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Hoʻohana nui ʻia nā ʻano hoʻopaʻa inoa pili Enzymatic e pili ana i nā esters i hoʻāla ʻia a i ʻole radical phenoxy no ka palapala ʻana i nā proteomes subcellular a me nā mea pili pūmua i loko o nā cell ola.Eia nō naʻe, ʻoi aku ka liʻiliʻi o nā esters i hoʻāla ʻia, ka hopena i ka radius lepili ākea, a hiki i nā radical phenoxy i hana ʻia e ka lāʻau peroxide ke hoʻopilikia i nā ala redox.Ma ʻaneʻi, hōʻike mākou i kahi ala pili i ka hoʻopili ʻana i ka photoactivation (PDPL) i hoʻomohala ʻia e ka hoʻopili genetically i ka miniSOG photosensitizer protein i kahi protein hoihoi.Hoʻomaka ʻia e ke kukui uliuli a hoʻomalu ʻia e ka manawa hoʻolaha, hoʻokumu ʻia ka oxygen singlet a laila hoʻoholo ʻia ka lepili ʻana o nā koena histidine e ka aniline probe.Hōʻike mākou i kona kūpaʻa kiʻekiʻe ma o ka palapala proteome kikoʻī organelle.ʻO ka hoʻohālikelike ʻaoʻao ʻaoʻao o PDPL me TurboID e hōʻike ana i kahi uhi proteomic kikoʻī a piha o PDPL.Ma hope aʻe, ua noi mākou i ka PDPL i ka coactivator transcriptional pili i ka maʻi BRD4 a me E3 Parkin ligase a loaʻa i nā mea pili i ʻike ʻole ʻia.Ma ka overexpression screening, ʻelua mau mea ʻike ʻole ʻia, Ssu72 a me SNW1, i ʻike ʻia no Parkin, nona ka hoʻohaʻahaʻa ʻia e ke ala ubiquitination-proteasome.
ʻO ka hōʻike pololei ʻana o nā ʻupena protein e hoʻokumu i nā kaʻina cellular kumu.No laila, ʻo ka palapala ʻāina spatiotemporal pololei loa o nā pilina pūmua e hāʻawi i kahi kumu molekala no ka wehewehe ʻana i nā ala olaola, pathology maʻi, a hoʻopau i kēia mau pilina no nā kumu therapeutic.No kēia hopena, makemake nui ʻia nā ala e hiki ai ke ʻike i nā pilina kino i loko o nā cell ola a i ʻole nā ​​kikoʻī.Ua hoʻohana mua ʻia ʻo Affinity Purification Mass Spectrometry (AP-MS) e ʻike i nā hoa pili o nā protein of interest (POI).Me ka hoʻomohala ʻana o nā ʻano proteomic quantitative, ua hoʻokumu ʻia ʻo Bioplex3.0, ka waihona nui loa o nā pūnaewele protein e pili ana i ka AP-MS.ʻOiai he ikaika loa ka AP-MS, ʻo ka lysis cell a me nā ʻanuʻu hoʻoheheʻe i loko o ka workflow e pili ana i nā pilina paʻa nāwaliwali a transient a hoʻolauna i nā mea kiʻi post-lysis e like me nā hoa pili hoʻopunipuni i nele i ka compartmentalization ma mua o ka lysis.
No ka hoʻoponopono ʻana i kēia mau pilikia, ua hoʻomohala ʻia nā ʻakika amino kūlohelohe (UAA) me nā hui crosslinking a me nā paepae enzymatic kokoke i ka lepili (PL) (e laʻa me APEX a me BioID)5.ʻOiai ua hoʻohana maikaʻi ʻia ke ʻano UAA i nā hiʻohiʻona he nui a hāʻawi i ka ʻike e pili ana i nā mea hoʻopili protein pololei, koi ʻia ka hoʻonui ʻana i ka pūnaewele hoʻokomo UAA.ʻO ka mea nui, ʻo ia ke ʻano hōʻailona stoichiometric i nele i ka hoʻohuli catalytic o nā hanana lepili.ʻO ka ʻokoʻa, hoʻohui i nā ʻano PL enzymatic, e like me ke ʻano BioID, hoʻohui i ka ligase biotin i hana ʻia i POI7, a laila e hoʻāla i ka biotin e hana i ka biotinyl-AMP ester intermediate.No laila, hoʻolalelale ka enzyme a hoʻokuʻu i kahi "cloud" biotin i hoʻāla ʻia e hōʻailona i nā koena lysine proximal.Eia naʻe, koi ʻo BioID ma mua o 12 mau hola e loaʻa ai kahi hōʻailona hōʻailona kūpono, kahi e pale ai i ka hoʻohana ʻana me ka hoʻonā kino.Me ka hoʻohana ʻana i ka hoʻomohala kuhikuhi ʻia e pili ana i ka hōʻike hū, ua hoʻolālā ʻia ʻo TurboID e pili ana i ka BioID i ʻoi aku ka maikaʻi, e ʻae i ka hoʻopaʻa inoa ʻana me ka biotin i loko o 10 mau minuke, e ʻae i nā kaʻina hana ikaika e aʻo ʻia.Ma muli o ka ikaika loa o TurboID a ua lawa nā pae biotin endogenous no ka hōʻailona haʻahaʻa haʻahaʻa, lilo ka lepili hope i mea pilikia ke koi ʻia ka hoʻonui ʻia ʻana a me ka hoʻopaʻa ʻana i ka manawa e ka hoʻohui ʻana i ka biotin exogenous.Eia kekahi, ʻaʻole maikaʻi ka hana ʻana o nā esters i hoʻāla ʻia (t1/2 ~ 5 min), hiki ke alakaʻi i kahi radius lepili nui, ʻoi aku ma hope o ka saturation o nā protein e pili ana me ka biotin 5. Ma kahi ʻano ʻē aʻe, ka hui pū ʻana o ka engineered ascorbate peroxidase (ie biotin- phenol radicals a hiki i ka lepili pūmua i loko o hoʻokahi minute9,10. Hoʻohana nui ʻia ʻo APEX e ʻike i nā proteomes subcellular, membrane protein complexes, a me cytosolic signaling protein complexes11,12. Eia nō naʻe, hiki i ka pono o nā kiʻekiʻe kiʻekiʻe o nā peroxide ke hoʻopili i nā protein redox a i ʻole nā ​​ala, e hoʻopilikia ai. nā hana kelepona.
No laila, ʻo kahi ala hou e hiki ai ke hoʻohua i nā ʻano mea hoʻopaʻa lepili-radius reactive me ka pololei spatial a me ke kino me ka ʻole o ka hoʻopilikia nui ʻana i nā ala kelepona he mea hoʻohui koʻikoʻi i nā kaʻina hana. Ma waena o nā ʻano reactive, ua hoʻāla ka oxygen singlet i ko mākou manaʻo ma muli o kona ola pōkole a me ka palena o ka diffusion radius (t1/2 <0.6 µs i nā cell)13. Ma waena o nā ʻano reactive, ua hoʻāla ka oxygen singlet i ko mākou manaʻo ma muli o kona ola pōkole a me ka palena o ka diffusion radius (t1/2 <0.6 µs i nā cell)13. LIKE LIKE LIKE LIKE Ma waena o nā ʻano hana, ua huki ka oxygen singlet i ko mākou nānā ʻana ma muli o kona ola pōkole a me ka radius diffusion liʻiliʻi (t1/2 <0.6 µs i nā cell)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）走起们起们起们起中可能他們。 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 времени жизни и ограница / 6 злендный кислород. Ma waena o nā ʻano hana, hoʻokaʻawale ka oxygen singlet i ko mākou manaʻo no ka pōkole o kona ola a me ka palena o ka diffusion radius (t1/2 <0.6 μs i nā cell).Ua hōʻike ʻia ʻo Singlet oxygen e hoʻoheheʻe i ka methionine, tyrosine, histidine a me tryptophan, e lilo ia i polar 14,15 no ka hoʻopili ʻana i ka amine a i ʻole thiol e pili ana i nā probes16,17.ʻOiai ua hoʻohana ʻia ka oxygen singlet e hoʻopaʻa inoa i ka compartment subcellular RNA, ʻaʻole i ʻike ʻia nā hoʻolālā no ka hoʻihoʻi hou ʻana i nā māka kokoke POI endogenous.Ma ʻaneʻi, hōʻike mākou i kahi kahua i kapa ʻia ʻo photoactivation-dependent proximity labeling (PDPL), kahi e hoʻohana ai mākou i ke kukui polū e hoʻomālamalama i nā POI i hui pū ʻia me kahi miniSOG photosensitizer a hoʻoulu i ka hanauna oxygen singlet e hoʻoneʻe i nā koena proximal, a ukali ʻia e nā hoʻololi i loko o ka amine e hoʻoheheʻe i nā ʻimi kemika i loko. nā mea ola waena..Ua hoʻāʻo mākou i kahi pūʻulu o nā noiʻi kemika e hoʻonui i ka kikoʻī kikoʻī a ʻike ʻia nā wahi hoʻololi me ka hoʻohana ʻana i kahi workflow proteomic open.ʻO ka hoʻohālikelike ʻaoʻao ʻaoʻao o PDPL me TurboID e hōʻike ana i kahi uhi proteomic kikoʻī a piha o PDPL.Ua hoʻohana mākou i kēia ala i nā hōʻailona kikoʻī o organelle o ka proteome subcellular a me ka ʻike proteome maʻamau o nā hoa paʻa no ka protein epigenetic regulatory protein BRD4 pili i ka maʻi kanesa a me ka E3 ligase Parkin pili i ka maʻi o Parkinson, ka mea i hōʻoia i ka ʻike a me kahi pūnaewele ʻike ʻole o ka protein. nā pilina..ʻO ka hiki i ka PDPL ke ʻike i nā substrates E3 i loko o nā paʻakikī protein nui e hōʻike ana i kahi kūlana e koi ʻia ai ka ʻike ʻana i nā mea hoʻopaʻa ʻole.ʻElua mau papa parkin ʻike ʻole ʻia e ubiquitination-proteasome i hoʻopaʻa ʻia ma kahi.
