Xov xwm

Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv CSS kev txhawb nqa.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis lov tes taw Compatibility Hom hauv Internet Explorer).Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav ua rau lub xaib tsis muaj qauv thiab JavaScript.
Enzymatic proximity labeling txoj kev raws li activated esters los yog phenoxy radicals yog dav siv los qhia subcellular proteomes thiab protein interactors nyob rau hauv lub hlwb.Txawm li cas los xij, activated esters tsis tshua muaj kev cuam tshuam, ua rau lub voj voog dav dav, thiab phenoxy radicals tsim los ntawm kev kho peroxide tuaj yeem cuam tshuam rau txoj hauv kev redox.Ntawm no peb qhia txog qhov sib thooj labeling dependent photoactivation (PDPL) txoj kev tsim los ntawm genetic linking miniSOG photosensitizer protein rau cov protein uas txaus siab.Tig los ntawm lub teeb xiav thiab tswj los ntawm lub sij hawm raug, singlet oxygen yog generated thiab ces spatiotemporally daws cov ntawv lo ntawm histidine residues los ntawm aniline sojntsuam yog tiav.Peb ua kom pom nws txoj kev ncaj ncees los ntawm organelle-specific proteome mapping.Kev sib piv ntawm ib sab ntawm PDPL nrog TurboID qhia tau hais tias muaj ntau qhov tshwj xeeb thiab muaj txiaj ntsig zoo ntawm PDPL.Tom ntej no, peb tau thov PDPL rau cov kab mob sib txuas nrog cov kab mob sib kis BRD4 thiab E3 Parkin ligase thiab pom cov neeg tsis paub yav dhau los.Los ntawm kev tshuaj ntsuam ntau dhau, ob lub substrates tsis paub, Ssu72 thiab SNW1, tau txheeb xyuas rau Parkin, uas nws qhov kev puas tsuaj yog kho los ntawm txoj kev ubiquitination-proteasome.
Qhov tseeb tus yam ntxwv ntawm cov tes hauj lwm protein nyob rau hauv ntau lub hauv paus txheej txheem ntawm tes.Yog li ntawd, qhov tseeb spatiotemporal daim ntawv qhia ntawm cov protein sib cuam tshuam yuav muab lub hauv paus molecular rau deciphering biological pathology, kab mob pathology, thiab cuam tshuam cov kev sib cuam tshuam rau kev kho mob.Txog rau qhov kawg no, cov txheej txheem muaj peev xwm tshawb pom kev sib cuam tshuam ntawm lub cev hauv cov hlwb lossis cov ntaub so ntswg yog qhov xav tau.Affinity Purification Mass Spectrometry (AP-MS) tau siv keeb kwm los txheeb xyuas cov neeg koom tes ntawm cov protein uas txaus siab (POIs).Nrog rau txoj kev loj hlob ntawm ntau yam proteomics txoj kev, Bioplex3.0 tau tsim, qhov loj tshaj plaws databases ntawm cov tes hauj lwm protein raws li AP-MS.Txawm hais tias AP-MS muaj zog heev, lub cell lysis thiab dilution cov kauj ruam hauv kev ua haujlwm yog kev sib tw rau kev tsis muaj zog thiab kev sib cuam tshuam kev sib tshuam thiab qhia cov khoom siv tom qab lysis xws li kev sib cuam tshuam tsis zoo uas tsis muaj kev sib koom ua ntej lysis.
Txhawm rau daws cov teeb meem no, unnatural amino acids (UAA) nrog crosslinking pawg thiab enzymatic nyob ze daim ntawv lo (PL) platforms (xws li APEX thiab BioID)5 tau tsim.Txawm hais tias UAA txoj kev tau ua tiav hauv ntau qhov xwm txheej thiab muab cov ntaub ntawv ntawm cov nplaum nplaum ncaj qha, kev ua kom zoo dua ntawm UAA qhov chaw nkag yog tseem xav tau.Qhov tseem ceeb tshaj, nws yog ib txoj kev sau npe stoichiometric uas tsis muaj qhov cuam tshuam ntawm kev rov qab los ntawm kev sau cov xwm txheej.Nyob rau hauv sib piv, enzymatic PL txoj kev, xws li txoj kev BioID, fuse tus engineered biotin ligase rau POI7, uas tom qab activates biotin los ua ib tug reactive biotinyl-AMP ester intermediate.Lub enzyme yog li catalyzes thiab tso tawm ib qho activated biotin "huab" uas sau cov lysine residues.Txawm li cas los xij, BioID xav tau ntau dua 12 teev kom tau txais cov cim cim npe txaus, uas txwv tsis pub siv nrog kev daws teeb meem ntawm lub cev.Siv cov lus qhia evolution raws li cov poov xab zaub, TurboID tau tsim los ntawm BioID kom ua tau zoo dua, tso cai rau cov ntawv sau nrog biotin tsis pub dhau 10 feeb, tso cai rau kev kawm ntau dua.Vim hais tias TurboID yog cov nquag nquag thiab endogenous biotin qib txaus rau cov ntawv sau qis, cov ntawv sau keeb kwm yav dhau los dhau los ua teeb meem tshwm sim thaum muaj kev txhim kho siab heev thiab lub sijhawm sau ntawv yuav tsum tau los ntawm kev ntxiv ntawm exogenous biotin.Tsis tas li ntawd, activated esters tsis zoo reactive (t1/2 ~ 5 min), uas tuaj yeem ua rau lub vojvoog loj, tshwj xeeb tshaj yog tom qab saturation ntawm cov protein nyob sib ze nrog biotin 5. Hauv lwm txoj hauv kev, genetic fusion ntawm engineered ascorbate peroxidase (ie biotin- phenol radicals thiab tso cai rau cov ntawv sau protein nyob rau hauv ib feeb9,10. APEX tau dav siv los txheeb xyuas cov subcellular proteomes, membrane protein complexes, thiab cytosolic signaling protein complexes11,12. Txawm li cas los xij, qhov xav tau ntawm cov peroxides siab tuaj yeem cuam tshuam rau redox proteins lossis txoj hauv kev, cuam tshuam. cellular txheej txheem.
Yog li, ib txoj hauv kev tshiab uas muaj peev xwm tsim tau ntau dua reactive labeled-radius suppression hom nrog siab spatial thiab temporal raug yam tsis muaj kev cuam tshuam loj cellular txoj kev yuav yog ib qho tseem ceeb ntxiv rau txoj kev uas twb muaj lawm. Ntawm cov hom reactive, singlet oxygen aroused peb mloog vim nws lub neej luv luv thiab txwv diffusion radius (t1/2 < 0.6 µs hauv hlwb)13. Ntawm cov hom reactive, singlet oxygen aroused peb mloog vim nws lub neej luv luv thiab txwv diffusion radius (t1/2 < 0.6 µs hauv hlwb)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жифте тривлек и чрулород из-за его короткого времени жифео жизни и чрулани ограни Ntawm cov ntaub ntawv nquag, singlet oxygen nyiam peb cov xim vim nws lub neej luv luv thiab tsis tshua muaj lub vojvoog diffusion (t1/2 < 0.6 µs hauv hlwb) 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t的态氧因其寿命短和扩散半径有限（细胞中的晭的态氧因其寿命短和扩散半。 1/2 < 0.6 µs) 而引起了我们的注意13 Среди активных форм наше внимание привлекает синглетный кислород из-за его короткого времание привлекает синглетный кислород из-за его короткого времанио дремени жизни у чана огранга и чрана ограна и чрана огранога Ntawm cov ntaub ntawv nquag, singlet oxygen nyiam peb cov xim vim nws lub neej luv luv thiab txwv tsis pub muaj qhov sib txawv (t1/2 < 0.6 μs hauv hlwb).Singlet oxygen tau raug tshaj tawm rau randomly oxidize methionine, tyrosine, histidine thiab tryptophan, ua rau nws polar 14,15 rau txuas rau amine los yog thiol raws li probes16,17.Txawm hais tias singlet oxygen tau siv los sau npe subcellular compartment RNA, cov tswv yim rau kev rov ua dua cov cim POI ze ze tseem tsis tau tshawb nrhiav.Ntawm no, peb nthuav tawm lub platform hu ua photoactivation-dependent proximity labeling (PDPL), qhov twg peb siv lub teeb xiav los teeb POIs fused nrog miniSOG photosensitizer thiab ua rau singlet oxygen tiam los oxidize proximal residues, ua raws li amine-muaj kev hloov kho rau oxidize tshuaj soj ntsuam rau hauv. nruab nrab cov hlwb..Peb tau sim ib pab pawg tshuaj ntsuam xyuas kom ua kom pom qhov tshwj xeeb thiab txheeb xyuas qhov chaw hloov kho siv qhov qhib proteomics workflow.Kev sib piv ntawm ib sab ntawm PDPL nrog TurboID qhia tau hais tias muaj ntau qhov tshwj xeeb thiab muaj txiaj ntsig zoo ntawm PDPL.Peb tau siv txoj hauv kev no rau cov cim tshwj xeeb ntawm cov kab mob ntawm cov kab mob hauv cov cellular thiab cov kev qhia dav dav ntawm cov neeg koom tes sib koom ua ke rau cov kab mob qog noj ntshav uas cuam tshuam rau cov kab mob qog noj ntshav BRD4 thiab Parkinson tus kab mob-koom nrog E3 ligase Parkin, uas tau lees paub ob qho tib si paub thiab tsis paub txog cov protein. kev sib tham..Lub peev xwm ntawm PDPL kom paub txog E3 substrates nyob rau hauv cov protein ntau complexes sawv cev rau ib qho teeb meem uas yuav tsum tau paub txog indirect binders.Ob tug tsis paub parkin substrates kho los ntawm ubiquitination-proteasome tau lees paub nyob rau hauv situ.
Photodynamic therapy (PDT)19 thiab chromophore-pab laser inactivation (CALI)20, nyob rau hauv uas lub teeb irradiation nrog photosensitizers generates singlet oxygen, muaj peev xwm inactivate lub hom phiaj proteins los yog ua rau cell tuag.Txij li thaum singlet oxygen yog ib yam khoom muaj zog heev nrog qhov theoretical diffusion nrug ntawm li 70 nm, spatially txwv oxidation nyob ib ncig ntawm lub photosensitizer tuaj yeem tswj tau.Raws li lub tswv yim no, peb tau txiav txim siab siv cov pa oxygen singlet kom ua tiav cov ntawv sau ze ntawm cov protein ntau hauv cov hlwb nyob.Peb tau tsim ib txoj hauv kev PDPL chemoproteomic kom ua tiav plaub lub luag haujlwm: (1) txhawm rau ua kom muaj zog ntawm singlet oxygen zoo ib yam li PL enzymatic mus kom ze;(2) muab lub sij hawm daws teeb meem daim ntawv lo thaum lub teeb pib;(3) los ntawm kev hloov pauv (4) Tsis txhob siv cov tshuaj endogenous cofactors (xws li biotin) los txo cov keeb kwm yav dhau los, lossis siv cov tshuaj exogenous reagents (xws li peroxides) kom txo tau cov cell raug rau ib puag ncig kev ntxhov siab.
Photosensitizers tuaj yeem muab faib ua ob pawg xws li me me molecular hnyav fluorophores (xws li rose bengal, methylene xiav) 22 thiab genetically encoded me proteins (xws li miniSOG, KillerRed)23.Txhawm rau ua tiav cov qauv tsim qauv, peb tau tsim thawj tiam PDPL platform los ntawm kev ntxiv cov proteins photosensitizer (PS) rau POI24,25 (Daim duab 1a).Thaum irradiated nrog lub teeb xiav, singlet oxygen oxidizes proximal nucleophilic amino acid residues, uas ua rau lub umpolung polarity uas yog electrophilic thiab muaj peev xwm ntxiv react nrog amine sojntsuam nucleophiles16,17.Kev sojntsuam yog tsim los nrog tus kov alkyne kom tso cai rau nyem chemistry thiab rub rau LC / MS / MS characterization.
