswa, uye mapuroteni akasefa kuti awane chiyero chekuwanikwa chenhema che <1%. Yakasvibiswa hits yakabviswa, uye mapuroteni akasefa kuti awane chiyero chekuwanikwa chenhema che <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэффициент ложного обнаружения <1%. Yakasvibiswa hits yakabviswa uye mapuroteni akasefa kuti ape chiyero chekuona chenhema che <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%. Yakasvibiswa hits yakabviswa uye mapuroteni akasefa kuti awane chiyero chenhema che <1%.Pakuongorora kwehuwandu pasina kushandiswa kwemavara, yakajairwa mapuroteni emukati kubva kutatu biological kudzokorora kwakashandiswa.Protein subcellular localization analysis yakaitwa pachishandiswa Gene Ontology (GO) ongororo kubva kuna DAVID Bioinformatics Resources, MitoCarta 3.0 uye dhatabhesi rakanyorwa uye rakadhindwa neboka reAlice Ting.Mepu yegomo rinoputika yakawanikwa kubva kuPerseus (v1.6.15.0). Protein abundance fold shanduko yakaedzwa kuti ione kukosha kwenhamba uchishandisa maviri-side t-test, uye mapuroteni hits akaonekwa nekuwanda shanduko> 2 (kunze kwekunge zvataurwa neimwe nzira) uye p kukosha <0.05. Protein abundance fold shanduko yakaedzwa kuti ione kukosha kwenhamba uchishandisa maviri-side t-test, uye mapuroteni hits akaonekwa nekuwanda shanduko> 2 (kunze kwekunge zvataurwa neimwe nzira) uye p kukosha <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. Protein yemukati kupeta shanduko yakaedzwa kuti ione kukosha kwenhamba uchishandisa maviri-tailed t-test, uye mapuroteni machisi akaonekwa aine shanduko yemukati> 2 (kunze kwekunge zvacherechedzwa neimwe nzira) uye ap kukosha <0.05.10使用 双边 t 检验 测试 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 倍数 变化 的统计 显着性 并 定 蛋白质 倍数 变化 的统计 显着性 并 确定 蛋白质 中明 中 中 古 5 Статистическую значимость кратных изменений содержания белка проверяли с использованием двустороннего t-критерия, а совпадения белков определяли для изменений содержания >2 (если не указано иное) и p-значений <0,05. Kukosha kwehuwandu hwekupeta shanduko mune zvemukati mapuroteni kwakaedzwa uchishandisa maviri-tailed t-bvunzo, uye mapuroteni machisi akatemerwa shanduko yemukati> 2 (kunze kwekunge zvaratidzwa) uye p-tsika <0.05.Kuongororwa kweprotein yekudyidzana kwakaitwa pachishandiswa GO ongororo pamwe chete neString database.
Zvitatu zvebiological replicate zvakaitwa nemhedzisiro yakafanana.Kuongorora kwenhamba kwakaitwa neGraphPad Prism (GraphPad software) uye mapundu emakomo akagadzirwa nePerseus (v1.6.15.0).Kuenzanisa mapoka maviri aya, p-tsika dzakatemerwa uchishandisa t-test yeMudzidzi ine miswe miviri.Mapuroteni esingleton chete akaonekwa kanenge kaviri muboka rekuyedza akaverengerwa mumakomo anoputika, uye anowirirana akashaikwa muboka rekutonga akatsiviwa nePerseus kubva mukugovaniswa kwakajairika kuti p-value iverengerwe.Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.Mukuongorora kweproteomic yekuongorora kwenhamba, kuwanda kwemapuroteni aionekwa mune inokwana maviri biological replicates akachengetwa.Statistical nzira hadzina kushandiswa kufanoona saizi yemuenzaniso.Miedzo haina kungoitika.Vatsvakurudzi vakanga vasina mapofu kumabasa panguva yekuedza uye kuongororwa kwemigumisiro.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Nature Research Report abstract yakabatana nechinyorwa ichi.
Iyo yakawanda spectrometry data yakawanikwa muchidzidzo ichi yakaendeswa kuProteomeXchange Consortium kuburikidza neiyo iProX57 shamwari repository pasi pedataset ID PXD034811 (PDPL-MS dataset).Raw data inopiwa nenzira yemafaira e data.Ichi chinyorwa chinopa data rekutanga.
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