Photodynamic therapy (PDT) 19 a me ka chromophore-assisted laser inactivation (CALI)20, kahi e hoʻoulu ai ka hoʻomālamalama māmā me nā photosensitizers i ka oxygen singlet, hiki ke hoʻopau i nā protein target a i ʻole ke kumu o ka make cell.No ka mea, ʻo ka oxygen singlet he mea hoʻoikaika nui loa me kahi mamao diffusion theoretical ma kahi o 70 nm, hiki ke hoʻomalu ʻia ka hoʻokahe ʻana i kaupalena ʻia a puni ka photosensitizer.Ma muli o kēia manaʻo, ua hoʻoholo mākou e hoʻohana i ka oxygen singlet no ka hoʻokō ʻana i ka hōʻailona kokoke ʻana i nā paʻakikī protein i nā cell ola.Ua hoʻomohala mākou i kahi ala chemoproteomic PDPL e hoʻokō ai i ʻehā mau hana: (1) e hoʻolalelale i ka hana o ka oxygen singlet ikaika e like me ka PL enzymatic approach;(2) hāʻawi i ka lepili i hoʻoholo ʻia i ka manawa ma ka hoʻomaka māmā;(3) ma ka hoʻololi ʻana (4) Hōʻalo i ka hoʻohana ʻana i nā cofactor endogenous (e like me ka biotin) e hōʻemi i ka hope, a i ʻole e hoʻohana i nā reagents exogenous hoʻopilikia loa (e like me peroxides) e hōʻemi i ka ʻike ʻana o ke kelepona i ke koʻikoʻi o ke kaiapuni.
Hiki ke hoʻokaʻawale ʻia nā Photosensitizers i ʻelua ʻāpana e like me nā fluorophores paona molekala liʻiliʻi (e laʻa i ka rose bengal, methylene blue)22 a me nā protein liʻiliʻi i hoʻopaʻa ʻia ma ke ʻano genetically (eg miniSOG, KillerRed)23.No ka hoʻokō ʻana i kahi hoʻolālā modular, hoʻomohala mākou i ka pae mua o ka PDPL ma o ka hoʻohui ʻana i nā protein photosensitizer (PS) i POI24,25 (Figure 1a).Ke hoʻomālamalama ʻia me ke kukui polū, hoʻoneʻe ka oxygen singlet i nā koena nucleophilic amino acid proximal, ka hopena i kahi polarity umpolung i electrophilic a hiki ke hana hou me ka amine probe nucleophiles16,17.Hoʻolālā ʻia ka ʻimi noiʻi me kahi paʻa alkyne e ʻae i ka chemistry kaomi a huki i lalo no ka hōʻailona LC/MS/MS.
Hōʻike hoʻohālike o ka hōʻailona ʻana i nā paʻakikī protein i hoʻopili ʻia e miniSOG.Ke ʻike ʻia i ke kukui polū, hoʻopuka nā cell e hōʻike ana i ka miniSOG-POI i ka oxygen singlet, kahi e hoʻololi ai i nā protein pili akā ʻaʻole nā ​​​​protein paʻa ʻole.Hoʻopili ʻia nā huahana waena o ka photooxidation e nā lepili relay o ka ʻimi chemical amine e hana i nā hoʻohui covalent.ʻO ka pūʻulu alkynyl ma ka ʻimi kemika hiki iā ʻoe ke kaomi kemika no ka hoʻonui ʻana ma ka huki-iho a ukali ʻia e ka helu LC-MS/MS.b Ka hoʻonohonoho kemika o nā ʻimi amine 1-4.c Lunamaka'āinana fluorescent gel ka nānā 'ana o ka mitochondrial localized miniSOG-mediated proteomic markers me ka hoʻohana 'ana i ka probes 1-4 a me ka helu pili e pili ana i ka gel densitometry.Ua loiloi ʻia ka ratio hōʻailona-i-background o nā ʻimi kemika me ka hoʻohana ʻana i nā hoʻokolohua hoʻokele maikaʻi ʻole me ka ʻole o ke kukui polū a i ʻole ka hoʻohana ʻana i nā cell HEK293T me ka ʻole o ka hōʻike miniSOG.n = 2 mau laʻana kūʻokoʻa olaola.Hōʻike kēlā me kēia kiko i kahi kope olaola.d Ka ʻike makamaka a me ka helu ʻana o PDPL me ka hoʻohana ʻana i ka probe 3 i ʻike ʻia ma ke alo a i ʻole ka ʻole o nā ʻāpana PDPL i hōʻike ʻia e like me c.n = 3 mau laʻana kūʻokoʻa olaola.Hōʻike kēlā me kēia kiko i kahi kope olaola.Hōʻike nā laina waena a me nā ʻumiʻumi i ka mean a me ± deviation maʻamau.CBB: Coomassie Brilliant Blue.e Kiʻi kiʻi confocal o ka oxygen singlet me ka ʻulaʻula nui Si-DMA stain.ʻO ka pā unahi: 10 µm.Ua hana hou ʻia nā hoʻokolohua gel imaging a me confocal ma kahi o ʻelua mau hopena me nā hopena like.
Ua hoʻāʻo mua mākou i ka hiki o nā photosensitizers makua miniSOG26 a me KillerRed23, i hōʻike paʻa ʻia ma HEK293T, e hoʻopili i ka lepili propargylamine o ka proteome ma ke ʻano he ʻimi kemika (Supplementary Fig. 1a).Hōʻike ka hōʻike ʻana o ka gel fluorescence ua hoʻokō ʻia ka hōʻailona proteome holoʻokoʻa me ka hoʻohana ʻana i ka miniSOG a me ka irradiation kukui uliuli, ʻoiai ʻaʻole i ʻike ʻia kahi huahana lepili ʻike ʻia me KillerRed.No ka hoʻomaikaʻi ʻana i ka ratio hōʻailona-i-background, a laila hoʻāʻo mākou i kahi pūʻulu o nā ʻimi kemika i loaʻa i ka aniline (1 a me 3), propylamine (2), a i ʻole benzylamine (4).Ua ʻike mākou he ʻoi aku ka kiʻekiʻe o nā pūnaewele HEK293T i hoʻohālikelike ʻia me ke kukui uliuli ʻole, ma muli paha o ka endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27. Ua hāʻawi ʻo Aniline-based chemical probes 1 a me 3 i ka kikoʻī ʻoi aku ka maikaʻi, me HEK293T e hōʻike mau ana i ka miniSOG i ka mitochondria e hōʻike ana i ka hoʻonui ʻana o ka hōʻailona he 8-fold no ka probe 3, aʻo ka probe 2 i hoʻohana ʻia i ke ʻano RNA-labeling method CAP-seq wale nō e hōʻike ana ~2.5- ʻO ka piʻi ʻana o ka hōʻailona paʻi, ma muli paha o nā makemake ʻokoʻa ma waena o ka RNA a me ka protein (Fig. 1b, c). Ua hāʻawi ʻo Aniline-based chemical probes 1 a me 3 i ka kikoʻī ʻoi aku ka maikaʻi, me HEK293T e hōʻike mau ana i ka miniSOG i ka mitochondria e hōʻike ana i ka hoʻonui ʻana o ka hōʻailona he 8-fold no ka probe 3, aʻo ka probe 2 i hoʻohana ʻia i ke ʻano RNA-labeling method CAP-seq wale nō e hōʻike ana ~2.5- ʻO ka piʻi ʻana o ka hōʻailona paʻi, ma muli paha o nā makemake ʻokoʻa ma waena o ka RNA a me ka protein (Fig. 1b, c).Ua hōʻike ʻo Aniline-based chemical probes 1 a me 3 i kahi kikoʻī ʻoi aku ka maikaʻi: HEK293T, ka mea e hōʻike paʻa i ka miniSOG i ka mitochondria, hōʻike ʻoi aku ma mua o ka piʻi ʻana o 8-fold i ka hōʻailona no ka probe 3, aʻo ka probe 2, i hoʻohana ʻia i ke ʻano lepili RNA CAP-seq, wale nō. hōʻike i ka ~ 2.5-fold ka piʻi ʻana o ka hōʻailona, ​​ma muli paha o nā makemake reactivity like ʻole ma waena o RNA a me ka protein (Fig. 1b, c).基于 苯胺 的 和化学 具有 a 具有, he pink93t 探针 增加 表达 稳定 稳定 稳定 表达 表达 方法 探针 的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）。基于基于 的 化学化学 具有化学 具有特异性 的 具有, Ankd 在特异性 表达 表达 增加 MPAN-8-2. 仅 ~ ~ 2.) -倍信号增加，可能是由于RNAUa ʻoi aku ka maikaʻi o ka ʻike kikoʻī ʻo Aniline-based chemical probes 1 a me 3, HEK293T i hōʻike paʻa i ka miniSOG i ka mitochondria, a ʻo ka probe 3 i ʻoi aku ma mua o 8-fold ka piʻi ʻana o ka hōʻailona, ​​aʻo ka probe 2 no ke ʻano hōʻailona CAP-seq RNA i hōʻike ʻia he ~2.5-fold wale nō.i ka hōʻailona, ​​ma muli paha o nā manaʻo like ʻole ma waena o RNA a me ka protein (Fig. 1b, c).Eia hou, ua ho'āʻoʻia kaʻimi 3 isomers a me ka hydrazine probes (probes 5, 6, 7), e hōʻoiaʻiʻo ana i ka optimization o ka probe 3 (Supplementary Fig. 1b, c).Pēlā nō, hōʻike ʻia ka nānā ʻana o ka fluorescence in-gel i nā ʻāpana hoʻokolohua ʻē aʻe: ka lōʻihi o ka hawewe irradiation (460 nm), ka ʻimi noiʻi kemika (1 mM), a me ka manawa irradiation (20 min) (Supplementary Fig. 2a–c).ʻO ka haʻalele ʻana i kekahi ʻāpana a i ʻole ka pae i ka protocol PDPL i hopena i ka hoʻohuli ʻana i ka hōʻailona koʻikoʻi i ke kua (Fig. 1d).ʻO ka mea nui, ua hōʻemi nui ʻia ka hōʻailona protein ma ke alo o ka sodium azide a i ʻole trolox, i ʻike ʻia e kinai i ka oxygen singlet.ʻO ka hiki ʻana o D2O, ka mea i ʻike ʻia e hoʻopaʻa i ka oxygen singlet, hoʻonui i ka hōʻailona hōʻailona.No ka noiʻi ʻana i ka hāʻawi ʻana o nā ʻano oxygen reactive ʻē aʻe i ka lepili, ua hoʻohui ʻia ka mannitol a me ka huaora C e hoʻokumu i ka hydroxyl a me ka superoxide radical scavengers, kēlā me kēia, 18, 29, akā ʻaʻole i ʻike ʻia e hōʻemi i ka lepili.ʻO ka hoʻohui ʻana o H2O2, ʻaʻole naʻe i hoʻomālamalama ʻia, ʻaʻole i hopena i ka lepili (Supplementary Fig. 3a).Ua hōʻoia ʻo Fluorescence singlet oxygen imaging me Si-DMA probes i ka loaʻa ʻana o ka oxygen singlet i ka uea HEK293T-miniSOG, akā ʻaʻole i ka uea HEK293T kumu.Eia kekahi, ʻaʻole hiki i ka mitoSOX Red ke ʻike i ka hana superoxide ma hope o ka hoʻomālamalama ʻana (Fig. 1e a me ka Fig.Ua loiloi ʻia ka Cytotoxicity o PDPL me ka hoʻomālamalama ʻana o ka kukui polū a me nā ʻimi kemika, a ʻaʻole i ʻike ʻia ka cytotoxicity nui (Supplementary Fig. 4a).