Schematic illustration of labeling protein complexes mediated by miniSOG.Thaum raug lub teeb xiav, cov hlwb uas qhia txog miniSOG-POI tsim cov pa oxygen singlet, uas hloov cov protein sib cuam tshuam tab sis tsis yog cov protein uas tsis yog khi.Cov khoom nruab nrab ntawm photooxidation yog cuam tshuam los ntawm cov ntawv xa tawm ntawm cov tshuaj amine tshuaj ntsuam xyuas los tsim covalent adducts.Cov pab pawg alkynyl ntawm kev soj ntsuam chemistry tso cai rau nyem chemistry rau kev txhawb nqa los ntawm rub-down ua raws li LC-MS / MS quantitation.b Cov qauv tshuaj ntawm amine probes 1-4.c Cov neeg sawv cev fluorescent gel tsom xam ntawm mitochondrial localized miniSOG-mediated proteomic markers siv probes 1-4 thiab txheeb ze kom muaj nuj nqis raws li gel densitometry.Lub teeb liab-rau-tom qab piv ntawm cov tshuaj ntsuam xyuas tau soj ntsuam siv cov kev sim tswj tsis zoo tsis suav nrog lub teeb xiav lossis siv HEK293T hlwb yam tsis muaj miniSOG qhia.n = 2 cov qauv tsis muaj tshuaj lom neeg lom neeg.Txhua lub dot sawv cev rau cov khoom siv roj ntsha.d Cov neeg sawv cev nrhiav kom tau thiab kom muaj nuj nqis ntawm PDPL siv optimized sojntsuam 3 nyob rau hauv lub xub ntiag los yog tsis muaj cov qhia PDPL Cheebtsam xws li c.n = 3 cov qauv kev ywj pheej.Txhua lub dot sawv cev rau cov khoom siv roj ntsha.Centerlines thiab whiskers sawv cev qhov txhais tau tias thiab ± tus qauv sib txawv.CBB: Coomassie Brilliant Blue.e Confocal imaging ntawm singlet oxygen nrog deb-liab Si-DMA stain.Scale bar: 10 µm.Gel imaging thiab confocal thwmsim tau ntawm nws tus kheej rov ua dua tsawg kawg ob zaug nrog cov txiaj ntsig zoo sib xws.
Peb thawj zaug kuaj lub peev xwm ntawm cov paub tab photosensitizers miniSOG26 thiab KillerRed23, stably qhia nyob rau hauv HEK293T, los kho kom haum propargylamine labeling ntawm lub proteome raws li ib tug tshuaj sojntsuam (Ntxiv daim duab 1a).Gel fluorescence tsom xam pom tias tag nrho cov ntawv cim proteome tau ua tiav siv miniSOG thiab xiav lub teeb irradiation, thaum tsis pom cov ntawv cim npe tau pom nrog KillerRed.Txhawm rau txhim kho lub teeb liab-rau-tom qab qhov sib piv, peb mam li sim cov txheej txheem tshuaj ntsuam xyuas uas muaj aniline (1 thiab 3), propylamine (2), lossis benzylamine (4).Peb tau pom tias HEK293T hlwb lawv tus kheej muaj cov teeb liab ntau dua piv rau tsis muaj lub teeb xiav, tej zaum yog vim muaj cov endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27 . Aniline-raws li tshuaj ntsuam xyuas 1 thiab 3 muab qhov tshwj xeeb zoo dua, nrog HEK293T ruaj khov qhia miniSOG hauv mitochondria nthuav tawm> 8-fold nce hauv lub teeb liab rau kev sojntsuam 3, thaum sojntsuam 2 siv hauv RNA-labeling method CAP-seq tsuas yog tso tawm ~ 2.5- fold teeb liab nce, tej zaum yog vim qhov sib txawv reactivity nyiam ntawm RNA thiab protein (Fig. 1b, c). Aniline-raws li tshuaj ntsuam xyuas 1 thiab 3 muab qhov tshwj xeeb zoo dua, nrog HEK293T ruaj khov qhia miniSOG hauv mitochondria nthuav tawm> 8-fold nce hauv lub teeb liab rau kev sojntsuam 3, thaum sojntsuam 2 siv hauv RNA-labeling method CAP-seq tsuas yog tso tawm ~ 2.5- fold teeb liab nce, tej zaum yog vim qhov sib txawv reactivity nyiam ntawm RNA thiab protein (Fig. 1b, c).Aniline-raws li tshuaj ntsuam xyuas 1 thiab 3 tau pom qhov tshwj xeeb zoo dua: HEK293T, uas ruaj khov qhia miniSOG hauv mitochondria, qhia ntau dua 8-fold nce hauv lub teeb liab rau kev sojntsuam 3, thaum sojntsuam 2, siv hauv CAP-seq RNA labeling method, nkaus xwb. qhia tau hais tias ~ 2.5-fold teeb liab nce, tej zaum yog vim txawv reactivity nyiam ntawm RNA thiab protein (Fig. 1b, c).基于 苯胺 化学 探针 探针 探针 1 具有 好 好 好 好 特异性 特异性 特异性 特异性 表达 粒体 中 稳定 稳定 中 稳定 增加表达og, 倍 用 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 于 的 的 探针 探针 探针 探针 探针 探针 ~ 显示 ~ ~ 2.5-倍信号增加，可能是由于RNA 和蛋白质之间的不同反应偏好 (图1b, c).基于 苯胺 的 化学 探针 1 和 3 具有 更 的 特异性 ， hek293t 在 粒体 中 稳定 表达 minisog ， 探针 3 的 信号 增加 增加> 8 倍 而 于 于 rna 标记 cap-eq 的 2 仅 ~ ~ ~ ~ 2.5 - 倍信号增加，可能是由于RNAAniline-raws li tshuaj ntsuam xyuas 1 thiab 3 muaj qhov tshwj xeeb zoo dua, HEK293T ruaj khov qhia miniSOG hauv mitochondria, thiab kev sojntsuam 3 muaj ntau dua 8-fold nce hauv lub teeb liab, thaum sojntsuam 2 rau CAP-seq RNA labeling method pom tsuas yog ~ 2.5-fold nce.nyob rau hauv lub teeb liab, tej zaum yog vim txawv cov tshuaj tiv thaiv nyiam ntawm RNA thiab protein (Fig. 1b, c).Tsis tas li ntawd, kev sojntsuam 3 isomers thiab hydrazine probes (probes 5, 6, 7) raug kuaj, lees paub qhov kev ua kom zoo ntawm kev sojntsuam 3 (Ntxiv daim duab 1b,c).Ib yam li ntawd, nyob rau hauv-gel fluorescence tsom xam qhia lwm yam optimized sim tsis: irradiation wavelength (460 nm), tshuaj ntsuam xyuas concentration (1 mM), thiab irradiation lub sij hawm (20 min) (Suab ntxiv Fig. 2a–c).Kev tshem tawm ib qho kev tiv thaiv lossis cov kauj ruam hauv PDPL raws tu qauv ua rau muaj teeb meem cuam tshuam rau keeb kwm yav dhau (Daim duab 1d).Qhov tseem ceeb, cov ntawv sau cov protein tau txo qis hauv qhov muaj sodium azide lossis trolox, uas paub tias tua cov pa oxygen.Lub xub ntiag ntawm D2O, uas paub tias ruaj khov singlet oxygen, txhim kho cov cim cim npe.Txhawm rau tshawb xyuas qhov kev koom tes ntawm lwm hom pa oxygen reactive rau kev sau npe, mannitol thiab vitamin C tau ntxiv los tsim hydroxyl thiab superoxide radical scavengers, raws li, 18, 29, tab sis lawv tsis pom tias txo cov ntawv sau.Ntxiv ntawm H2O2, tab sis tsis muaj teeb pom kev zoo, tsis ua rau kev sau npe (Ntxiv daim duab 3a).Fluorescence singlet oxygen imaging nrog Si-DMA probes tau lees paub tias muaj cov pa oxygen singlet hauv HEK293T-miniSOG hlau, tab sis tsis nyob hauv thawj HEK293T hlau.Tsis tas li ntawd, mitoSOX Liab tsis tuaj yeem ntes superoxide ntau lawm tom qab illumination (Fig. 1e thiab Supplementary Fig. 3b) 30. Cov ntaub ntawv no qhia tau hais tias singlet oxygen yog lub ntsiab reactive oxygen hom lub luag hauj lwm rau tom ntej proteomic labeling.Cytotoxicity ntawm PDPL raug soj ntsuam nrog rau lub teeb xiav irradiation thiab tshuaj ntsuam xyuas, thiab tsis muaj qhov tseem ceeb cytotoxicity tau soj ntsuam (Ntxiv Fig. 4a).
Txhawm rau kawm txog cov txheej txheem sau npe thiab ua kom muaj kev txheeb xyuas cov protein ntau siv LC-MS / MS, peb yuav tsum xub txiav txim siab seb cov amino acids twg raug hloov kho thiab cov delta pawg ntawm cov ntawv sojntsuam.Methionine, histidine, tryptophan thiab tyrosine tau tshaj tawm tias tau hloov kho los ntawm singlet oxygen14,15.Peb sib koom ua ke TOP-ABPP31 kev ua haujlwm nrog kev tsis ncaj ncees qhib kev tshawb nrhiav los ntawm FragPipe xam platform raws li MSFragger32.Tom qab singlet oxygen hloov kho thiab tshuaj ntsuam xyuas cov ntawv sau npe, nyem chemistry tau ua los ntawm kev siv lub biotin txo daim ntawv lo uas muaj cov cleavable linker, ua raws li neutravidin stretching thiab trypsin digestion.Cov peptide hloov kho, tseem khi rau cov resin, yog photocleaved rau LC-MS/MS tsom xam (Daim duab 2a thiab Cov Ntaub Ntawv Ntxiv 1).Ntau qhov kev hloov kho tau tshwm sim thoob plaws hauv lub proteome nrog ntau tshaj 50 daim ntawv qhia peptide (PSM) qhov sib tw (Fig. 2b).Kuj ceeb tias, peb tsuas pom kev hloov kho ntawm histidine, tej zaum yog vim qhov siab dua ntawm oxidized histidine ntawm aniline probes dua lwm cov amino acids.Raws li cov txheej txheem luam tawm ntawm histidine oxidation los ntawm singlet oxygen, 21,33 lub tswv yim delta-mass qauv ntawm +229 Da sib raug rau adduct ntawm kev sojntsuam 3 nrog 2-oxo-histidine tom qab ob oxidation, thaum +247 Da yog cov khoom siv hydrolysis. ntawm +229 Da (Ntxiv daim duab 5).Kev ntsuam xyuas ntawm MS2 spectrum tau pom muaj kev ntseeg siab ntawm kev txheeb xyuas feem ntau ntawm cov y thiab b ions, suav nrog kev txheeb xyuas qhov hloov pauv cov fragment ions (y thiab b) (Daim duab 2c).Kev txheeb xyuas cov ntsiab lus ntawm cov kab ke hauv zos ntawm PDPL-hloov histidines tau nthuav tawm qhov kev nyiam me ntsis rau cov dej hydrophobic me me ntawm ± 1 txoj haujlwm (Ntxiv daim duab 4b).Qhov nruab nrab, 1.4 histidines tau txheeb xyuas ib qho protein, thiab cov chaw ntawm cov cim no tau txiav txim siab los ntawm qhov hnyav nkag mus tau hauv cheeb tsam (SASA) thiab txheeb ze cov kuab tshuaj muaj nyob (RSA) kev tshuaj ntsuam (Suab ntxiv Fig. 4c,d).