I mea e aʻo ai i ka mīkini lepili a hiki i ka ʻike proteomic o nā paʻakikī protein me ka hoʻohana ʻana i ka LC-MS/MS, pono mākou e hoʻoholo mua i nā amino acids i hoʻololi ʻia a me ka delta mass o nā lepili probe.ʻO ka methionine, histidine, tryptophan a me tyrosine ua hōʻike ʻia e hoʻololi ʻia e ka oxygen singlet14,15.Hoʻohui mākou i ke kahe hana TOP-ABPP31 me ka huli wehe ʻole i hāʻawi ʻia e ka FragPipe computing platform ma muli o MSFragger32.Ma hope o ka hoʻololi ʻana o ka oxygen singlet a me ka hōʻailona ʻana i ke kinikini, ua hoʻokō ʻia ke kaomi kemika me ka hoʻohana ʻana i kahi lepili hoʻemi biotin i loaʻa kahi loulou hiki ke hoʻokaʻawale ʻia, a ukali ʻia e neutravidin stretching a me trypsin digestion.ʻO ka peptide i hoʻololi ʻia, i hoʻopaʻa ʻia i ka resin, ua kiʻi ʻia no ka nānā ʻana o LC-MS / MS (Figure 2a a me ka ʻikepili Hoʻohui 1).Nui nā hoʻololi i loaʻa i loko o ka proteome me ka ʻoi aku o 50 peptide map (PSM) pāʻani i helu ʻia (Fig. 2b).ʻO ka mea kupanaha, ʻike wale mākou i ka hoʻololi ʻana o ka histidine, ma muli paha o ke kiʻekiʻe o ka reactivity o ka histidine oxidized i ka aniline probes ma mua o nā amino acids.E like me ka mea i paʻi ʻia o ka histidine oxidation e ka oxygen singlet, 21,33 ka mea i manaʻo ʻia ʻo ka delta-mass structure o +229 Da e pili ana i ka hoʻohui ʻana o ka probe 3 me 2-oxo-histidine ma hope o ʻelua oxidations, aʻo +247 Da ka huahana hydrolysis. o +229 Da (Hoʻohui Fig. 5).ʻO ka loiloi o ka MS2 spectrum i hōʻike i ka hilinaʻi kiʻekiʻe o ka ʻike ʻana i ka hapa nui o nā ion y a me b, me ka ʻike ʻana i nā ʻāpana ʻāpana i hoʻololi ʻia (y a me b) (Fig. 2c).ʻO ka nānā ʻana i ka ʻatikala o ke kaʻina kūloko o nā histidines i hoʻololi ʻia e PDPL i hōʻike ʻia kahi makemake kumu kūpono no nā koena hydrophobic liʻiliʻi ma ± 1 kūlana (Supplementary Fig. 4b).Ma ka awelika, ua ʻike ʻia nā histidines 1.4 i kēlā me kēia protein, a ua hoʻoholo ʻia nā pūnaewele o kēia mau māka e ka solvent accessible surface area (SASA) a me ka hoʻopili ʻana i ka loaʻa ʻana o ka solvent (RSA) (Supplementary Fig. 4c,d).
He kaʻina hana ʻole no ke aʻo ʻana i ke koena koho me ka hoʻohana ʻana i ka platform computing FragPipe i hoʻohana ʻia e MSFragger.Hoʻohana ʻia nā loulou Cleavable ma Click chemistry e ʻae i ka photocleavage o nā peptides i hoʻololi ʻia mai ka resin streptavidin.Ua hoʻomaka ʻia kahi ʻimi ākea e ʻike i nā hoʻololi he nui, a me nā koena pili.b E hāʻawi i ka nui o nā hoʻololi i hana ʻia i loko o ka proteome.Palapala peptide PSM.c MS2 spectral annotation o ka histidine paena i hoololiia me ka probe 3. Ma ke ano he la'ana, ua ho'ohui 'ia ka hopena covalent me ka probe 3 +229.0938 Da i ka waikawa amino i ho'ololi 'ia.d Hoʻohana ʻia ka hoʻololi ʻana no ka hoʻāʻo ʻana no nā māka PDPL.Ua hoʻololi ʻia ʻo PRDX3 (H155A, H225A) a me PRDX1 (H10A, H81A, H169A) me nā plasmids ʻano hihiu no ka ʻike anti-Flag.e Hoʻopili ʻia ka peptide synthetic me ka miniSOG i hoʻomaʻemaʻe ʻia i mua o ka probe 3 a me nā huahana pili me Δm +247 a me +229 i ʻike ʻia i ka spectrum LC-MS.f In vitro protein-to-protein launa pū ʻana me ka miniSOG-6xHis-tag a me anti-6xHis antibody.Antibiotin (streptavidin-HRP) a me anti-mouse Western blot analysis of miniSOG-6xHis/anti-6xHis antibody complexes i kapa ʻia me ka probe 3, e pili ana i ka manawa o ka ʻike ʻana i ka mālamalama.Hōʻike ʻia nā lepili no nā protein pākahi i ke kaumaha molekala like: LC kaulahao māmā antibody, HC antibody kaulahao kaumaha.Ua hana hou ʻia kēia mau hoʻokolohua ma ka liʻiliʻi ʻelua me nā hopena like.
No ka hōʻoia biochemical o ka pae lepili, ua hoʻololi ʻia ʻo PRDX3 a me PRDX1 i ʻike ʻia e ka mass spectrometry mai ka histidine a i ka alanine a hoʻohālikelike ʻia me nā ʻano ʻāhiu i ka hoʻololi ʻana.Ua hōʻike nā hopena PDPL i ka hoʻohaʻahaʻa nui ʻana o ka hoʻololi ʻana i ka lepili (Fig. 2d).I kēia manawa, ua hoʻohui ʻia nā ʻāpana peptide i ʻike ʻia i ka ʻimi ākea a hana ʻia i loko o ka vitro me ka miniSOG i hoʻomaʻemaʻe ʻia i mua o ka probe 3 a me ke kukui polū, e hāʻawi ana i nā huahana me kahi hoʻololi nui o +247 a me +229 Da ke ʻike ʻia e LC-MS (Fig). . 2e).).No ka hoʻāʻo ʻana inā hiki ke hoʻopaʻa inoa ʻia nā protein proximal i loko o ka vitro i ka pane ʻana i ka miniSOG photoactivation, ua hoʻolālā mākou i kahi hōʻike hoʻohālikelike kūlohelohe e ka pilina ma waena o ka miniSOG-6xHis protein a me kahi anti-His monoclonal antibody in vitro (Figure 2f).Ma kēia ho'āʻo, manaʻo mākou i ka lepili kokoke i nā kaulahao kaumaha a me nā māmā me ka miniSOG.ʻO kaʻoiaʻiʻo, anti-mouse (ʻike i nā kaulahao kaumaha a me ka māmā o ka anti-6xHis-labeled antibody) a me streptavidin Western blots i hōʻike i ka biotinylation ikaika o nā kaulahao kaumaha a māmā.ʻO ka mea nui, ua ʻike mākou i ka miniSOG autobiotinylation ma muli o ka 6xHis tag a me nā loulou i waena o nā kaulahao māmā a me ke kaumaha, e pili ana paha i ka lua i wehewehe mua ʻia ma waena o lysine a me 2-oxo-histidine proximal pane.I ka hopena, hoʻoholo mākou e hoʻololi ʻo PDPL i ka histidine ma kahi ʻano pili pili.
ʻO kā mākou pahuhopu aʻe, ʻo ia ke ʻano o ka subcellular proteome e hoʻāʻo i ka kikoʻī o ka hōʻailona in situ.No laila, ua hōʻike paʻa mākou i ka miniSOG i loko o ka nucleus, mitochondrial matrix, a i ʻole membrane ER waho o nā pūnaewele HEK293T (Fig. 3a).Ua hōʻike ʻia ka hōʻike ʻana o ka gel fluorescence i ka nui o nā kaula i hoʻopaʻa ʻia ma ʻekolu mau wahi subcellular a me nā ʻano lepili like ʻole (Fig. 3b).Ua hōʻike ʻia ka ʻike kiʻi fluorescence i ka kikoʻī kiʻekiʻe o PDPL (Fig. 3c).Ua ukali ʻia ka holo kaʻina hana PDPL e kaomi i nā hopena me nā dyes rhodamine e wehewehe i nā proteomes subcellular me ka hoʻohana ʻana i ka microscopy fluorescence, a ua colocalized nā hōʻailona PDPL me DAPI, mitochondrial trackers, a i ʻole ER trackers, e hōʻoia ana i ka hilinaʻi kiʻekiʻe o PDPL.No nā wahi organelle ʻekolu, ʻo ka hoʻohālikelike ʻaoʻao ʻaoʻao o PDPL me TurboID me ka hoʻohana ʻana i avidin western blot i hōʻike ʻia ua hōʻailona ʻia ʻo PDPL i hoʻohālikelike ʻia i kā lākou mau mana.Ma lalo o nā kūlana PDPL, ua ʻike ʻia nā pūʻulu i hōʻailona ʻia, e hōʻike ana i nā protein i kapa ʻia ʻo PDPL (Supplementary Fig. 6a-d).
kahi hōʻike Schematic o ka miniSOG-mediated organelle-specific proteome labeling.Hoʻolālā ka miniSOG i ka matrix mitochondrial ma o ka hui ʻana i ka N-terminal 23 amino acids o ke kanaka COX4 (mito-miniSOG), ka nucleus ma o ka hui ʻana iā H2B (nucleus-miniSOG), a me Sec61β ma o ka ʻaoʻao cytoplasmic o ka membrane ER (ER-miniSOG). ).ʻO nā hōʻailona ke kiʻi gel, ke kiʻi confocal, a me ka spectrometry lehulehu.b Nā kiʻi ʻelele o ʻekolu mau kikoʻī PDPL kikoʻī organelle.ʻO CBB Coomassie ʻulaʻula uliuli.c Nā kiʻi huikau o HEK293T e hōʻike paʻa ana i ka miniSOG me nā ʻāpana subcellular like ʻole i ʻike ʻia e ka antibody i kapa ʻia V5 (ʻulaʻula).Hoʻohana ʻia nā māka subcellular no ka mitochondria a me ER ('ōmaʻomaʻo).Aia i ka PDPL workflow ka ʻike ʻana o ka miniSOG (melemele) i hoʻopaʻa ʻia i nā proteomes subcellular me ka hoʻohana ʻana i ka kemika kaomi Cy3-azide.ʻO ka pā unahi: 10 µm.d Nā ʻāpana pele o nā proteomes i hoʻopaʻa ʻia e PDPL i loko o nā ʻāpana like ʻole i helu ʻia e ka helu helu ʻole ʻia (n = 3 mau hoʻokolohua kūlohelohe kūʻokoʻa).Ua hoʻohana ʻia ka hoʻāʻo-t a ka Haumāna huelo ʻelua ma nā pā lua pele.Ua hoʻohana ʻia ka ʻano ʻāhiu HEK293T ma ke ʻano he mana maikaʻi ʻole. Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻokoʻa ʻokoʻa o ka ion. Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻokoʻa ʻokoʻa o ka ion. Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻelua o ka ʻokoʻa o ka ion).显着变化的蛋白质以红色突出显示（p < 0.05 和> 2 倍离子强度差异）。显着变化的蛋白质以红色突出显示（p < 0.05和> 2 Значительно измененные белки выделены красным цветом (p < 0,05 и > 2-кратная разница в ионной силе). Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa 2-fold i ka ikaika ionic).Hōʻike ʻia nā protein pili i ka HEK293T-miniSOG akā ʻaʻole koʻikoʻi no HEK293T i ka ʻōmaʻomaʻo.e Ka nānā 'ana i ke kiko'ī o nā waihona proteomic mai nā ho'okolohua d.Hōʻailona ʻia ka heluna nui o nā polokina koʻikoʻi ma kēlā me kēia organelle (nā kiko ʻulaʻula a me ka ʻōmaʻomaʻo) ma luna.Hōʻike nā histograms i nā protein i hoʻopaʻa ʻia i loko o nā organelles e pili ana i ka MitoCarta 3.0, ka loiloi GO a me A. Ting et al.kanaka.E hoʻokaʻawale i nā ʻikepili no ka mitochondria, nuclei, a me ER.Ua hana hou ʻia kēia mau hoʻokolohua ma ka liʻiliʻi ʻelua me nā hopena like.Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.
Hoʻoikaika ʻia e ka gel a me nā hopena kiʻi, ua hoʻohana ʻia ka helu helu ʻole e helu i ka proteome i ʻike ʻia i kēlā me kēia organelle (Supplementary Data 2).Ua hoʻohana ʻia ʻo HEK293T i hoʻololi ʻole ʻia ma ke ʻano he mana maikaʻi ʻole e unuhi i nā māka hope. Ua hōʻike ʻia ka nānā ʻana o ka lua pele i nā protein i hoʻonui nui ʻia (p <0.05 a me >2-fold ion intensity) a me nā protein singleton i loaʻa wale i nā laina miniSOG-expressing (Fig. 3d ʻulaʻula a me nā kiko'ōmaʻomaʻo). Ua hōʻike ʻia ka nānā ʻana o ka lua pele i nā protein i hoʻonui nui ʻia (p <0.05 a me >2-fold ion intensity) a me nā protein singleton i loaʻa wale i nā laina miniSOG-expressing (Fig. 3d ʻulaʻula a me nā kiko'ōmaʻomaʻo). 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). Ua hōʻike ʻia ka nānā ʻana o ka lua pele i nā protein i hoʻonui nui ʻia (p<0.05 a me > 2-fold ion intensity) a me nā protein hoʻokahi i loaʻa wale i nā laina miniSOG-expressing (Fig. 3d, nā kiko ʻulaʻula a me ka ʻōmaʻomaʻo).火山火山 a 分析 显示 poay 显着 蛋白质 (蛋白质 (p 7 图 蛋白质 强度) 红色 和 绿色点) 红色 和 (图 3 和 (图 3 和火山 火山出 分析 to 0 p <р5 图> 2 系 蛋白质 在于 (图 存 ...)))))))))))))))))))))))) 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). Ua hōʻike ʻia ka nānā ʻana o ka lua pele i nā protein i hoʻonui nui ʻia (p<0.05 a me >2x ionic ikaika) a me nā protein hoʻokahi aia wale nō ma ka laina hōʻike miniSOG (nā kiko ʻulaʻula a me ka ʻōmaʻomaʻo ma Fig. 3d).I ka hui pū ʻana i kēia mau ʻikepili, ua ʻike mākou i ka 1364, 461, a me 911 ka nui o ka nuklea, mitochondrial, a me ER o waho membrane protein, kēlā me kēia.No ke kālailai ʻana i ka pololei o ka organelle-localized PDPL, ua hoʻohana mākou i ka MitoCarta 3.0, Gene Ontology (GO), a me A. Ting et al.ua hoʻohana ʻia kahi data set8 no ka mitochondria, nucleus, a me ER e hoʻāʻo i ke kikoʻī o ka organelle o nā protein i ʻike ʻia, e pili ana i ka pololei o 73.4, 78.5, a me 73.0% (Fig. 3e).ʻO ka kikoʻī o PDPL e hōʻoia i ka PDPL he mea hana kūpono no ka ʻike ʻana i nā proteomes kikoʻī organelle.ʻO ka mea nui, ua hōʻike ʻia ka loiloi submitochondrial o nā protein mitochondrial i ʻike ʻia ua māhele nui ʻia ka proteome i hoʻopaʻa ʻia i loko o ka matrix a me ka membrane i loko (226 a me 106, kēlā me kēia), helu no 91.7% (362) o ka huina o nā protein mitochondrial i ʻike ʻia.ua hōʻoia hou ʻia kahi kiʻekiʻe kiʻekiʻe o PDPL (Supplementary Fig. 7a).Pēlā nō, hōʻike ʻia ka nānā ʻana subnuclear ua puʻunaue nui ʻia ka proteome i hopu ʻia i loko o ka nucleus, nucleoplasm, a me nucleolus (Supplementary Fig. 7b).Nukelea proteomic kālailai me ka nukelea localization hōʻailona peptide (3xNLS) hōʻike like pololei me ka H2B kūkulu (Supplementary Fig. 7c-h).No ka hoʻoholo ʻana i ka kikoʻī o ka māka PDPL, ua koho ʻia ka laminin nuklea A ma ke ʻano he pahele POI7 i ʻike ʻia.Ua ʻike ʻia ʻo PDPL he 36 mau protein i hoʻonui nui ʻia, ʻo ia ka 12 proteins (30.0% me ka lamin A) i hōʻike maikaʻi ʻia i ka lamin A i ʻōlelo ʻia e ka String database, me ka hapa kiʻekiʻe ma mua o ke ʻano BioID (122 proteins) 28 o 28. , 22.9 %) 7. Ua ʻike ʻia ka liʻiliʻi o nā protein i kā mākou ʻano, ma muli paha o ka liʻiliʻi ʻana o nā wahi lepili, i hiki ke hana ʻia e ka oxygen singlet ʻoi aku ka ikaika.Hōʻike ka hōʻike ʻana ʻo GO i ka nui o nā protein i ʻike ʻia i loko o ka nucleoplasm (26), nuclear membrane (10), nuclear membrane (9), a me nā nuklea pores (5).ʻO ka hui pū ʻana, ua helu ʻia kēia mau protein nuklea-localized no 80% o nā protein i hoʻonui ʻia, e hōʻike hou ana i ka kikoʻī o PDPL (Supplementary Fig. 8a-d).
Ma hope o ka hoʻokumu ʻana i ka hiki o PDPL e hana i ka hōʻailona kokoke ʻana i nā organelles, a laila hoʻāʻo mākou inā hiki ke hoʻohana ʻia ʻo PDPL e nānā i nā hoa paʻa POI.Ma keʻano kūikawā, ua ʻimi mākou e wehewehe i ka nānā ʻana i ka PDPL o nā protein cytosolic, i manaʻo ʻia he ʻoi aku ka paʻakikī ma mua o ko lākou mau membrane-localized counterparts ma muli o ko lākou ʻano ikaika.ʻO ka bromodomain a me ka extraterminal (BET) protein BRD4 ua huki mākou i ko mākou manaʻo no kāna kuleana nui i nā maʻi like ʻole 35, 36.ʻO ka paʻakikī i hana ʻia e BRD4 he coactivator transcriptional a me kahi pahuhopu therapeutic koʻikoʻi.Ma ka hooponopono ana i ka hoike ana o c-myc a me Wnt5a kumu transcription, BRD4 ua manaoia he kumu nui o ka acute myeloid leukemia (AML), myeloma lehulehu, Burkitt's lymphoma, colon cancer and inflammatory diseases37,38.Eia kekahi, ke kuhikuhi nei kekahi mau maʻi iā BRD4 e hoʻoponopono i ka transcription viral a me ka cellular, e like me ka papillomavirus, HIV, a me SARS-CoV-236,39.