Ib qho kev ua haujlwm tsis ncaj ncees rau kev kawm txog kev xaiv seem uas siv FragPipe xam platform siv los ntawm MSFragger.Cleavable linkers yog siv hauv Click chemistry kom tso cai rau photocleavage ntawm hloov peptides los ntawm streptavidin resin.Kev tshawb nrhiav qhib tau pib txhawm rau txheeb xyuas ntau yam kev hloov kho, nrog rau cov seem tseem ceeb.b Muab qhov loj ntawm kev hloov kho uas tshwm sim thoob plaws hauv lub proteome.Peptide daim ntawv qhia PSM.c MS2 spectral annotation ntawm histidine qhov chaw hloov kho nrog kev sojntsuam 3. Raws li tus neeg sawv cev piv txwv, covalent cov tshuaj tiv thaiv nrog kev sojntsuam 3 ntxiv +229.0938 Da rau cov hloov kho amino acid.d Mutation assay siv los kuaj rau PDPL cov cim.PRDX3 (H155A, H225A) thiab PRDX1 (H10A, H81A, H169A) tau kis nrog cov tsiaj qus plasmids rau kev tiv thaiv chij.e Cov hluavtaws peptide tau hnov ​​​​mob nrog purified miniSOG nyob rau hauv lub xub ntiag ntawm kev sojntsuam 3 thiab cov khoom siv nrog Δm +247 thiab +229 tau sau tseg hauv LC-MS spectrum.f Hauv vitro protein-rau-protein kev sib cuam tshuam ua qauv nrog miniSOG-6xHis-tag thiab anti-6xHis antibody.Antibiotin (streptavidin-HRP) thiab anti-nas Western blot tsom xam ntawm miniSOG-6xHis / anti-6xHis cov tshuaj tiv thaiv kab mob sib sau ua ke nrog kev sojntsuam 3, nyob ntawm lub sijhawm raug rau lub teeb.Cov ntawv lo rau ib tus neeg cov protein tau qhia nyob rau hauv cov khoom hnyav molecular: LC antibody light chain, HC antibody heavy chain.Cov kev sim no tau rov ua nws tus kheej tsawg kawg ob zaug nrog cov txiaj ntsig zoo sib xws.
Rau kev txheeb xyuas biochemical ntawm qhov chaw sau npe, PRDX3 thiab PRDX1 tau txheeb xyuas los ntawm huab hwm coj spectrometry tau hloov pauv los ntawm histidine rau alanine thiab muab piv rau hom tsiaj qus hauv kev kuaj mob.Cov txiaj ntsig ntawm PDPL tau pom tias qhov kev hloov pauv tau txo qis kev sau npe (Daim duab 2d).Lub caij no, cov peptide sequences uas tau txheeb xyuas nyob rau hauv qhov qhib kev tshawb nrhiav tau synthesized thiab reacted nyob rau hauv vitro nrog purified miniSOG nyob rau hauv lub xub ntiag ntawm kev sojntsuam 3 thiab xiav lub teeb, yielding khoom nrog ib tug loj hloov ntawm +247 thiab +229 Da thaum kuaj pom los ntawm LC-MS (Fig. . 2e).).Txhawm rau kuaj seb puas muaj cov protein sib cuam tshuam tuaj yeem raug sau npe hauv vitro hauv cov lus teb rau miniSOG photoactivation, peb tau tsim qhov kev soj ntsuam ze ze los ntawm kev sib cuam tshuam ntawm miniSOG-6xHis protein thiab anti-Nws monoclonal antibody hauv vitro (Daim duab 2f).Hauv qhov kev ntsuam xyuas no, peb xav kom muaj cov ntawv sau sib thooj ntawm cov tshuaj tiv thaiv hnyav thiab lub teeb saw nrog miniSOG.Qhov tseeb, los tiv thaiv nas (paub txog qhov hnyav thiab lub teeb chains ntawm anti-6xHis-labeled antibody) thiab streptavidin Western blots pom muaj zog biotinylation ntawm cov hnyav thiab lub teeb chains.Qhov tseem ceeb, peb pom miniSOG autobiotinylation vim yog 6xHis tag thiab kev sib txuas ntawm lub teeb thiab hnyav chains, uas tej zaum yuav cuam tshuam rau qhov sib txawv ntawm cov lus piav qhia yav dhau los ntawm lysine thiab 2-oxo-histidine proximal teb.Hauv kev xaus, peb txiav txim siab tias PDPL hloov kho histidine nyob rau hauv qhov sib thooj.
Peb lub hom phiaj tom ntej yog los ua tus cwj pwm ntawm subcellular proteome los ntsuas qhov tshwj xeeb ntawm cov ntawv cim npe.Yog li ntawd, peb stably qhia miniSOG nyob rau hauv lub nucleus, mitochondrial matrix, los yog txheej ER membrane ntawm HEK293T hlwb (Fig. 3a).Gel fluorescence tsom xam pom muaj ntau daim ntawv sau npe ntawm peb qhov chaw subcellular nrog rau cov ntawv cim sib txawv (Fig. 3b).Fluorescence imaging tsom pom qhov tshwj xeeb ntawm PDPL (Fig. 3c).PDPL kev ua haujlwm tau ua raws li cov kev cuam tshuam nrog rhodamine dyes rau delineate subcellular proteomes siv fluorescence microscopy, thiab PDPL cov teeb liab tau colocalized nrog DAPI, mitochondrial trackers, lossis ER trackers, lees paub qhov siab ncaj ncees ntawm PDPL.Rau peb qhov chaw organelle, kev sib piv ib sab ntawm PDPL nrog TurboID siv avidin sab hnub poob blot pom tias PDPL tau sau npe tshwj xeeb dua piv rau lawv cov kev tswj hwm.Raws li PDPL tej yam kev mob, ntau daim ntawv sau npe tau tshwm sim, qhia tias muaj ntau cov PDPL-labeled proteins (Supplementary Fig. 6a-d).
Schematic sawv cev ntawm miniSOG-mediated organelle-specific proteome labeling.miniSOG tsom rau mitochondrial matrix ntawm fusion mus rau N-terminal 23 amino acids ntawm tib neeg COX4 (mito-miniSOG), lub nucleus ntawm fusion rau H2B (nucleus-miniSOG), thiab Sec61β ntawm cytoplasmic sab ntawm ER membrane (ER-miniSOG). ).Indications muaj xws li gel duab, confocal imaging, thiab loj spectrometry.b Cov neeg sawv cev gel duab ntawm peb lub cev tshwj xeeb PDPL profiles.CBB Coomassie Brilliant Blue.c Cov duab sawv cev ntawm HEK293T hlwb stably qhia miniSOG nrog sib txawv subcellular localizations kuaj pom los ntawm cov tshuaj tiv thaiv kab mob V5 (liab).Subcellular markers yog siv rau mitochondria thiab ER (ntsuab).PDPL kev ua haujlwm suav nrog kev tshawb pom ntawm miniSOG (daj) sau npe subcellular proteomes siv Cy3-azide nyem chemistry.Scale bar: 10 µm.d Volcanic plots ntawm PDPL-tagged proteomes nyob rau hauv ntau yam organelles quantified los ntawm unlabeled quantification (n = 3 ywj siab biological thwmsim).Ob tug tub-ntxhais kawm ntawv qhov t-test tau siv rau ntawm thaj av hluav taws kub.HEK293T hom tsiaj qus tau siv los ua kev tswj tsis zoo. Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold ion siv sib txawv). Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold ion siv sib txawv). Значительно измененные белки выделены красным цветом (p <0,05 thiab >2-кратная разница в интенсивиност). Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv liab (p <0.05 thiab> 2-fold sib txawv hauv ion siv).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). Cov proteins uas tau hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold sib txawv hauv ionic zog).Cov proteins uas tseem ceeb rau HEK293T-miniSOG tab sis tsis tseem ceeb rau HEK293T yog pom hauv ntsuab.e Kev tshuaj xyuas qhov tshwj xeeb ntawm cov ntaub ntawv proteomic los ntawm kev sim d.Tag nrho cov naj npawb ntawm cov protein tseem ceeb hauv txhua lub cev (liab thiab ntsuab dots) yog cim rau saum.Histograms qhia cov proteins nyob rau hauv cov organelles raws li MitoCarta 3.0, GO tsom xam thiab A. Ting li al.neeg.Sib cais cov ntaub ntawv rau mitochondria, nuclei, thiab ER.Cov kev sim no tau rov ua nws tus kheej tsawg kawg ob zaug nrog cov txiaj ntsig zoo sib xws.Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Kev txhawb nqa los ntawm cov gel thiab cov txiaj ntsig tau pom, cov ntawv sau tsis pub muaj ntau npaum li cas los txheeb xyuas cov proteome hauv txhua lub cev (Cov Ntaub Ntawv Ntxiv 2).Untransfected HEK293T tau siv los ua kev tswj tsis zoo rau rho tawm cov cim keeb kwm yav dhau. Volcano zaj duab xis tsom pom cov protein ntau ntxiv (p <0.05 thiab> 2-fold ion siv) nrog rau singleton proteins uas tsuas muaj nyob rau hauv miniSOG-qhia kab (Fig. 3d liab thiab ntsuab dots). Volcano zaj duab xis tsom pom cov protein ntau ntxiv (p <0.05 thiab> 2-fold ion siv) nrog rau singleton proteins uas tsuas muaj nyob rau hauv miniSOG-qhia kab (Fig. 3d liab thiab ntsuab 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 zaj duab xis tsom xam pom cov protein ntau ntxiv (p <0.05 thiab> 2-fold ion siv) nrog rau cov proteins uas tsuas muaj nyob rau hauv miniSOG-expressing kab (Fig. 3d, liab thiab ntsuab dots).火山图 显示 出 出 显着 富集 富集 的 的 蛋白质 蛋白质 蛋白质 (p <0.05 和> 2 倍 离子 强度) 以及 仅 在于 在于在于og 表达 中系 的 的在于og 表达表达 的单一蛋白质 表达蛋白质 (图 3d 红色 绿色点).火山图 分析 显示 出 的 的 蛋白质 蛋白质 蛋白质 (p <0.05 和> 2 倍 在于 在于 在于 在于 在于在于og 表达 的 单一在于og 表达 的的 单一蛋白质蛋白质 蛋白质 (图 3d 红色红色蛋白质蛋白质 (图 3d 红色红色蛋白质蛋白质蛋白质 (图. 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 zaj duab xis tsom xam pom cov protein ntau ntxiv (p <0.05 thiab> 2x ionic zog) nrog rau cov proteins nkaus xwb tam sim no hauv miniSOG kab qhia (liab thiab ntsuab dots hauv daim duab 3d).Ua ke cov ntaub ntawv no, peb tau txheeb xyuas 1364, 461, thiab 911 statistically tseem ceeb nuclear, mitochondrial, thiab ER txheej membrane proteins, feem.Txhawm rau txheeb xyuas qhov tseeb ntawm organelle-localized PDPL, peb siv MitoCarta 3.0, Gene Ontology (GO) tsom, thiab A. Ting li al.ib cov ntaub ntawv set8 tau siv rau mitochondria, nucleus, thiab ER los ntsuam xyuas lub organelle tshwj xeeb ntawm cov proteins uas kuaj pom, sib raug rau qhov raug ntawm 73.4, 78.5, thiab 73.0% (Fig. 3e).Qhov tshwj xeeb ntawm PDPL lees paub tias PDPL yog cov cuab yeej zoo tshaj plaws rau kev txheeb xyuas cov kab mob tshwj xeeb ntawm cov kab mob.Qhov tseem ceeb, kev tsom xam ntawm cov mitochondrial cov proteins uas tau txheeb xyuas pom tau hais tias cov khoom pov tseg tau muab faib ua feem ntau hauv cov matrix thiab sab hauv daim nyias nyias (226 thiab 106, raws li), suav txog 91.7% (362) ntawm tag nrho cov naj npawb ntawm cov mitochondrial proteins.Ib qib siab ntawm PDPL tau raug lees paub ntxiv (Ntxiv daim duab 7a).Ib yam li ntawd, kev tshuaj xyuas subnuclear tau pom tias cov proteome uas ntes tau feem ntau yog muab faib rau hauv cov nucleus, nucleoplasm, thiab nucleolus (Ntxiv daim duab 7b).Nuclear proteomic tsom xam nrog lub teeb liab peptide peptide (3xNLS) pom qhov tseeb zoo sib xws rau H2B tsim (Ntxiv Fig. 7c–h).Txhawm rau txiav txim siab qhov tshwj xeeb ntawm PDPL tus cim, nuclear laminin A tau raug xaiv los ua qhov tsis sib haum xeeb hauv cheeb tsam POI7 ntxiab.PDPL tau txheeb xyuas 36 cov protein ntau ntxiv, ntawm 12 cov proteins (30.0% suav nrog lamin A) yog cov yam ntxwv zoo lamin A cuam tshuam cov proteins sau tseg los ntawm String database, nrog rau feem pua ​​​​ntawm cov txheej txheem BioID (122 proteins) 28 ntawm 28. , 22.9 %) 7. Peb txoj kev tau txheeb xyuas cov protein tsawg dua, tej zaum yog vim muaj qhov txwv tsis pub sau npe, uas ua tau los ntawm kev ua haujlwm ntau dua singlet oxygen.GO tsom xam pom tias cov proteins uas tau txheeb xyuas tau nyob rau hauv lub nucleoplasm (26), nuclear membrane (10), nuclear membrane (9), thiab nuclear pores (5).Sib sau ua ke, cov proteins uas muaj nyob hauv nuclear tau suav txog 80% ntawm cov proteins uas muaj txiaj ntsig zoo, ntxiv rau qhov qhia txog qhov tshwj xeeb ntawm PDPL (Cov Lus Qhia Ntxiv 8a–d).