No ka palapala ʻana i ka pilina BRD4 me ka hoʻohana ʻana iā PDPL, ua hui mākou i ka miniSOG me kahi isoform N- a i ʻole C-terminal pōkole o BRD4.Ua hōʻike ʻia nā hopena proteomic i kahi kiʻekiʻe o ka overlap ma waena o nā kūkulu ʻelua (Supplementary Fig. 9a).ʻO ka proteome nuklea i ʻike ʻia me ka miniSOG-H2B e uhi i ka 77.6% o nā protein e pili ana me BRD4 (Supplementary Fig. 9b).A laila, hoʻohanaʻia nā manawa likeʻole o ka hoʻomālamalamaʻana (2, 5, 10, 20 min) e hoʻololi i ka radius marker (Fig. 4a a me nāʻikepili hou 3).Manaʻo mākou ma nā photoperiods pōkole, e hoʻopaʻa inoa ʻo PDPL i nā hoa paʻa pololei, aʻo nā manawa lōʻihi e hoʻokomo ʻia nā protein i ʻike ʻia i ka wā pōkole o ka photoactivation a me nā pahuhopu indirect i nā paʻakikī lepili.ʻO kaʻoiaʻiʻo, ua loaʻa iā mākou ka hoʻopili ikaika ma waena o nā wahi manawa pili (84.6% no 2 a me 5 min; 87.7% no 5 a me 10 min; 98.7% no 10 a me 20 min) (Fig. 4b a me ka Fig 9c).Ma nā pūʻulu hoʻokolohua a pau, ʻaʻole mākou i ʻike i ka hōʻailona ponoʻī ʻo BRD4 wale nō, akā i kekahi mau pahuhopu i ʻike ʻia e like me MED1, CHD8, BICRA, NIPBL, SMC1A, a me HMGB1 i hōʻike ʻia ma ka waihona string.ʻO ka ikaika ionic o kēia mau pahuhopu e like me ka manawa hoʻolaha (Fig. 4c a me Supplementary Fig. 9d).ʻO ka loiloi GO o nā protein i ʻike ʻia i loko o ka hui 2-minuke i hōʻike ʻia ua ʻike ʻia nā protein i ʻike ʻia i loko o ka nucleus a ua komo i ka chromatin remodeling a me ka hana RNA polymerase.Ua hoʻonuiʻia ka hana molekala o ka protein i ka chromatin binding a iʻole ka transcriptional coactivation, e like me ka hana BRD4 (Fig. 4d).Ua hōʻike ʻia ka hōʻike ʻana i ka pilina pili pūmua i ka ʻikepili i hoʻohana ʻia i kahi pae mua o nā pilina pili ʻole ma waena o BRD4 a me HDAC ʻohana e pili ana i nā paʻakikī e like me SIN3A, NCOR2, BCOR, a me SAP130 (Fig. 4e a me ka Fig. ..Eia kekahi, ua hoʻokūpaʻa ʻia nā pahuhopu ʻelele i ʻike ʻia e LC-MS/MS, me Sin3A, NSUN2, Fus, a me SFPQ, e ka Western blotting (Fig. 4f).I kēia mau lā, ua hōʻike ʻia ka isoform pōkole o BRD4 e hana i ka nuclei me nā waiwai hoʻokaʻawale wai-wai (LLPS).Hoʻopili ka RNA binding protein Fus a me SFPQ i ka LLPS o nā kaʻina kelepona like ʻole a ua ʻike ʻia ma aneʻi he mau protein paʻa ʻole BRD4.Ua hōʻoia ʻia ka pilina ma waena o BRD4 a me SFPQ e nā hoʻokolohua co-immunoprecipitation (co-IP) (Figure 4g), e hōʻike ana i kahi ʻano hana ʻē aʻe no ka hoʻokaʻawale ʻana o ka wai-wai-wai-wai i hoʻokaʻawale ʻia e BRD4 e pono ai ka hoʻokolokolo hou ʻana.Hoʻohui pū ʻia, hōʻike kēia mau hopena he kahua kūpono ka PDPL no ka ʻike ʻana i ka pilina BRD4 i ʻike ʻia a me nā protein paʻa ʻike ʻole.
kahi hōʻike Schematic o ka miniSOG-mediated BRD4 hōʻailona kokoke, nā manawa hōʻike: 2, 5, 10, a me 20 min.b Ka uhi ʻana o nā protein i ʻike ʻia i nā manawa hoʻomālamalama like ʻole.ʻO ka hoʻonui ʻia ʻana o ka protein i ʻike ʻia ma HEK293T-miniSOG-BRD4 he mea koʻikoʻi i ka helu helu hoʻohālikelike ʻia me HEK293T ʻano hihiu.c Ka ikaika o ka Ion i ka helu ʻana i ka ʻelele i ʻike ʻole ʻia i ʻike ʻia i nā protein BRD4-binding i ka manawa i ʻike ʻia.n = 3 mau laʻana kūʻokoʻa olaola.Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± deviation maʻamau.d Gene ontological analysis (GO) o nā protein i ʻike ʻia ma ka hui 2-minuke.Ua helu ʻia nā huaʻōlelo mua he ʻumi GO.Hoʻokaʻawale ʻia nā pōpō e like me ke kāʻei huaʻōlelo GO, a ua like ka nui o ka bubble i ka helu o nā protein i loaʻa i kēlā me kēia huaʻōlelo.e Ka nānā ʻana i ke kaula o nā protein e pili ana me BRD4.ʻO nā pōʻai melemele ke kāpili pololei a ʻo nā pōʻai hina ka papa mua o ke kāʻei pololei ʻole.Hōʻike nā laina ʻulaʻula i nā pilina hoʻokolohua i hoʻoholo ʻia a ʻo nā laina polū e hōʻike ana i nā pilina wānana.f Ua hōʻoia ʻia nā pahuhopu paʻa BRD4 Lunamakaʻāinana i ʻike ʻia ma LC-MS/MS e ka blotting Western.g Hōʻoia nā hoʻokolohua Co-immunoprecipitation i ka pilina ma waena o SFPQ a me BRD4.Ua hana hou ʻia kēia mau hoʻokolohua ma ka liʻiliʻi ʻelua me nā hopena like.Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.
Ma waho aʻe o ka ʻike ʻana i nā pahuhopu pili POI ʻaʻole i kākau inoa ʻia, manaʻo mākou e kūpono ka PDPL no ka ʻike ʻana i nā substrate no nā enzymes, kahi e koi ai i ke ʻano o nā protein paʻa ʻole i loko o nā paʻakikī nui e hōʻike i nā substrate i kākau ʻole ʻia.ʻO Parkin (i hoʻopaʻa ʻia e PARK2) he ligase E3 a ua ʻike ʻia nā hoʻololi ʻana o ka parkin i mea e hoʻoulu ai i ka maʻi ʻo Parkinson's juvenile autosomal recessive (AR-JP)42.Eia kekahi, ua wehewehe ʻia ka parkin he mea nui no ka mitophagy (mitochondrial autophagy) a me ka wehe ʻana i nā ʻano oxygen reactive.Eia naʻe, ʻoiai ua ʻike ʻia kekahi mau substrate parkin, ʻaʻole maopopo ke kuleana o ka parkin i kēia maʻi.No ka hōʻike ʻana i kāna mau substrates uncharacterized, ua hoʻāʻo ʻia ʻo PDPL ma ka hoʻohui ʻana i ka miniSOG i ka N- a i ʻole C-terminus o parkin.Ua mālama ʻia nā kelepona me ka carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) e hoʻāla i ka parkin ma o ke ala PINK1-Parkin.Hoʻohālikelike ʻia i kā mākou hopena BRD4 PDPL, ua hōʻike ʻo parkin N-terminus fusion i kahi hoʻonohonoho nui o nā protein target, ʻoiai ua uhi ʻia i kahi hapa nui o ka C-terminus (177 mai 210) (Figure 5a, b a me Supplementary Data 4).Ua kūlike ka hopena me nā hōʻike e hiki ai i nā hōʻailona N-terminal ke hoʻōla iā Parkin44.ʻO ka mea kupaianaha, aia wale nō he 18 mau protein i kā mākou ʻikepili me nā hopena AP-MS i paʻi ʻia no Parkin43, ma muli paha o nā ʻokoʻa ma waena o nā laina kelepona a me nā kahe hana proteomics.Ma waho aʻe o ʻehā mau protein i ʻike ʻia (ARDM1, HSPA8, PSMD14, a me PSMC3) i ʻike ʻia e nā ala ʻelua (Fig. 5c)43.No ka hōʻoia hou ʻana i nā hopena o ka LC-MS/MS, ua hoʻohana ʻia ka mālama ʻana i ka PDPL a me ka blotting Western e hoʻohālikelike i nā hopena o ka HEK293T makua cell assay a me ka laina paʻa N-terminal parkin.Ua hoʻāʻo ʻia nā pahuhopu i ʻike ʻole ʻia ma mua CDK2, DUT, CTBP1, a me PSMC4 me kahi mea hoʻopili i ʻike ʻia, DNAJB1 (Fig. 5d).
ʻO ka lua pele o nā pūmua e pili ana i ka parkin i loko o nā keena HEK293T me ka miniSOG i hōʻike paʻa ʻia i hoʻohui ʻia i ka N- a i ʻole C-terminus o parkin (n = 3 mau hoʻokolohua kūlohelohe kūʻokoʻa).Ua hoʻohana ʻia ka hoʻāʻo-t a ka Haumāna huelo ʻelua ma nā pā lua pele.Ua hoʻohana ʻia ʻo HEK293T ma ke ʻano he mana ʻino. Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻokoʻa ʻokoʻa o ka ion. Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻokoʻa ʻokoʻa o ka ion. Значительно измененные белки выделены красным цветом (p < 0,05 и >2-кратная разница в интенсивности ионов). Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa ʻelua o ka ʻokoʻa o ka ion).显着变化的蛋白质以红色突出显示（p < 0.05 和> 2 倍离子强度差异）。显着变化的蛋白质以红色突出显示（p < 0.05和> 2 Значительно измененные белки выделены красным цветом (p < 0,05 и > 2-кратная разница в ионной силе). Hōʻike ʻia nā protein i hoʻololi nui ʻia i ka ʻulaʻula (p <0.05 a me ka ʻokoʻa 2-fold i ka ikaika ionic).Hōʻike ʻia nā protein pili i ka HEK293T-miniSOG akā ʻaʻole koʻikoʻi no HEK293T i ka ʻōmaʻomaʻo.b Venn kiʻi e hōʻike ana i nā polokina hili ma waena o N-terminal a me C-terminal kūkulu.Hiki i nā hōʻailona N-terminal ke hoʻoikaika i ka parkin a loaʻa i nā protein i ʻike ʻia.c kiʻi Venn e hōʻike ana i nā polokina e kau ana ma waena o PDPL a me AP-MS.Hoʻopaʻa ʻia nā mea kamaʻilio i ʻike ʻia, me 4 o 18 mau protein overlapping a me 11 o 159 mau protein i ʻike ʻia ma PDPL.d Ua hōʻoia ʻia nā pahuhopu lunamakaʻāinana i ʻike ʻia e LC-MS/MS e ka Western blotting.Ua ʻike ʻia ʻo Ssu72 a me SNW1 he mau substrate parkin i kākau ʻole ʻia.Ua hoʻololi ʻia kēia mau plasmid protein i kā FLAG i HEK293T a me HEK293T-Parkin-miniSOG i ukali ʻia e ka mālama CCCP i nā manawa like ʻole.Ua ʻoi aku ka maikaʻi o ka hoʻohaʻahaʻa ʻana ma ka laina overexpression Parkin.f Me ka hoʻohana ʻana i ka mea hoʻopale proteasome MG132, ua hōʻoia ʻia ua hoʻopili ʻia ke kaʻina hana degradation o Ssu72 a me SNW1 e ka proteasome-ubiquitination.Ua hana hou ʻia kēia mau hoʻokolohua ma ka liʻiliʻi ʻelua me nā hopena like.Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.
ʻO ka mea nui, ʻo nā protein i ʻike ʻia e PDPL pono e hoʻokomo i nā protein paʻa parkin a me kā lākou substrates.No ka ʻike ʻana i nā substrate parkin i kākau ʻole ʻia, ua koho mākou i ʻehiku mau protein i ʻike ʻia (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 a me SNW1) a me nā plasmids i hoʻololi ʻia e hōʻike i kēia mau genes i ka HEK293T maʻamau a hōʻike paʻa i ka miniSOG-Parkin's HEK293T a ukali ʻia e CCCP lapaʻau.Ua hoʻemi nui ʻia nā pae o nā protein Ssu72 a me SNW1 i ka laina miniSOG-Parkin paʻa (Fig. 5e).ʻO ka hoʻomaʻamaʻa ʻana me CCCP no 12 mau hola i hopena i ka hōʻino nui loa o nā substrates ʻelua.No ka noiʻi ʻana inā hoʻoponopono ʻia ka degradation o Ssu72 a me SNW1 e ka proteasome-ubiquitination, ua hoʻohui ʻia ka mea hoʻopuʻi proteasome MG132 e kāohi i ka hana proteasome, a ʻoiaʻiʻo ua ʻike mākou ua kāohi ʻia kā lākou hana hoʻohaʻahaʻa (Fig. 5f).Ua hoʻopaʻa ʻia nā pahuhopu non-substrate hou e like me nā mea hoʻopili Parkin me ka hoʻohana ʻana i ka Western blotting (Supplementary Fig. 10), i hōʻike i nā hopena kūlike me LC-MS/MS.I ka hopena, ʻo ka hoʻohui ʻana o ka workflow PDPL me ka hōʻoia transfection protein target e hiki ai ke ʻike i nā substrates ligase E3 i hoʻopaʻa ʻole ʻia.
Ua hoʻomohala mākou i kahi kahua hōʻailona hoʻokokoke maʻamau e hiki ai iā ʻoe ke ʻike i nā POI e pili ana i ka lewa a me ka manawa.Hoʻokumu ʻia ka paepae ma ka miniSOG photosensitizer protein, ʻo ia wale nō ma kahi o 12 kDa, ʻoi aku ma mua o ka hapalua o ka nui o ka enzyme APEX2 makua (27 kDa) a me ka hapakolu o ka nui o TurboID (35 kDa).ʻO ka liʻiliʻi liʻiliʻi e hoʻonui nui i ka laulā o nā noi no ke aʻo ʻana i nā pilina protein liʻiliʻi.Pono e ʻimi hou aʻe i nā photosensitizer hou, inā he protein i hoʻopaʻa ʻia a i ʻole nā ​​molekole liʻiliʻi, e hoʻonui i ka hua nui o ka oxygen singlet a hoʻonui i ka naʻau o kēia ala.No ka mana o kēia manawa o ka miniSOG, hiki ke hoʻokō ʻia ka hoʻonā kiʻekiʻe me ka hoʻohana ʻana i ka kukui polū e hoʻāla i nā māka kokoke.Eia hou, ua hoʻokuʻu ʻia ka "kapua" nui o ka oxygen singlet i ka manawa lōʻihi, e hopena i ka hoʻololi ʻana i nā koena histidine distal, hoʻonui i ka radius lepili, a me ka hiki ke hoʻoponopono i ka hoʻonā spatial PDPL.Ua hoʻāʻo pū mākou i ʻehiku ʻimi kemika no ka hoʻonui ʻana i ka ratio hōʻailona-i-background a ʻimi i ka mīkini molekala ma hope o kēia ala.Ua hōʻoia ka TOP-ABPP workflow i hui pū ʻia me ka ʻimi wehe ʻole ʻia i nā histidines wale nō ka hoʻololi ʻana a ʻaʻohe microenvironment kūlike i ʻike ʻia no ka hoʻonui ʻana i ka histidine hoʻololi, koe wale nō ka makemake haʻahaʻa no nā histidines ma ka ʻāpana loop.
Ua hoʻohana pū ʻia ʻo PDPL e hōʻike i nā proteome subcellular me ka kikoʻī proteome a me ka uhi ʻana i ka liʻiliʻi loa e hoʻohālikelike ʻia me nā ʻano hoʻopaʻa inoa kokoke a me nā ʻano hana hoʻokolohua kemika kikoʻī organelle.Ua hoʻohana maikaʻi ʻia nā māka kokoke e ʻike i ka ʻili, lysosomal, a me nā proteomes e pili ana i kahi huna46,47.Manaʻo mākou e kūpono ana ka PDPL me kēia mau ʻāpana subcellular.Eia kekahi, ua hoʻokūkū mākou i ka PDPL ma ka ʻike ʻana i nā pahuhopu no ka cytosolic protein paʻa i ʻoi aku ka paʻakikī ma mua o nā protein i hoʻopaʻa ʻia me ka membrane ma muli o ko lākou mau waiwai ikaika a me ke komo ʻana i nā pilina pili kino.Ua hoʻohana ʻia ka PDPL i ʻelua mau protein, ka coactivator transcriptional BRD4 a me ka ligase E3 Parkin pili i ka maʻi.Ua koho ʻia kēia mau protein ʻelua ʻaʻole wale no kā lākou hana koʻikoʻi koʻikoʻi, akā no ko lākou pili pono ʻana a me ka hiki ke therapeutic.No kēia mau POI ʻelua, ua ʻike ʻia nā hoa paʻa kaulana a me nā pahuhopu i kākau inoa ʻole ʻia.ʻO ka mea nui, ua hōʻoia ʻia ka protein SFPQ pili i ka māhele hoʻokaʻawale e ka co-IP, e hōʻike ana i kahi hana hou e hoʻoponopono ai ʻo BRD4 (short isoform) i ka LLPS.I ka manawa like, ke manaʻoʻiʻo nei mākou ʻo ka ʻike ʻana i nā substrates Parkin kahi hiʻohiʻona e koi ʻia ai ka ʻike ʻana o nā mea hoʻopili indirect.Ua ʻike mākou i ʻelua mau papa parkin i ʻike ʻole ʻia a hōʻoia i ko lākou hōʻino ʻana ma ke ala ubiquitination-proteasome.I kēia mau lā, ua hoʻomohala ʻia kahi hoʻolālā hoʻopaʻapaʻa e pili ana i ka mīkini e ʻike i nā substrate hydrolase ma o ka hoʻopaʻa ʻana iā lākou me nā enzymes.ʻOiai he ʻano ikaika loa kēia, ʻaʻole kūpono ia no ka nānā ʻana i nā substrates e pili ana i ka hoʻokumu ʻana i nā complexes nui a koi i ka hoʻokumu ʻana i nā paʻa covalent ma waena o ka enzyme a me ka substrate.Manaʻo mākou e hiki ke hoʻonui ʻia ka PDPL i ke aʻo ʻana i nā ʻohana protein a me nā ʻohana enzyme, e like me ka deubiquitinase a me nā ʻohana metalloprotease.
Ua hoʻomohala ʻia kahi ʻano hou o miniSOG, i kapa ʻia ʻo SOPP3, me ka hoʻomaikaʻi ʻana i ka hana oxygen singlet.Ua hoʻohālikelike mākou i ka miniSOG me SOPP3 a loaʻa ka hoʻomaikaʻi ʻana i ka hana mākaʻikaʻi, ʻoiai ʻaʻole i hoʻololi ʻia ka ratio hōʻailona-a-noise (Supplementary Fig. 11).Manaʻo mākou ʻo ka hoʻonui ʻana i ka SOPP3 (e laʻa, ma o ka hoʻomohala kuhikuhi ʻia) e alakaʻi i nā protein photosensitizer ʻoi aku ka maikaʻi e koi ana i nā manawa māmā pōkole a no laila e ʻae ai i nā kaʻina kelepona ʻoi aku ka ikaika e hopu.ʻO ka mea nui, ua kaupalena ʻia ka mana o kēia manawa o PDPL i ke kaiapuni kelepona no ka mea e koi ana i ka hoʻomālamalama kukui polū a ʻaʻole hiki ke komo i nā ʻiʻo hohonu.Kāohi kēia hiʻohiʻona i kona hoʻohana ʻana i nā haʻawina hoʻohālike holoholona.Eia naʻe, hiki i ka hui pū ʻana o optogenetics me PDPL ke hāʻawi i kahi manawa no ka noiʻi holoholona, ​​ʻoi aku hoʻi i ka lolo.Eia kekahi, hoʻoneʻe pū kekahi i nā photosensitizer infrared ʻē aʻe i kēia palena.Ke holo nei ka noiʻi ma kēia wahi.
Loaʻa ka laina kelepona HEK293T mai ATCC (CRL-3216).Ua ho'āʻo maikaʻi ʻia ka laina kelepona no ka maʻi mycoplasma a ua hana ʻia ma DMEM (Thermo, #C11995500BT) i hoʻohui ʻia me 10% fetal bovine serum (FBS, Vistech, #SE100-B) a me 1% penicillin/streptomycin (Hyclone, #SV30010).ulu i loko.
3-Aminophenylene (la'ana 3) a me (4-ethynylphenyl) metanamine (la'ana 4) i kū'ai 'ia mai Bidepharm.Ua kūʻai ʻia mai ka Propylamine (probe 2) mai Energy-chemicals.N-(2-Aminophenyl)pent-4-ynamide (probe 1) ua synthesized e like me nā hana i paʻi ʻia.
Hōʻike ʻia ka papa helu 1 i nā kūkulu genetic i hoʻohana ʻia i kēia haʻawina.Ua cloned ka miniSOG a me KillerRed sequences mai kahi makana plasmid mai P. Zou (Peking University).Ua kiʻi ʻia ke kaʻina kuhi mitochondrial matrix mai ka 23 N-terminal amino acids o COX4 a cloned i loko o nā vectors i hōʻike ʻia me ka hoʻohana ʻana i kahi hui Gibson (Beyotime, #D7010S).No ka huli ʻana i ka membrane a me ka nucleus o ka endoplasmic reticulum, SEC61B DNA kanaka (NM_006808.3) (NEB, #M0491L) i hoʻonui ʻia e PCR mai kahi waihona cDNA o HEK293T cell, a me H2B DNA (hāʻawi ʻia e D. Lin, Shenzhen Bay Laboratory) a cloned , e like me ka mea i ʻōlelo ʻia ma luna.Inā ʻaʻole i hōʻike ʻia, ua hoʻonui ʻia ka PCR mai ka waihona HEK293T cell cDNA.Ua hoʻohana ʻia ʻo G3S (GGGS) a me G4S (GGGGS) i mea hoʻopili ma waena o ka protein maunu a me ka miniSOG.Ua hoʻohui ʻia kahi hōʻailona epitope V5 (GKPIPNPLLGLDST) i kēia mau hana fusion.No ka hōʻike ʻana i nā mammals a no ka hoʻokumu ʻana i kahi laina kelepona paʻa, ua hoʻopili ʻia ka miniSOG fusion construct i loko o ka pLX304 lentiviral vector.No ka hōʻike maʻi bacteria, ua hoʻopili ʻia ka miniSOG i ka vector pET21a i kapa ʻia ʻo 6xHis ma ka C-terminus.