Tom qab tsim lub peev xwm ntawm PDPL los ua qhov sib thooj cim hauv cov kab mob, peb mam li sim seb PDPL puas tuaj yeem siv los txheeb xyuas POI cov neeg koom tes.Tshwj xeeb, peb tau nrhiav los txhais PDPL kev tshuaj xyuas ntawm cytosolic proteins, uas yog suav tias yog lub hom phiaj nyuaj dua li lawv cov membrane-localized counterparts vim lawv qhov muaj zog heev.Lub bromodomain thiab extraterminal (BET) protein BRD4 tau nyiam peb cov xim rau nws lub luag haujlwm tseem ceeb hauv ntau yam kab mob 35, 36.Lub complex tsim los ntawm BRD4 yog ib tug transcriptional coactivator thiab ib tug tseem ceeb kho lub hom phiaj.Los ntawm kev tswj cov kev qhia ntawm c-myc thiab Wnt5a cov ntaub ntawv hloov pauv, BRD4 tau xav tias yog qhov tseem ceeb ntawm kev txiav txim siab ntawm tus mob myeloid leukemia (AML), ntau yam myeloma, Burkitt's lymphoma, mob qog noj ntshav thiab inflammatory kab mob37,38.Tsis tas li ntawd, qee cov kab mob tsom BRD4 los tswj cov kab mob thiab cov cellular transcription, xws li papillomavirus, HIV, thiab SARS-CoV-236,39.
Txhawm rau daim duab qhia kev sib cuam tshuam BRD4 siv PDPL, peb ua ke miniSOG nrog luv N- lossis C-terminal isoform ntawm BRD4.Cov txiaj ntsig Proteomic tau nthuav tawm ntau qhov sib tshooj ntawm ob qho kev tsim kho (Ntxiv daim duab 9a).Lub nuclear proteome tau txheeb xyuas nrog miniSOG-H2B npog 77.6% ntawm cov proteins cuam tshuam nrog BRD4 (Ntxiv daim duab 9b).Tom qab ntawd, lub sijhawm sib txawv ntawm lub teeb pom kev zoo (2, 5, 10, 20 min) tau siv los kho lub vojvoog cim (daim duab 4a thiab cov ntaub ntawv ntxiv 3).Peb xaus lus tias ntawm luv luv photoperiods, PDPL feem ntau yuav sau npe ncaj qha rau cov neeg koom tes, thaum lub sij hawm ntev yuav suav nrog cov proteins uas tau txheeb xyuas thaum lub sij hawm luv luv photoactivation nrog rau cov hom phiaj tsis ncaj hauv cov ntawv sau npe.Qhov tseeb, peb pom muaj zog sib tshooj ntawm cov ntsiab lus nyob ib sab (84.6% rau 2 thiab 5 min; 87.7% rau 5 thiab 10 min; 98.7% rau 10 thiab 20 min) (Fig. 4b thiab Supplementary Fig. 9c).Hauv txhua pab pawg sim, peb pom tsis yog BRD4 tus kheej-labeling, tab sis ob peb lub hom phiaj paub xws li MED1, CHD8, BICRA, NIPBL, SMC1A, thiab HMGB1 annotated nyob rau hauv cov hlua database.Lub zog ionic ntawm cov hom phiaj no yog proportional rau lub sij hawm raug (Fig. 4c thiab Ntxiv Fig. 9d).GO tsom xam ntawm cov proteins uas tau txheeb xyuas hauv 2-feeb pab pawg tau pom tias cov proteins uas tau txheeb xyuas tau nyob hauv lub nucleus thiab tau koom nrog kev hloov kho chromatin thiab RNA polymerase muaj nuj nqi.Lub molecular muaj nuj nqi ntawm cov protein tau enriched nyob rau hauv chromatin binding los yog transcriptional coactivation, raws li zoo raws li BRD4 muaj nuj nqi (Fig. 4d).String database-enabled protein interaction analysis tau nthuav tawm thawj theem ntawm kev sib cuam tshuam ncaj qha ntawm BRD4 thiab HDAC tsev neeg sib cuam tshuam xws li SIN3A, NOR2, BCOR, thiab SAP130 (Fig. 4e thiab Supplementary Fig. 9e), zoo ib yam nrog BRD4 thiab HDAC binding acetylated histones ..Tsis tas li ntawd, cov neeg sawv cev lub hom phiaj tau txheeb xyuas los ntawm LC-MS/MS, suav nrog Sin3A, NSUN2, Fus, thiab SFPQ, tau lees paub los ntawm Western blotting (Fig. 4f).Tsis ntev los no, luv luv isoform ntawm BRD4 tau tshaj tawm los tsim cov nuclei nrog cov kua-kua theem sib cais (LLPS) cov khoom.RNA binding proteins Fus thiab SFPQ kho cov LLPS ntawm ntau cov txheej txheem ntawm tes thiab tau raug txheeb xyuas ntawm no yog BRD4 binding proteins uas tsis tau sau tseg.Kev sib cuam tshuam ntawm BRD4 thiab SFPQ tau lees paub los ntawm kev sim co-immunoprecipitation (co-IP) (Daim duab 4g), qhia lwm txoj hauv kev rau BRD4-mediated kua-kua theem sib cais tsim nyog tau txais kev tshawb nrhiav ntxiv.Ua ke, cov txiaj ntsig no qhia tias PDPL yog lub platform zoo tshaj plaws rau kev txheeb xyuas qhov paub txog BRD4 cuam tshuam nrog rau cov protein tsis paub txog kev sib txuas.
Schematic sawv cev ntawm miniSOG-mediated BRD4 qhov sib thooj cim, lub sij hawm raug: 2, 5, 10, thiab 20 min.b Kev sib tshooj ntawm cov protein uas tau txheeb xyuas thaum lub sijhawm teeb pom kev sib txawv.Protein enrichment pom nyob rau hauv HEK293T-miniSOG-BRD4 yog qhov tseem ceeb ntawm cov ntaub ntawv piv rau cov tsiaj qus HEK293T.c Ion siv zog thaum ntsuas cov neeg sawv cev tsis muaj npe paub BRD4-binding proteins thaum lub sij hawm raug teev tseg.n = 3 cov qauv kev ywj pheej.Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± tus qauv sib txawv.d Gene ontological tsom xam (GO) ntawm cov proteins pom nyob rau hauv pawg 2-feeb.Thawj kaum nqe lus GO tau teev tseg.Cov npuas yog xim raws li GO lub sij hawm qeb, thiab npuas loj yog proportional rau cov proteins uas pom nyob rau hauv txhua lub sij hawm.e Txoj hlua tsom xam ntawm cov protein sib cuam tshuam nrog BRD4.Lub voj voog daj yog cov kua nplaum ncaj qha thiab cov voj voog grey yog thawj txheej ntawm cov kua nplaum tsis ncaj.Cov kab liab sawv cev rau qhov kev sim txiav txim siab sib cuam tshuam thiab cov kab xiav sawv cev rau kev kwv yees kev sib cuam tshuam.f Tus Neeg Sawv Cev BRD4 khi lub hom phiaj pom hauv LC-MS/MS tau txheeb xyuas los ntawm Western blotting.g Co-immunoprecipitation thwmsim paub meej tias kev sib cuam tshuam ntawm SFPQ thiab BRD4.Cov kev sim no tau rov ua nws tus kheej tsawg kawg ob zaug nrog cov txiaj ntsig zoo sib xws.Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Ntxiv rau kev txheeb xyuas cov hom phiaj uas tsis tau sau npe POI koom nrog, peb xav tias PDPL yuav tsim nyog rau kev txheeb xyuas cov substrates rau cov enzymes, uas yuav xav tau cov yam ntxwv ntawm kev sib txuas tsis ncaj ntawm cov proteins hauv cov complexes loj los piav qhia cov substrates uas tsis tau sau npe.Parkin (encoded los ntawm PARK2) yog E3 ligase thiab kev hloov pauv hauv parkin paub tias ua rau autosomal recessive juvenile Parkinson's disease (AR-JP)42.Tsis tas li ntawd, parkin tau piav qhia tias yog qhov tseem ceeb rau mitophagy (mitochondrial autophagy) thiab tshem tawm cov pa oxygen reactive.Txawm li cas los xij, txawm hais tias ntau qhov chaw nres tsheb tau raug txheeb xyuas, lub luag haujlwm ntawm parkin hauv tus kab mob no tseem tsis tau meej.Txhawm rau piav qhia nws cov substrates uas tsis muaj qhov tsis zoo, PDPL tau sim los ntawm kev ntxiv miniSOG rau N- lossis C-terminus ntawm parkin.Cov hlwb raug kho nrog carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) kom qhib qhov chaw nres tsheb ntawm txoj kev PINK1-Parkin.Piv nrog rau peb cov txiaj ntsig BRD4 PDPL, parkin N-terminus fusion tau nthuav tawm cov txheej txheem loj ntawm cov hom phiaj protein, txawm hais tias nws tau them ib feem loj ntawm C-terminus (177 tawm ntawm 210) (Daim duab 5a, b thiab Cov Ntaub Ntawv Ntxiv 4).Cov txiaj ntsig tau zoo ib yam nrog cov lus ceeb toom tias N-terminal tags tuaj yeem ua rau Parkin44 tsis zoo.Kuj ceeb tias, tsuas muaj 18 qhov sib tshooj ntawm cov proteins hauv peb cov ntaub ntawv uas tau luam tawm AP-MS cov txiaj ntsig rau Parkin43, yuav yog vim qhov sib txawv ntawm cov kab ntawm tes thiab cov kev ua haujlwm proteomics.Ntxiv rau plaub yam proteins uas paub (ARDM1, HSPA8, PSMD14, thiab PSMC3) tau txheeb xyuas los ntawm ob txoj kev (Fig. 5c)43.Txhawm rau kom ua tiav cov txiaj ntsig ntawm LC-MS / MS, PDPL kev kho mob thiab tom qab Western blotting tau siv los sib piv cov txiaj ntsig ntawm HEK293T niam txiv ntawm tes ntsuas thiab ruaj khov N-terminal parkin kab.Yav dhau los tsis paub lub hom phiaj CDK2, DUT, CTBP1, thiab PSMC4 tau raug sim nrog cov ntaub ntawv paub, DNAJB1 (Fig. 5d).