Ua kanu ʻia nā cell HEK293T ma 2.0 x 105 cell i kēlā me kēia lua i loko o nā papa ʻeono-well a hoʻololi ʻia 24 mau hola ma hope me ka recombinant lentiviral plasmids (2.4 μg pLX304) a me nā plasmids packaging viral (1.5 μg psPAX2 a me 1.2 μg pMD2.G00) , #C0533), ma kahi o 80% hui.Ma hope o ka hoʻololi ʻana i ka pō, ua hoʻololi ʻia ke ʻano a hoʻomoʻa ʻia no 24 mau hola.Ua lawe ʻia ka hōʻiliʻili o ka maʻi ma hope o 24, 48 a me 72 mau hola.Ma mua o ka maʻi o nā laina kelepona i hoʻopaʻa ʻia, ua kānana ʻia ka media viral ma o kahi kānana 0.8 μm (Merck, #millex-GP) a me polybrene (Solarbio, #H8761) i hoʻohui ʻia i kahi ʻano o 8 μg / ml.Ma hope o 24 mau hola, ua ʻae ʻia nā cell e ola hou ma ka hoʻololi ʻana i ke ʻano.Ua koho ʻia nā kelepona me ka hoʻohana ʻana i ka 5 μg / ml blasticidin (Solarbio, #3513-03-9) no nā pauku mua ʻekolu ma ke ʻano he koho haʻahaʻa haʻahaʻa.A laila hoʻohana ʻia ka 20 μg/ml ma ke ʻano he regimen koʻikoʻi no nā pauku ʻekolu e hiki mai ana.
Ua kanu ʻia nā kelepona i loko o nā keʻena 12-well (Ibidi, #81201) ma kahi mānoanoa ma kahi o 20,000 mau pūnāwai no ka luawai.No ka hoʻomaikaʻi ʻana i ka hoʻopili ʻana o nā cell HEK293T, hoʻohui i ka 50 µg/ml fibronectin (Corning, #356008) i hoʻoheheʻe ʻia i loko o ka paʻakai phosphate buffered (PBS, Sangon, #B640435) ma 37°C.Hoʻomaʻamaʻa mua ʻia nā keʻena no 1 hola a laila wehe ʻia me ka PBS.Ma hope o 24 h, holoi ʻia nā cell i hoʻokahi manawa me ka PBS, incubated me 1 mM probe 3 i loko o ka wai paʻakai paʻakai paʻakai hou o Hanks (HBSS, Gibco, #14025092) no 1 h ma 37 ° C, a laila hoʻomoʻi ʻia me kahi LED polū (460 nm. ).) ua hoʻomālamalama ʻia no 10 min ma ka lumi wela.Ma hope o kēlā, ua holoiʻia nā pūnaewele iʻelua manawa me ka PBS a hoʻopaʻaʻia me ka 4% formaldehyde i PBS (Sangon, #E672002) no nā minuke 15 i ka mahana wela.Ua hoʻoneʻe ʻia ka formaldehyde nui mai nā keena paʻa ma ka holoi ʻana i ʻekolu manawa me ka PBS.Ua hoʻopili ʻia nā kelepona me 0.5% Triton X-100 (Sangon, #A600198) ma PBS a holoi ʻia ʻo 3 mau manawa me PBS.A laila wehe i ke keʻena a hoʻohui i kēlā me kēia hāpana 25 µl o kahi hui hoʻohui kaomi i loaʻa ka 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, #A603008), 1 mM BTTAA (Confluore, #BDJ-4) a me ka 0.5 mg / ml sodium ascorbate (Aladdin, no. S105024) a hoʻokomoʻia no nā minuke 30 i ka mahana wela.Ma hope o ka pane ʻana, ua holoi ʻia nā cell i ʻeono manawa me ka PBS i loaʻa ka 0.05% Tween-20 (Sangon, #A600560) (PBST) a laila pāpā ʻia me 5% BSA (Abcone, #B24726) i PBST no 30 mau minuke i ka lumi wela.
No ka immunostaining colocalization, ua hoʻokomo ʻia nā cell me nā antibodies mua e like me nā kūlana i hōʻike ʻia: anti-V5 tag mAb (1:500, CST, #80076), rabbit anti-Hsp60 mAb (1:1000), ABclonal, #A0564), rabbit polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) a i ʻole rabbit anti-lamin A/C monoclonal antibody (1:500; CST, #2032) ma 4 °C i ka pō.Ma hope o ka holoi ʻana i 3 mau manawa me ka PBST, ua hoʻomoʻa ʻia nā cell me nā antibodies lua: kao anti-rabbit Alexa Fluor 488 (Thermo, #A11034) diluted 1:1000, kao anti-mouse Alexa Fluor 594 (CST, #8889) diluted 1:1000.dilution E hoʻoheheʻe i ka lumi wela no 30 mau minuke.Holoi ʻia nā kelepona i 3 mau manawa me PBST a counterstained me DAPI (Thermo, #D1306) ma PBS no 10 mau minuke i ka lumi wela.Ma hope o 3 holoi ʻana me PBS, ua hoʻopaʻa ʻia nā cell i ka 50% glycerol (Sangon, #A600232) ma PBS no ke kiʻi.Loaʻa nā kiʻi immunofluorescent me ka ZEISS LSM 900 Airyscan2 confocal microscope a me ka polokalamu ZNE 3.5.
No ke kiʻi ʻana o ka oxygen fluorescent, ua holoi ʻia ʻelua mau mea me ka paʻa Hanks HEPES ma mua o ka hoʻohui ʻana i ka 100 nM Si-DMA ma Hanks HEPES buffer (DOJINDO, #MT05).Ma hope o kaʻikeʻana i ka mālamalama, ua hoʻokomoʻia nā pūnaewele i loko o ka CO2 incubator ma 37 ° C no 45 mau minuke.Holoi ʻelua ʻia nā kelepona me Hanks 'HEPES buffer a counterstained me Hoechst i Hanks' HEPES buffer no 10 mau minuke ma ka lumi wela a nānā ʻia me ka ZEISS LSM 900 confocal microscope., #M36008) i loko o ka HBSS buffer loaʻa ka calcium a me ka magnesium.Ma hope o kaʻikeʻana i ka mālamalama a iʻole ka doxorubicin (MCE, #HY-15142A), ua hoʻokomoʻia nā pūnaewele i loko o ka CO2 incubator ma 37 ° C. no nā minuke 10, holoiʻia iʻelua manawa me ka HBSS buffer, a hoʻokomoʻia me Hoechst i ka HBSS buffer ma ka lumi wela.minuke.Ua hoʻohana ʻia ʻo Doxorubicin ma ke ʻano he mana hoʻokolohua maikaʻi kahi i mālama ʻia ai nā cell me 20 μM doxorubicin ma HBSS i loaʻa ka 1% BSA no 30 min.Loaʻa nā kiʻi immunofluorescent me ka hoʻohana ʻana i kahi microscope confocal Zeiss LSM 900.
ʻO nā pūnaewele HEK293T e hōʻike mau ana i ka mito-miniSOG i kanu ʻia ma kahi kikoʻī o 30% i loko o nā kīʻaha 15 cm.Ma hope o nā hola 48, i ka wā i loaʻa ai ka 80% confluence, ua holoi ʻia nā cell i hoʻokahi manawa me ka PBS, i hoʻopili ʻia me 1 mM Probe 3 i loko o ka HBSS buffer hou no 1 hola ma 37 ° C a laila hoʻomālamalama ʻia me kahi LED polū no 10 mau minuke ma ka lumi. mahana wela..Ma hope mai, ua holoi ʻia nā keena me ka PBS, ʻoki ʻia a hoʻopaʻa hou ʻia i loko o ka pahu hau-anu PBS i loaʻa i nā mea hoʻopaʻa protease manuahi ʻole EDTA (MCE, #HY-K0011).Ua lysed nā pūnaewele ma ka sonicating i ka piko no 1 minuke (1 kekona a me 1 kekona ma 35% amplitude).ʻO ka hopena o ka huiʻana i centrifuged ma 15,871 xg no 10 min ma 4 ° C no ka weheʻana i nā'ōpala, a ua hoʻololiʻia ka manaʻo nui i ka 4 mg / mL me ka hoʻohanaʻana i ka BCA protein assay kit (Beyotime, #P0009).Hoʻohui i ka 1 ml o ka lysate i luna me 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), a me 1 mM CuSO4 incubator me lalo. hoʻololi i 1 hola ma ka lumi wela.Ma hope o ka hoʻololi ʻana, e hoʻohui i ka hui ʻana i ka hopena i hui mua ʻia (MeOH:CHCl3:H2O = 4 ml:1 ml:3 ml) i loko o kahi hue aniani 10 ml.Hoʻopili ʻia nā laʻana a hoʻopaʻa ʻia i ka 4500 g no 10 mau minuke ma ke ana wela.Ua hoʻolei ʻia nā haʻina haʻahaʻa a me luna, holoi ʻia ka precipitate i ʻelua mau manawa me 1 ml o ka methanol a centrifuged ma 15871 × g no 5 min ma 4 ° C.E hoʻohui i 1 ml o 8 M urea (Aladdin, no. U111902) i 25 mM ammonium bicarbonate (ABC, Aladdin, no. A110539) e hoʻoheheʻe i ka wai.Hoʻohui hou ʻia nā laʻana me 10 mM dithiothreitol (Sangon, #A100281 ma 25 mM ABC) no 40 mau minuke ma 55 ° C a ukali ʻia e ka hoʻohui ʻana o 15 mM hou iodoacetamide (Sangon, #A600539) i ka lumi wela i ka pōʻeleʻele.Alkylation i loko o 30 minuke..Hoʻohui ʻia kahi 5 mM dithiothreitol e hoʻopau i ka hopena.E hoʻomākaukau ma kahi o 100 µl NeutrAvidin agarose (Thermo, #29202) no kēlā me kēia hāpana ma ka holoi ʻana i 3 manawa me 1 ml PBS.ʻO ka hopena proteome ma luna nei i hoʻoheheʻe ʻia me 5 ml PBS a hoʻomoʻi ʻia me nā beads NeutrAvidin agarose i holoi mua ʻia no 4 mau hola ma ke ana wela.A laila holoi ʻia nā pahu i 3 mau manawa me 5 ml PBS i loaʻa ka 0.2% SDS (Sangon, #A600485), 3 mau manawa me 5 ml PBS i loaʻa ka 1M urea, a me 3 mau manawa me 5 ml ddH2O.ʻOhi ʻia nā pahu e ka centrifugation a hoʻopaʻa hou ʻia i 200 μl o 25 mM ABC i loaʻa ka 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) a me 20 ng / μl trypsin (Promega, #V5280).Trypsinize i ka pō ma 37 ° C me ka hoʻololi.Ua kāpae ʻia ka hopena ma ka hoʻohui ʻana i ka waika formic (Thermo, # A117-50) a hiki i ka pH i 2-3.Ua holoi ʻia nā pēke i 3 mau manawa me 1 ml PBS i loaʻa ka 0.2% SDS, 3 mau manawa me 1 ml PBS i loaʻa ka 1 M urea, a laila 3 mau manawa me 1 ml o ka wai distilled.Ua hoʻokuʻu ʻia nā peptides i hoʻololi ʻia e ka lysis māmā (365 nm) no 90 min me ka hoʻohana ʻana i 200 μl o 70% MeOH.Ma hope o ka centrifugation, ua hōʻiliʻili ʻia ka supernatant.Holoi ʻia nā pahu i hoʻokahi manawa me 100 μl o 70% MeOH a ua hui ʻia nā supernatants.Hoʻomaloʻo ʻia nā laʻana i loko o kahi mea hoʻomaʻamaʻa Speedvac a mālama ʻia ma -20 ° C a hiki i ka nānā ʻana.