Volcano zaj duab xis ntawm parkin-interacting proteins nyob rau hauv HEK293T hlwb nrog stably qhia miniSOG fused rau lub N- los yog C-terminus ntawm parkin (n = 3 ywj siab biological thwmsim).Ob tug tub-ntxhais kawm ntawv qhov t-test tau siv rau ntawm thaj av hluav taws kub.HEK293T tau siv los ua kev tswj tsis zoo. Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold ion siv sib txawv). Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold ion siv sib txawv). Значительно измененные белки выделены красным цветом (p <0,05 thiab >2-кратная разница в интенсивиност). Cov protein hloov pauv tseem ceeb yog qhov tseem ceeb hauv liab (p <0.05 thiab> 2-fold sib txawv hauv ion siv).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). Cov proteins uas tau hloov pauv tseem ceeb yog qhov tseem ceeb hauv xim liab (p <0.05 thiab> 2-fold sib txawv hauv ionic zog).Cov proteins uas tseem ceeb rau HEK293T-miniSOG tab sis tsis tseem ceeb rau HEK293T yog pom hauv ntsuab.b Venn daim duab qhia txog cov protein sib tshooj ntawm N-terminal thiab C-terminal tsim.N-terminal tags tuaj yeem ua rau tsis zoo rau parkin thiab ua rau muaj cov protein ntau dua.c Venn daim duab qhia txog cov protein sib tshooj ntawm PDPL thiab AP-MS.Paub cov neeg sib tham tau teev tseg, suav nrog 4 ntawm 18 cov protein sib tshooj thiab 11 ntawm 159 cov protein tshwj xeeb tau txheeb xyuas hauv PDPL.d Cov neeg sawv cev lub hom phiaj tau txheeb xyuas los ntawm LC-MS / MS tau txheeb xyuas los ntawm Western blotting.e Ssu72 thiab SNW1 tau raug txheeb xyuas tias tsis tau sau npe chaw nres tsheb substrates.Cov FLAG-tagged protein plasmids tau kis mus rau HEK293T thiab HEK293T-Parkin-miniSOG ua raws li kev kho mob CCCP ntawm ntau lub sijhawm.Qhov degradation tau tshaj tawm hauv Parkin overexpression kab.f Siv cov proteasome inhibitor MG132, nws tau lees paub tias cov txheej txheem degradation ntawm Ssu72 thiab SNW1 yog kho los ntawm proteasome-ubiquitination.Cov kev sim no tau rov ua nws tus kheej tsawg kawg ob zaug nrog cov txiaj ntsig zoo sib xws.Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Qhov tseem ceeb, cov proteins uas tau txheeb xyuas los ntawm PDPL yuav tsum suav nrog cov proteins uas muaj cov kab mob Parkin-binding thiab lawv cov substrates.Txhawm rau kuaj xyuas cov kab mob Parkin uas tsis tau sau npe, peb tau xaiv xya cov proteins uas tau txheeb xyuas (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 thiab SNW1) thiab kis plasmids kom nthuav tawm cov noob no rau ib txwm HEK293T thiab khov kho qhia miniSOG-Parkin's HEK293T.Cov qib ntawm Ssu72 thiab SNW1 cov proteins tau txo qis hauv cov kab ruaj khov miniSOG-Parkin (Fig. 5e).Kev kho mob nrog CCCP rau 12 teev ua rau muaj kev puas tsuaj loj tshaj plaws ntawm ob lub substrates.Txhawm rau tshawb xyuas seb qhov degradation ntawm Ssu72 thiab SNW1 puas yog tswj hwm los ntawm proteasome-ubiquitination, proteasome inhibitor MG132 tau ntxiv rau inhibit proteasome kev ua si, thiab qhov tseeb peb pom tias lawv cov txheej txheem degradation yog inhibited (Fig. 5f).Ntxiv cov hom phiaj uas tsis yog-substrate tau lees paub tias yog Parkin interactors siv Western blotting (Ntxiv daim duab. 10), uas qhia tau zoo ib yam nrog LC-MS/MS.Hauv kev xaus, kev sib koom ua ke ntawm PDPL kev ua haujlwm nrog lub hom phiaj kev hloov pauv cov ntaub ntawv pov thawj tso cai rau kev txheeb xyuas cov tsis tau sau npe E3 ligase substrates.
Peb tau tsim ib qho chaw sib thooj sib thooj uas tso cai rau koj txheeb xyuas qhov chaw thiab lub sijhawm cuam tshuam POIs.Lub platform yog raws li cov miniSOG photosensitizer protein, uas tsuas yog hais txog 12 kDa, tsawg dua ib nrab ntawm qhov loj ntawm APEX2 enzyme (27 kDa) thiab ib feem peb qhov loj ntawm TurboID (35 kDa).Qhov me me yuav tsum nthuav dav ntau ntawm cov ntawv thov rau kev kawm me me protein interactomes.Kev tshawb nrhiav ntxiv ntawm cov photosensitizers ntxiv, txawm tias cov noob caj noob ces cov proteins lossis cov molecules me, yuav tsum tau ua kom cov quantum tawm los ntawm singlet oxygen thiab nthuav dav qhov kev nkag siab ntawm txoj hauv kev no.Rau cov qauv tam sim no ntawm miniSOG, kev daws teeb meem siab ntawm lub cev tuaj yeem ua tiav siv lub teeb pom kev xiav kom qhib cov cim sib thooj.Tsis tas li ntawd, lub sij hawm raug tso tawm ntev dua "huab" ntawm singlet oxygen, uas ua rau muaj kev hloov kho ntawm ntau qhov seem histidine residues, nce lub vojvoog ntawm daim ntawv lo, thiab lub peev xwm los kho qhov PDPL spatial daws teeb meem.Peb kuj tau sim xya qhov tshuaj ntsuam xyuas kom nce cov teeb liab-rau-tom qab piv thiab tshawb xyuas cov txheej txheem molecular tom qab txoj hauv kev no.TOP-ABPP kev ua haujlwm ua ke nrog kev tshawb nrhiav tsis txaus ntseeg tau lees paub tias kev hloov kho tsuas yog tshwm sim hauv histidines thiab tsis muaj kev cuam tshuam microenvironment tau pom zoo rau kev hloov kho histidine, tshwj tsis yog rau kev nyiam me ntsis rau histidines hauv cheeb tsam voj.
PDPL kuj tau siv los ua tus cwj pwm subcellular proteomes nrog proteome tshwj xeeb thiab kev pab them nqi tsawg kawg yog piv rau lwm cov ntawv sib thooj thiab cov txheej txheem tshuaj organelle tshwj xeeb.Cov cim sib thooj kuj tau siv los ua tus yam ntxwv ntawm qhov chaw, lysosomal, thiab secretome-associated proteomes46,47.Peb ntseeg tias PDPL yuav sib haum nrog cov subcellular organelles.Tsis tas li ntawd, peb tau sib tw PDPL los ntawm kev txheeb xyuas lub hom phiaj rau cytosolic protein binding uas nyuaj tshaj li daim nyias nyias cov proteins vim lawv cov khoom muaj zog thiab kev koom tes hauv kev sib cuam tshuam ntawm lub cev.PDPL tau siv rau ob lub proteins, transcriptional coactivator BRD4 thiab cov kab mob sib txuas ligase E3 Parkin.Ob qho proteins no tau raug xaiv tsis yog rau lawv qhov tseem ceeb ntawm kev ua haujlwm lom neeg, tab sis kuj yog rau lawv qhov chaw kho mob thiab cov peev xwm kho mob.Rau ob lub POIs no, cov neeg koom tes paub zoo nrog rau cov hom phiaj tsis tau sau npe tau txheeb xyuas.Qhov tshwj xeeb tshaj yog, theem kev sib cais-sib koom nrog cov protein SFPQ tau lees paub los ntawm co-IP, uas yuav qhia tau tias muaj cov txheej txheem tshiab uas BRD4 (luv isoform) tswj LLPS.Nyob rau tib lub sijhawm, peb ntseeg tias kev txheeb xyuas ntawm Parkin substrates yog qhov xwm txheej uas yuav tsum tau txheeb xyuas cov nplaum tsis ncaj.Peb tau txheeb xyuas ob qhov tsis paub qhov chaw nres tsheb substrates thiab paub tseeb tias lawv qhov degradation raws txoj hauv kev ubiquitination-proteasome.Tsis ntev los no, ib lub tshuab kev siv tshuab-ua cov cuab yeej ua kom zoo nkauj tau tsim los tshawb pom hiilolase sastrates los ntawm cuab lawv nrog enzymes.Txawm hais tias qhov no yog ib txoj kev muaj zog heev, nws tsis haum rau kev tsom xam ntawm cov substrates koom nrog hauv kev tsim cov complexes loj thiab yuav tsum tau tsim cov kev sib txuas ntawm cov enzyme thiab cov substrate.Peb cia siab tias PDPL tuaj yeem txuas ntxiv mus kawm lwm yam protein complexes thiab enzyme tsev neeg, xws li cov tsev neeg deubiquitinase thiab metalloprotease.
Ib daim ntawv tshiab ntawm miniSOG, hu ua SOPP3, tau tsim nrog kev txhim kho singlet oxygen ntau lawm.Peb piv miniSOG nrog SOPP3 thiab pom cov cim kev ua tau zoo dua qub, txawm hais tias qhov sib piv ntawm cov teeb liab-rau-suab nrov tseem tsis hloov pauv (Cov duab ntxiv 11).Peb xav tias kev ua kom zoo dua ntawm SOPP3 (piv txwv li, los ntawm kev qhia txog kev hloov pauv) yuav ua rau cov protein ntau dua photosensitizer uas xav tau lub sijhawm luv luv thiab yog li tso cai rau cov txheej txheem cellular ntau dua.Qhov tseem ceeb, tam sim no version ntawm PDPL yog txwv rau lub cellular ib puag ncig raws li nws xav tau lub teeb xiav thiab tsis tuaj yeem nkag mus rau hauv cov ntaub so ntswg sib sib zog nqus.Qhov no feature txwv tsis pub nws siv hauv kev tshawb fawb tsiaj.Txawm li cas los xij, kev sib xyaw ntawm optogenetics nrog PDPL tuaj yeem muab sijhawm rau kev tshawb fawb tsiaj, tshwj xeeb hauv lub hlwb.Tsis tas li ntawd, lwm cov engineered infrared photosensitizers kuj tshem tawm qhov kev txwv no.Kev tshawb nrhiav tam sim no tab tom ua hauv cheeb tsam no.
HEK293T xov tooj ntawm tes tau txais los ntawm ATCC (CRL-3216).Cov kab xov tooj ntawm tes tau kuaj pom tsis zoo rau tus kab mob mycoplasma thiab tau coj mus kuaj hauv DMEM (Thermo, #C11995500BT) ntxiv nrog 10% fetal bovine serum (FBS, Vistech, #SE100-B) thiab 1% penicillin / streptomycin (Hyclone, #SV30010).loj hlob hauv.
3-Aminophenylene (piv txwv 3) thiab (4-ethynylphenyl) methanamine (piv txwv 4) tau yuav los ntawm Bidepharm.Propylamine (kev sojntsuam 2) tau yuav los ntawm Energy-chemicals.N-(2-Aminophenyl) pent-4-ynamide (kev sojntsuam 1) tau tsim los raws li kev tshaj tawm.
Cov Lus Qhia Ntxiv 1 teev cov caj ces tsim siv hauv txoj kev tshawb no.Cov miniSOG thiab KillerRed ib ntus tau cloned los ntawm khoom plig plasmid los ntawm P. Zou (Peking University).Lub mitochondrial matrix targeting sequence tau muab los ntawm 23 N-terminal amino acids ntawm COX4 thiab cloned rau hauv cov vectors qhia siv Gibson los ua ke (Beyotime, #D7010S).Txhawm rau tsom rau daim nyias nyias thiab cov nucleus ntawm endoplasmic reticulum, SEC61B tib neeg DNA (NM_006808.3) (NEB, #M0491L) nthuav dav los ntawm PCR los ntawm lub tsev qiv ntawv cDNA ntawm HEK293T hlwb, thiab H2B DNA (nyiaj los ntawm D. Lin, Shenzhen Bay Laboratory) thiab cloned, raws li tau hais los saum toj no.Tshwj tsis yog hais tias muaj lwm yam qhia, lwm cov noob caj noob ces siv rau kev hloov pauv thiab kev tsim kho ntawm cov kab cell ruaj khov yog PCR amplified los ntawm HEK293T cell cDNA tsev qiv ntawv.G3S (GGGS) thiab G4S (GGGGS) tau siv los ua kev sib txuas ntawm cov khoom noj protein thiab miniSOG.A V5 epitope tag (GKPINPNPLLGLDST) tau ntxiv rau cov fusion tsim.Rau kev nthuav qhia hauv cov tsiaj nyeg thiab tsim kom muaj kab ke ruaj khov, miniSOG fusion tsim tau subcloned rau hauv pLX304 lentiviral vector.Rau cov kab mob qhia, miniSOG tau cloned rau hauv pET21a vector sau 6xHis ntawm C-terminus.
HEK293T hlwb tau noob ntawm 2.0 x 105 hlwb rau ib qhov dej hauv 6-zoo daim hlau thiab kis tau 24 teev tom qab nrog recombinant lentiviral plasmids (2.4 μg pLX304) thiab viral ntim plasmids (1.5 μg siv psPAX2 thiab 1.2e μg) , #C0533), txog 80% fusion.Tom qab ib hmos transfection, qhov nruab nrab tau hloov thiab incubated rau lwm 24 teev.Kev sau tus kab mob no tau ua tom qab 24, 48 thiab 72 teev.Ua ntej kis kab mob ntawm lub hom phiaj ntawm tes, cov kab mob nruab nrab tau lim los ntawm 0.8 μm lim (Merck, #millex-GP) thiab polybene (Solarbio, #H8761) tau ntxiv rau qhov concentration ntawm 8 μg / ml.Tom qab 24 teev, cov hlwb raug tso cai kom rov zoo los ntawm kev hloov qhov nruab nrab.Cells raug xaiv siv 5 μg / ml blasticidin (Solarbio, #3513-03-9) rau thawj peb nqe lus raws li kev xaiv qis dua.Tom qab ntawd siv 20 μg / ml raws li kev tswj hwm nruj dua rau peb nqe lus tom ntej.
Cells tau noob nyob rau hauv 12-zoo chav (Ibidi, #81201) ntawm ib tug ceev ntawm kwv yees li 20,000 hlwb ntawm ib qhov dej.Txhawm rau txhim kho adhesion ntawm HEK293T hlwb, ntxiv 50 µg / ml fibronectin (Corning, #356008) diluted hauv phosphate buffered saline (PBS, Sangon, #B640435) ntawm 37 ° C.Cov chav tau pretreated rau 1 teev thiab tom qab ntawd tshem tawm nrog PBS.Tom qab 24 teev, cov hlwb raug ntxuav ib zaug nrog PBS, incubated nrog 1 mM sojntsuam 3 nyob rau hauv tshiab Hanks 'balanced ntsev tov (HBSS, Gibco, #14025092) rau 1 h ntawm 37 ° C, thiab ces incubated nrog ib tug xiav LED (460 nm. ).) tau irradiated rau 10 min ntawm chav tsev kub.Tom qab ntawd, cov hlwb raug ntxuav ob zaug nrog PBS thiab kho nrog 4% formaldehyde hauv PBS (Sangon, #E672002) rau 15 feeb ntawm chav tsev kub.Tshaj formaldehyde raug tshem tawm los ntawm cov hlwb ruaj khov los ntawm kev ntxuav peb zaug nrog PBS.Cells tau permeabilized nrog 0.5% Triton X-100 (Sangon, #A600198) hauv PBS thiab ntxuav 3 zaug nrog PBS.Tom qab ntawd tshem tawm lub chamber thiab ntxiv rau txhua tus qauv 25 µl ntawm cov tshuaj tiv thaiv nyem uas muaj 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) thiab 0.5 mg/ml sodium ascorbate (Aladdin, no. S105024) thiab incubated rau 30 feeb ntawm chav tsev kub.Tom qab cov tshuaj tiv thaiv snap, cov hlwb raug ntxuav rau 6 zaug nrog PBS uas muaj 0.05% Tween-20 (Sangon, #A600560) (PBST) thiab tom qab ntawd thaiv nrog 5% BSA (Abcone, #B24726) hauv PBST rau 30 feeb ntawm chav sov.
Rau colocalization immunostaining, hlwb tau incubated nrog thawj cov tshuaj tiv thaiv raws li cov kev mob qhia: nas anti-V5 tag mAb (1:500, CST, #80076), luav anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), luav polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) los yog luav anti-lamin A/C monoclonal antibody (1:500; CST, #2032) ntawm 4 °C thaum hmo ntuj.Tom qab ntxuav 3 zaug nrog PBST, cov hlwb tau incubated nrog cov tshuaj tiv thaiv thib ob: tshis tiv thaiv luav Alexa Fluor 488 (Thermo, #A11034) diluted 1: 1000, tshis tiv thaiv nas Alexa Fluor 594 (CST, # 8889) diluted 1: 1000.diluted nyob rau hauv chav tsev kub rau 30 feeb.Cells raug ntxuav 3 zaug nrog PBST thiab counterstained nrog DAPI (Thermo, #D1306) hauv PBS rau 10 feeb ntawm chav tsev kub.Tom qab 3 ntxuav nrog PBS, cov hlwb raug kaw hauv 50% glycerol (Sangon, #A600232) hauv PBS rau kev yees duab.Cov duab Immunofluorescent tau txais siv ZEISS LSM 900 Airyscan2 confocal microscope thiab ZNE 3.5 software.
Rau singlet oxygen fluorescent duab, hlwb raug ntxuav ob zaug nrog Hanks HEPES tsis ua ntej ntxiv 100 nM Si-DMA hauv Hanks HEPES tsis (DOJINDO, #MT05).Tom qab raug rau lub teeb, lub hlwb raug incubated nyob rau hauv ib tug CO2 incubator ntawm 37 ° C rau 45 feeb.Cells raug ntxuav ob zaug nrog Hanks 'HEPES tsis thiab suav nrog Hoechst hauv Hanks 'HEPES tsis nyob rau 10 feeb ntawm chav tsev kub thiab pom tau siv ZEISS LSM 900 confocal microscope., #M36008) hauv HBSS tsis muaj calcium thiab magnesium.Tom qab raug lub teeb los yog doxorubicin (MCE, #HY-15142A), cov hlwb raug incubated hauv CO2 incubator ntawm 37 ° C. rau 10 feeb, ntxuav ob zaug nrog HBSS tsis, thiab incubated nrog Hoechst nyob rau hauv HBSS tsis nyob rau hauv chav tsev kub.feeb.Doxorubicin tau siv los ua qhov kev soj ntsuam zoo uas cov hlwb tau kho nrog 20 μM doxorubicin hauv HBSS uas muaj 1% BSA rau 30 min.Cov duab Immunofluorescent tau txais los ntawm Zeiss LSM 900 confocal microscope.
HEK293T hlwb stably qhia mito-miniSOG tau noob ntawm qhov ntom ntawm kwv yees li 30% hauv 15 cm lauj kaub tais diav.Tom qab 48 teev, thaum ~ 80% confluence tau mus txog, lub hlwb raug ntxuav ib zaug nrog PBS, incubated nrog 1 mM sojntsuam 3 nyob rau hauv tshiab HBSS tsis nyob rau hauv 1 teev ntawm 37 ° C thiab ces illuminated nrog ib tug xiav LED rau 10 feeb ntawm chav tsev. kub..Tom qab ntawd, cov hlwb raug ntxuav ob zaug nrog PBS, khawb thiab rov ua dua hauv cov dej khov txias PBS tsis muaj EDTA-free protease inhibitors (MCE, #HY-K0011).Cells tau lysed los ntawm sonicating lub ntsis rau 1 feeb (1 thib ob rau thiab 1 thib ob tawm ntawm 35% amplitude).Qhov sib xyaw ua ke tau centrifuged ntawm 15,871 xg rau 10 min ntawm 4 ° C kom tshem tawm cov khib nyiab, thiab cov concentration ntawm supernatant tau hloov mus rau 4 mg / mL siv cov khoom siv tshuaj BCA protein (Beyotime, #P0009).Muab 1 ml ntawm cov lysate saum toj no nrog 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), thiab 1 mM CuSO4 incub hauv qab tig mus rau 1 teev ntawm chav tsev kub.Tom qab cov tshuaj tiv thaiv snap, ntxiv qhov sib xyaw rau hauv cov tshuaj ua ntej sib xyaw (MeOH: CHCl3: H2O = 4 ml: 1 ml: 3 ml) hauv 10 ml iav vial.Cov qauv sib xyaw thiab centrifuged ntawm 4500 g rau 10 feeb ntawm chav tsev kub.Cov kev daws teeb meem qis thiab sab saud raug muab pov tseg, cov nag lossis daus tau ntxuav ob zaug nrog 1 ml ntawm methanol thiab centrifuged ntawm 15871 × g rau 5 min ntawm 4 ° C.Ntxiv 1 ml ntawm 8 M urea (Aladdin, no. U111902) nyob rau hauv 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) kom yaj cov precipitate.Cov qauv tau muab kho dua tshiab nrog 10 mM dithiothreitol (Sangon, #A100281 hauv 25 mM ABC) rau 40 feeb ntawm 55 ° C tom qab ntawd ntxiv 15 mM tshiab iodoacetamide (Sangon, #A600539) ntawm chav tsev kub hauv qhov tsaus ntuj.Alkylation hauv 30 feeb..Ib qho ntxiv 5 mM dithiothreitol tau ntxiv los nres cov tshuaj tiv thaiv.Npaj kwv yees li 100 µl NeutrAvidin agarose hlaws (Thermo, #29202) rau txhua tus qauv los ntawm kev ntxuav 3 zaug nrog 1 ml PBS.Cov tshuaj proteome saum toj no tau diluted nrog 5 ml PBS thiab incubated nrog pre-ntxuav NeutrAvidin agarose hlaws dai rau 4 teev ntawm chav tsev kub.Tom qab ntawd cov hlaws raug ntxuav 3 zaug nrog 5 ml PBS uas muaj 0.2% SDS (Sangon, #A600485), 3 zaug nrog 5 ml PBS uas muaj 1M urea, thiab 3 zaug nrog 5 ml ddH2O.Cov hlaws tau raug txiav los ntawm centrifugation thiab rov ua dua hauv 200 μl ntawm 25 mM ABC uas muaj 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) thiab 20 ng / μl trypsin (Promega, #V5280).Trypsinize ib hmos ntawm 37 ° C nrog kev sib hloov.Cov tshuaj tiv thaiv raug tso tseg los ntawm kev ntxiv cov tshuaj formic acid (Thermo, # A117-50) kom txog thaum pH mus txog 2-3.Cov hlaws dai tau ntxuav 3 zaug nrog 1 ml ntawm PBS uas muaj 0.2% SDS, 3 zaug nrog 1 ml ntawm PBS muaj 1 M urea, thiab tom qab ntawd 3 zaug nrog 1 ml ntawm dej distilled.Cov peptides hloov kho tau tso tawm los ntawm lub teeb lysis (365 nm) rau 90 min siv 200 μl ntawm 70% MeOH.Tom qab centrifugation, lub supernatant tau sau.Cov hlaws tom qab tau ntxuav ib zaug nrog 100 μl ntawm 70% MeOH thiab cov supernatants tau sib sau ua ke.Cov qauv tau qhuav rau hauv lub tshuab nqus tsev Speedvac thiab khaws cia ntawm -20 ° C kom txog thaum kev soj ntsuam.
Txhawm rau txheeb xyuas thiab txheeb xyuas cov pa oxygen hloov pauv peptides singlet, cov qauv tau muab rov ua dua hauv 0.1% formic acid thiab 1 μg ntawm peptides tau txheeb xyuas siv Orbitrap Fusion Lumos Tribrid huab hwm coj spectrometer nruab nrog nano ESI qhov chaw los ntawm Tune thiab Xcalibur los ntawm cov neeg muag khoom software 4.3.Cov qauv raug muab cais rau ntawm 75 µm × 15 cm sab hauv ntim capillary kem nrog 3 µm C18 khoom (ReproSil-pur, #r13.b9.) thiab txuas nrog EASY-nLC 1200 UHPLC system (Thermo).Cov peptides tau sib cais los ntawm linear 95 feeb gradient chromatography los ntawm 8% hnyav B mus rau 50% hnyav B (A = 0.1% formic acid hauv dej, B = 0.1% formic acid hauv 80% acetonitrile), ces linearly nce mus rau 98% B min. hauv 6 min ntawm tus nqi ntws ntawm 300 nl / min.Orbitrap Fusion Lumos sau cov ntaub ntawv hloov pauv ntawm tag nrho MS scan thiab MS2 scan nyob ntawm cov ntaub ntawv.Lub sputtering voltage tau teem rau 2.1 kV thiab qhov kub ntawm cov ion thauj capillary yog 320 ° C.MS spectra (350-2000 m / z) tau sau nrog kev daws teeb meem ntawm 120,000, AGC 4 × 105, thiab lub sijhawm nkag siab tshaj plaws ntawm 150 ms.10 feem ntau cov nqi them ua ntej nyob rau hauv txhua qhov kev tshuaj ntsuam tag nrho tau fragmented siv HCD nrog ib qho kev sib tsoo zog ntawm 30%, lub qhov rais quadrupole cais ntawm 1.6 m / z, thiab kev daws teeb meem ntawm 30,000.AGC lub hom phiaj rau tandem huab hwm coj spectrometry siv 5 × 104 thiab siab tshaj tawm lub sij hawm 150 ms.Qhov tshwj xeeb dynamic yog teem rau 30 vib nas this. Unassigned ions lossis cov uas them 1+ thiab > 7+ raug tsis lees paub rau MS/MS. Unassigned ions lossis cov uas them 1+ thiab > 7+ raug tsis lees paub rau MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Unassigned ions lossis ions nrog tus nqi ntawm 1+ thiab > 7+ raug tsis lees paub rau MS / MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于 MS/MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于 MS/MS. Неуказанные ионы или ионы с зарядами 1+ и >7+ были отклонены для МС/МС. Tsis muaj ions lossis ions nrog cov nqi ntawm 1+ thiab > 7+ raug tsis lees paub rau MS / MS.
Cov ntaub ntawv nyoos yog ua tiav siv FragPipe xam platform raws li MSFragger.Kev tsis ncaj ncees loj thiab sib raug cov amino acids tau txiav txim siab siv qhov qhib kev tshawb nrhiav algorithm nrog lub siab ua ntej ntawm -150 txog 500 Da.Hloov kho peptides tau txheeb xyuas siv cov kev hloov kho histidine nrog kev nce qib ntawm +229.0964 thiab +247.1069 Da hauv PD (Proteome Discoverer 2.5, Thermo).
Cells stably qhia cov fused miniSOG noob tau plated hauv 6 cm lauj kaub tais diav.Thaum ncav cuag ~ 80% confluence, hlwb raug ntxuav ib zaug nrog HBSS (Gibco, #14025092), ces incubated nrog tshuaj ntsuam xyuas nyob rau hauv HBSS rau 1 teev ntawm 37 ° C thiab illuminated nrog xiav lub teeb.10W LED rau 20 feeb ntawm chav tsev kub.Txhawm rau txiav txim siab seb hom pa oxygen reactive twg koom nrog PDPL, 0.5 mM vitamin 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 tau ntxiv rau cov hlwb ua cov tshuaj ntxiv.Tom qab ntxuav nrog txias PBS, cov hlwb raug khawb, sau rau hauv 1.5 ml centrifuge hlab, thiab sonicated nrog ib tug ntsis rau 1 min hauv 200 μl ntawm PBS nrog 1x protease inhibitor tsis muaj EDTA (1 s thiab 1 s tsis muaj, amplitude 35%).Qhov sib xyaw ua ke tau centrifuged ntawm 15,871 × g rau 10 min ntawm 4 ° C thiab qhov siab tshaj plaws tau hloov kho mus rau 1 mg / mL siv cov khoom kuaj BCA protein.Kwv yees li 50 µl ntawm cov lysate saum toj no tau tsim nrog 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, thiab 1 mM CuSO4 rau 1 teev ntawm chav tsev kub nrog kev sib hloov ntawm hauv qab mus rau saum.Tom qab nias cov tshuaj tiv thaiv, nag lossis daus nrog acetone tau ua los ntawm kev ntxiv 250 μl ntawm acetone ua ntej chilled rau cov qauv, incubating ntawm -20 ° C rau 20 min thiab centrifuging ntawm 6010 × g rau 10 min ntawm 4 ° C.Sau cov pellet thiab boil hauv 50 µl ntawm 1x Laemmli's buffer rau 10 min ntawm 95 ° C.Cov qauv tom qab ntawd tau txheeb xyuas ntawm SDS-PAGE cov gels ntev thiab pom tau siv Bio-rad ChemiDoc MP Touch imaging system nrog Image Lab Touch software.
Kev nthuav qhia thiab purification ntawm recombinant miniSOG-6x Nws cov protein tau ua raws li tau piav qhia dhau los.Luv luv, E. coli BL21(DE3) hlwb (TransGen, #CD701-02) tau hloov pauv nrog pET21a-miniSOG-6xHis thiab cov lus qhia protein raug ntxias nrog 0.5 mM IPTG (Sangon, #A600168).Tom qab cell lysis, cov proteins tau huv si siv Ni-NTA agarose hlaws (MCE, no. 70666), dialyzed tawm tsam PBS, thiab khaws cia ntawm -80 ° C.
Rau cov tshuaj tiv thaiv kab mob raws li hauv vitro daim ntawv lo sib ze, sib tov 100 μM purified miniSOG, 1 mM sojntsuam 3, thiab 1 μg anti-label nas monoclonal antibody (TransGen, #HT501-01) hauv PBS rau tag nrho cov tshuaj tiv thaiv ntim ntawm 50 μl..Cov tshuaj tiv thaiv sib xyaw yog irradiated nrog xiav LED teeb rau 0, 2, 5, 10, thiab 20 min ntawm chav tsev kub.Cov sib tov yog incubated nrog 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, thiab 1 mM CuSO4 rau 1 h ntawm chav tsev kub ntawm ib tug upward motion shaker.Tom qab cov tshuaj tiv thaiv snap, ntxiv 4x Laemmli's tsis ncaj qha mus rau qhov sib tov thiab boil ntawm 95 ° C rau 10 min.Cov qauv raug tshuaj xyuas ntawm SDS-PAGE gels thiab tshuaj xyuas los ntawm kev ua haujlwm sab hnub poob nrog streptavidin-HRP (1: 1000, Solarbio, #SE068).
Ib qho histidine-muaj hluavtaws peptide nrog C-terminal amidation (LHDALDAK-CONH2) tau siv los tshuaj xyuas peptide nyob ze-raws li hauv vitro labeling.Hauv qhov kev xeem no, 100 μM purified miniSOG, 10 mM sojntsuam 3 thiab 2 μg / ml hluavtaws peptide tau sib xyaw hauv PBS hauv tag nrho cov tshuaj tiv thaiv ntim ntawm 50 μl.Cov tshuaj tiv thaiv sib xyaw yog irradiated nrog xiav LED teeb rau 1 teev ntawm chav tsev kub.Ib qho microliter ntawm cov qauv tau txheeb xyuas siv LC-MS system (Dej, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer nrog MassLynx spectrum tsom xam software).
HEK293T hlwb stably qhia cov miniSOG fusion noob tau noob nyob rau hauv 10 cm lauj kaub tais diav rau kab nrog txawv organelle localization (Mito, ER, Nucleus) thiab 15 cm lauj kaub tais diav rau Parkin-miniSOG thiab BRD4-miniSOG kab.Thaum ncav cuag ~ 90% confluence, lub hlwb raug ntxuav ib zaug nrog HBSS, ces incubated nrog sojntsuam 3 nyob rau hauv HBSS rau 1 teev ntawm 37 ° C thiab illuminated nrog ib tug 10 W xiav LED ntawm chav tsev kub.Rau cov ntawv tsis sib cuag ntawm Parkin, 10 µM proton carbonyl cyanide cab kuj m-chlorophenylhydrazone CCCP (Solarbio, #C6700) nrog sojntsuam 3 hauv HBSS tau ntxiv rau 1 teev ntawm 37 ° C.Lub cell lysis, nyem chemistry, txo qis thiab alkylation cov kauj ruam yog tib yam li tau piav qhia saum toj no, tshwj tsis yog tias 2 mg ntawm lysate tau ntxiv thiab biotin PEG3 azide tau siv rau hauv cov tshuaj tiv thaiv nyem es tsis txhob photodegradable biotin azide.Tom qab kev txhim kho, cov hlaws dai tau ntxuav 3 zaug nrog 5 ml ntawm PBS uas muaj 0.2% SDS, 3 zaug nrog 5 ml ntawm PBS muaj 1 M urea, thiab 3 zaug nrog 5 ml ntawm PBS.Tom qab ntawd, 2 µg trypsin tau ntxiv rau 300 µl 25 mM ABC uas muaj 1 M urea kom tshem tawm cov protein thaum hmo ntuj ntawm 37 ° C.Cov tshuaj tiv thaiv tau nres los ntawm kev ntxiv formic acid kom txog thaum pH ntawm 2-3 mus txog.Tom qab trypsinization ntawm cov hlaws dai, cov tshuaj peptide tau muab tshem tawm siv SOLAµ HRP kem (Thermo, #60209-001) thiab qhuav hauv Speedvac lub tshuab nqus tsev twj tso kua mis.Peptides tau redissolved nyob rau hauv 0.1% formic acid thiab 500 ng ntawm peptides tau soj ntsuam siv Orbitrap Fusion Lumos Tribrid huab hwm coj spectrometer nruab nrog nano-ESI qhov chaw piav saum toj no.Peptides tau muab cais rau kev lag luam RP-HPLC precolumns (75 μm x 2 cm) (Thermo, no. 164946) thiab analytical RP-HPLC txhua (75 μm x 25 cm) (Thermo, no. 164941), ob qho tib si ntim nrog 2 μm.gradient los ntawm 8% mus rau 35% ACN hauv 60 feeb, ces linearly nce mus rau 98% B hauv 6 feeb ntawm tus nqi ntws ntawm 300 Nl / min.MS spectra (350-1500 m / z) tau sau nrog kev daws teeb meem ntawm 60,000, AGC 4 × 105, thiab lub sijhawm nkag siab tshaj plaws ntawm 50 ms.Cov ions xaiv tau raug faib ua ntu zus los ntawm HCD hauv 3 lub voj voog nrog lub zog sib tsoo ntawm 30%, lub qhov rais quadrupole cais ntawm 1.6 m / z, thiab kev daws teeb meem ntawm 15000. A 5 × 104 tandem huab hwm coj spectrometer AGC lub hom phiaj thiab lub sijhawm txhaj tshuaj siab tshaj plaws. siv 22ms.Qhov kev tshem tawm dynamic yog teem rau 45 vib nas this. Unassigned ions lossis cov uas them 1+ thiab > 7+ raug tsis lees paub rau MS/MS. Unassigned ions lossis cov uas them 1+ thiab > 7+ raug tsis lees paub rau MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Unassigned ions lossis ions nrog tus nqi ntawm 1+ thiab > 7+ raug tsis lees paub rau MS / MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于 MS/MS.未指定的离子或电荷为1+ 和>7+ 的离子被拒绝用于 MS/MS. Неуказанные ионы или ионы с зарядами 1+ и >7+ были отклонены для МС/МС. Tsis muaj ions lossis ions nrog cov nqi ntawm 1+ thiab > 7+ raug tsis lees paub rau MS / MS.
Cov qauv kev npaj nce mus txog qhov ua kom muaj txiaj ntsig ntawm NeutrAvidin hlaws tau zoo ib yam li hauv LC-MS / MS tsom xam tau piav qhia saum toj no.Kwv yees li ntawm 50 μg ntawm lysate tau siv los ua cov tswv yim rau kev tswj hwm kev thauj khoom thiab 2 mg ntawm lysate tau siv rau kev cuam tshuam nyem.Tom qab enrichment thiab ntxuav nrog neutravidin, cov bound proteins tau eluted los ntawm kev ntxiv 50 μl ntawm Laemmli's buffer rau agarose resin hlaws dai thiab boiling ntawm 95 ° C. rau 5 feeb.Kev tswj cov khoom nkag thiab cov hlaws ua kom zoo dua qub tau txheeb xyuas los ntawm SDS-PAGE thiab xa mus rau PVDF daim nyias nyias (Millipore, #ISEQ00010) los ntawm cov txheej txheem Western blot.Cov daim nyias nyias tau thaiv nrog 5% skim mis nyuj (Sangon, #A600669) hauv TBS muaj 0.1% tween-20 (TBST) thiab incubated sequentially nrog thawj thiab theem nrab cov tshuaj tiv thaiv.Thawj cov tshuaj tiv thaiv tau diluted 1: 1000 hauv 5% skim mis nyuj hauv TBST thiab incubated hmo ntuj ntawm 4 ° C.Cov tshuaj tiv thaiv thib ob tau siv hauv qhov sib piv ntawm 1: 5000 thiab incubated rau 1 teev ntawm chav tsev kub.Cov daim nyias nyias tau pom los ntawm chemiluminescence siv Chemidoc MP imaging system.Tag nrho cov ntawv tsis raug txiav ntawm blots thiab gels hauv daim duab tau nthuav tawm raws li cov ntaub ntawv nyoos.
Cov tshuaj tiv thaiv tseem ceeb siv hauv txoj kev tshawb no suav nrog luav anti-SFPQ monoclonal antibody (CST, no. 71992), luav anti-FUS monoclonal antibody (CST, no. 67840), luav anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1- AP), luav anti-mSin3A polyclonal antibody (Abcam, #ab3479), nas anti-tag monoclonal antibody (TransGen, #HT201-02), nas anti-β-actin monoclonal antibody (TransGen, #HC201-01), luav anti -CDK2 monoclonal antibody (ABclonal, #A0094), luav monoclonal antibody rau CTBP1 (ABclonal, #A11600), luav polyclonal antibody rau DUT (ABclonal, #A2901), luav polyclonal antibody rau PSMC4 (ABclonal, #A2505), luav tshuaj tiv thaiv DNAJB1 polyclonal antibody (ABclonal, # A5504).Cov tshuaj tiv thaiv no tau siv ntawm 1: 1000 dilution hauv 5% skim mis nyuj hauv TBST.Cov tshuaj tiv thaiv thib ob uas siv hauv txoj kev tshawb no suav nrog cov tshuaj tiv thaiv kab mob IgG (TransGen, #HS101-01), tiv thaiv nas IgG (TransGen, #HS201-01) ntawm 1: 5000 dilution.
Txhawm rau tshawb xyuas ntxiv seb BRD4 cuam tshuam nrog SFPQ, ruaj khov HEK293T thiab BRD4-miniSOG hlwb overexpressing HEK293T tau plated hauv 10 cm lauj kaub tais diav.Cells raug ntxuav nrog txias PBS thiab lysed nyob rau hauv 1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) nrog EDTA-free protease inhibitor rau 30 feeb ntawm 4 ° C.Tom qab ntawd, cov lysates tau sau rau hauv 1.5 ml centrifuge raj thiab centrifuged ntawm 15,871 xg rau 10 min ntawm 4 ° C.Lub supernatant tau sau thiab incubated nrog 5 µg ntawm anti-V5 sau npe nas monoclonal antibody (CST, #80076) hmo ntuj ntawm 4 ° C.Ntxuav kwv yees li 50 µl ntawm cov protein A/G sib nqus hlaws (MCE, #HY-K0202) ob zaug nrog PBS uas muaj 0.5% Tween-20.Tom qab ntawd cov cell lysates tau incubated nrog cov hlaws sib nqus rau 4 teev ntawm 4 ° C nrog kev sib hloov ntawm hauv qab mus rau saum.Tom qab ntawd cov hlaws tau raug ntxuav plaub zaug nrog 1 ml ntawm PBST tsis thiab boil ntawm 95 ° C rau 5 min.Cov qauv raug txheeb xyuas ntawm SDS-PAGE gels thiab xa mus rau PVDF daim nyias nyias siv cov txheej txheem Western blot.Cov membranes raug thaiv hauv 5% skim mis nyuj hauv TBST thiab incubated sequentially nrog thawj thiab theem nrab cov tshuaj tiv thaiv.Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) tau siv ntawm qhov sib piv ntawm 1: 1000 hauv 5% skim mis nyuj hauv TBST thiab incubated hmo ntuj ntawm 4 ° C.Anti-Lavbit IgG tau siv ntawm qhov sib piv ntawm 1: 5000 thiab incubated rau 1 teev ntawm chav tsev kub.Cov daim nyias nyias tau pom los ntawm chemiluminescence siv Chemidoc MP imaging system.
Tag nrho cov qauv siv rau Solvent Accessible Surface Area (SASA) tsom xam tau los ntawm Protein Data Bank (PDB)52 lossis AlphaFold Protein Structure Database53.Tsis muaj SASA raug xam rau txhua qhov seem uas siv FreeSASA program.Tsuas yog ua kom tiav thiab tsis paub meej SASA cov ntaub ntawv rau cov ntawv sau histidine thiab nws cov neeg nyob sib ze tau siv kom tau txais qhov txhais tau tias SASA rau txhua tus qauv.Tus txheeb ze cov kuab tshuaj nkag tau (RSA) rau txhua histidine tau xam los ntawm kev faib tus nqi SASA kiag li los ntawm qhov muaj peev xwm tshaj plaws ntawm qhov chaw seem uas muaj rau cov kuab tshuaj.Tag nrho cov histidines ces muab cais raws li zais yog qhov txhais tau tias RSA qis dua 20%, txwv tsis pub raug 56.
Cov ntaub ntawv nyoos tau txais hauv DDA hom tau tshawb fawb siv Proteome Discoverer (v2.5) lossis MSfragger (Fragpipe v15.0) hauv qhov tsim nyog SwissProt txheeb xyuas cov ntaub ntawv protein uas muaj cov kab mob sib kis.Cov peptides yuav tsum tau ua tiav trypsin nrog ob qhov chaw cleavage uas ploj lawm, carbamoyl methylation raws li kev hloov kho ruaj khov thiab methionine oxidation raws li kev hloov pauv hloov pauv.Precursor thiab fragment hnyav kam rau ua tau teem rau 10 ppm thiab 0.02 Da (MS2 Orbitrap), feem. Cov kab mob kis tau raug tshem tawm, thiab cov proteins raug lim kom tau txais qhov kev tshawb pom tsis tseeb ntawm <1%. Cov kab mob kis tau raug tshem tawm, thiab cov proteins raug lim kom tau txais qhov kev tshawb pom tsis tseeb ntawm <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэфафициент < ложнент < ложне %. Cov kab mob sib kis tau raug muab tshem tawm thiab cov proteins lim kom muab qhov ntsuas tsis tseeb ntawm <1%.去除污染物命中，过滤蛋白质以获得 <1%的错误发现率. <1% ntawm cov ntaub ntawv. Попадания загрязняющих веществ были удалены, а белки отфильтрованы для достижения уровня ложных <%. Cov kab mob sib kis tau raug tshem tawm thiab cov protein lim kom ua tiav qhov tsis zoo ntawm <1%.Rau kev soj ntsuam kom muaj nuj nqis yam tsis siv cov ntawv sau, cov ntsiab lus muaj protein ntau los ntawm peb qhov kev rov ua dua tshiab tau siv.Protein subcellular localization tsom xam tau ua los ntawm Gene Ontology (GO) tsom xam los ntawm DAVID Bioinformatics Resources, MitoCarta 3.0 thiab cov ntaub ntawv khaws cia thiab luam tawm los ntawm pawg Alice Ting.Daim ntawv qhia roob hluav taws tau los ntawm Perseus (v1.6.15.0). Protein abundance fold hloov tau raug kuaj xyuas qhov tseem ceeb ntawm kev siv ob sab t-test, thiab cov protein hits tau txheeb xyuas nrog kev hloov pauv ntau> 2 (tshwj tsis yog hais tias lwm yam) thiab p tus nqi <0.05. Protein abundance fold hloov tau raug kuaj xyuas qhov tseem ceeb ntawm kev siv ob sab t-test, thiab cov protein hits tau txheeb xyuas nrog kev hloov pauv ntau> 2 (tshwj tsis yog hais tias lwm yam) thiab p tus nqi <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 cov ntsiab lus hloov pauv tau raug kuaj xyuas qhov tseem ceeb ntawm kev siv ob-tailed t-test, thiab cov protein sib tw tau txheeb xyuas nrog cov ntsiab lus hloov>2 (tshwj tsis yog sau tseg) thiab tus nqi <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，并确定蛋白质员咭的。使用 t 检验 蛋白质 蛋白质 倍 数 的 的 统计 统计 并 并 确定 命 命 的 的 的 丰度 变化 变化 变化 变化 2 (另 有) 和 p 值 值 <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. Kev txheeb xyuas qhov tseem ceeb ntawm kev hloov pauv hauv cov ntsiab lus ntawm cov protein tau sim siv ob-tailed t-test, thiab cov protein sib tw tau txiav txim siab rau cov ntsiab lus hloov > 2 (tshwj tsis yog qhia tawm) thiab p-tus nqi <0.05.Kev soj ntsuam kev sib cuam tshuam ntawm cov protein tau ua tiav siv GO tsom xam nrog rau cov ntaub ntawv String.
Peb qhov kev hloov pauv roj ntsha tau ua tiav nrog cov txiaj ntsig zoo sib xws.Kev txheeb cais tau ua tiav nrog GraphPad Prism (GraphPad software) thiab cov phiaj xwm hluav taws xob tau tsim nrog Perseus (v1.6.15.0).Txhawm rau sib piv ob pawg, p-values ​​tau txiav txim siab siv ob-tailed Student's t-test.Tsuas yog singleton proteins uas tau txheeb xyuas tsawg kawg ob zaug hauv pawg sim tau suav nrog hauv cov phiaj xwm hluav taws xob, thiab cov txiaj ntsig uas ploj lawm hauv pawg tswj hwm tau hloov nrog Perseus los ntawm kev faib ib txwm ua kom cov p-value suav nrog.Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.Hauv kev tshuaj ntsuam xyuas proteomic rau kev txheeb xyuas kev txheeb cais, qhov ntau ntawm cov proteins uas tshwm sim hauv tsawg kawg yog ob qho kev hloov pauv lom neeg tau khaws cia.Cov txheej txheem txheeb cais tsis tau siv los txiav txim siab ua ntej tus qauv loj.Cov kev sim tsis yog random.Cov kws tshawb fawb tsis tau dig muag rau cov dej num thaum lub sij hawm sim thiab ntsuam xyuas cov txiaj ntsig.
Yog xav paub ntxiv txog kev tsim qauv, saib Nature Research Report abstract linked to this article.
Cov ntaub ntawv loj spectrometry tau txais nyob rau hauv txoj kev tshawb no tau xa mus rau ProteomeXchange Consortium ntawm iProX57 tus khub repository nyob rau hauv dataset ID PXD034811 (PDPL-MS dataset).Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.Kab lus no muab cov ntaub ntawv qub.
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