No ka ʻike ʻana a me ka helu ʻana i nā peptides i hoʻololi ʻia ka oxygen, ua hoʻoheheʻe ʻia nā laʻana i ka 0.1% formic acid a ua nānā ʻia ka 1 μg o nā peptides me ka hoʻohana ʻana i kahi spectrometer nui Orbitrap Fusion Lumos Tribrid i lako me kahi kumu nano ESI mai Tune a me Xcalibur mai ka polokalamu kūʻai 4.3.Ua hoʻokaʻawale ʻia nā laʻana ma kahi kolamu capillary 75 µm × 15 cm i hoʻopaʻa ʻia i loko me 3 µm C18 mea (ReproSil-pur, #r13.b9.) a pili i kahi ʻōnaehana EASY-nLC 1200 UHPLC (Thermo).Ua hoʻokaʻawale ʻia nā peptides e ka linear 95 minuke gradient chromatography mai 8% solvent B a i 50% solvent B (A = 0.1% formic acid i ka wai, B = 0.1% formic acid i 80% acetonitrile), a laila hoʻonui ʻia i ka 98% B min. i loko o 6 min ma kahi kahe kahe o 300 nl / min.ʻOhi ʻo Orbitrap Fusion Lumos i ka ʻikepili ma waena o ka MS scan piha a me ka MS2 scan ma muli o ka ʻikepili.Ua hoʻonohonoho ʻia ka volta sputtering i 2.1 kV a ʻo ka mahana o ka capillary transport ion he 320 ° C.Ua hōʻiliʻili ʻia ʻo MS spectra (350-2000 m/z) me ka hoʻonā o 120,000, AGC 4 × 105, a me ka manawa hoʻokomo kiʻekiʻe o 150 ms.ʻO ka 10 mau mea maʻamau i hoʻoili ʻia ma kēlā me kēia scan piha ua ʻāpana ʻia me ka HCD me ka ikehu collision maʻamau o 30%, kahi puka aniani kaʻawale quadrupole o 1.6 m/z, a me kahi hoʻonohonoho hoʻonā o 30,000.ʻO kahi pahuhopu AGC no ka tandem mass spectrometry e hoʻohana ana i ka 5×104 a me ka manawa hoʻokomo kiʻekiʻe 150 ms.Hoʻonohonoho ʻia ka ʻokoʻa ikaika i 30 kekona. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​mea nona ka uku o 1+ a me >7+ no MS/MS. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​mea nona ka uku o 1+ a me >7+ no MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​iona me ka uku o 1+ a me >7+ no 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. Ua hōʻole ʻia nā ʻiona i hōʻike ʻole ʻia a i ʻole nā ​​​​iona me nā koina o 1+ a me >7+ no MS/MS.
Hoʻohana ʻia ka ʻikepili maka me ka FragPipe computing platform ma muli o MSFragger.Ua hoʻoholo ʻia ka nui o ka nui a me nā waikawa amino e pili ana me ka hoʻohana ʻana i kahi algorithm hulina wehe me kahi precursor mass tolerance o -150 a 500 Da.Ua ʻike ʻia nā peptides i hoʻololi ʻia me ka hoʻololi ʻana i ka histidine me nā loaʻa nui o +229.0964 a me +247.1069 Da ma PD (Proteome Discoverer 2.5, Thermo).
Ua hoʻopaʻa ʻia nā pūnaewele e hōʻike ana i ka gen miniSOG i hoʻohui ʻia i loko o nā kīʻaha 6 cm.Ma ka hōʻea ʻana i ka ~ 80% hui, holoi ʻia nā cell i hoʻokahi manawa me HBSS (Gibco, #14025092), a laila hoʻomoʻi ʻia me nā probes kemika ma HBSS no 1 hola ma 37 ° C a hoʻomālamalama ʻia me ke kukui polū.10W LED no 20 mau minuke ma ka lumi wela.No ka hoʻoholo ʻana i ke ʻano o nā ʻano oxygen reactive i komo i PDPL, 0.5 mM vitamina 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 i hoʻohuiʻia i nā pūnaewele i mea hoʻonui.Ma hope o ka holoi ʻana me ka PBS anu, ua ʻoki ʻia nā pūnaewele, hōʻiliʻili ʻia i loko o 1.5 ml centrifuge tubes, a sonicated me ka piko no 1 min ma 200 μl o PBS me 1x protease inhibitor me ka EDTA (1 s a me 1 s me ka ʻole, amplitude 35%).ʻO ka hopena o ka hui ʻana ua centrifuged ma 15,871 × g no 10 min ma 4 ° C a ua hoʻoponopono ʻia ka manaʻo supernatant i 1 mg / mL me ka hoʻohana ʻana i kahi kit assay protein BCA.Ma kahi o 50 µl o ka lysate ma luna i hoʻokomo ʻia me 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, a me 1 mM CuSO4 no 1 hola ma ka lumi wela me ka hoʻololi ʻana mai lalo a luna.Ma hope o ke kaomi ʻana, ua hoʻokō ʻia ka ua me ka acetone ma ka hoʻohui ʻana i 250 μl o ka acetone pre-chilled i nā laʻana, incubating ma -20 ° C no 20 min a me ka centrifuging ma 6010 × g no 10 min ma 4 ° C.E hōʻiliʻili i ka pellet a hoʻolapalapa i loko o 50 µl o 1x paʻa Laemmli no 10 min ma 95 °C.Hoʻopili ʻia nā laʻana ma nā gels lōʻihi SDS-PAGE a ʻike ʻia me ka hoʻohana ʻana i ka ʻōnaehana kiʻi Bio-rad ChemiDoc MP Touch me ka polokalamu Image Lab Touch.
ʻO ka hōʻike a me ka hoʻomaʻemaʻe ʻana o ka recombinant miniSOG-6xHis protein i hana ʻia e like me ka mea i wehewehe mua ʻia.ʻO ka pōkole, ua hoʻololiʻia nā pūnaewele E. coli BL21 (DE3) (TransGen, # CD701-02) me pET21a-miniSOG-6xHis a ua hoʻokomoʻia ka'ōlelo protein me 0.5 mM IPTG (Sangon, #A600168).Ma hope o ka lysis cell, ua hoʻomaʻemaʻe ʻia nā protein me ka hoʻohana ʻana i nā beads Ni-NTA agarose (MCE, no. 70666), i hoʻopaʻa ʻia i ka PBS, a mālama ʻia ma -80 ° C.
No ka ho'āʻo kokoke i ka lepili in vitro i hoʻokumu ʻia i ka antibody, hoʻohui i ka 100 μM hoʻomaʻemaʻe miniSOG, 1 mM probe 3, a me 1 μg anti-label mouse monoclonal antibody (TransGen, #HT501-01) i PBS i ka huina hopena o 50 μl..Ua hoʻomālamalama ʻia ka hui ʻana me ke kukui LED polū no 0, 2, 5, 10, a me 20 min ma ka lumi wela.Hoʻokomo ʻia ka hui ʻana me 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, a me 1 mM CuSO4 no 1 h ma ka lumi wela ma luna o kahi mea hoʻoluliluli i luna.Ma hope o ka hoʻopili ʻana, e hoʻohui pololei i ka pahu 4x Laemmli i ka hui ʻana a e hoʻolapalapa ma 95 ° C no 10 min.Hoʻopili ʻia nā laʻana ma nā gels SDS-PAGE a nānā ʻia e ka blotting komohana me streptavidin-HRP (1:1000, Solarbio, #SE068).
Ua hoʻohana ʻia kahi histidine-containing synthetic peptide me C-terminal amidation (LHDALDAK-CONH2) no ka nānā ʻana i ka peptide-based in vitro labeling.I loko o kēia assay, 100 μM hoʻomaʻemaʻe miniSOG, 10 mM probe 3 a me 2 μg / ml synthetic peptide i hui pū ʻia i PBS i ka huina hopena o 50 μl.Ua hoʻomālamalama ʻia ka hui ʻana me ke kukui LED polū no 1 hola ma ke ana wela.Hoʻokahi microliter o ka laʻana i kālailai ʻia me ka ʻōnaehana LC-MS (Waters, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer me MassLynx spectrum analysis software).
ʻO nā pūnaewele HEK293T e hōʻike mau ana i ka miniSOG fusion gene i kanu ʻia i loko o nā kīʻaha 10 cm no nā laina me nā ʻano organelle localization (Mito, ER, Nucleus) a me nā kīʻaha 15 cm no nā laina Parkin-miniSOG a me BRD4-miniSOG.I ka hiki ʻana i ka ~ 90% confluence, ua holoi ʻia nā cell i hoʻokahi manawa me HBSS, a laila incubated me ka probe 3 i HBSS no 1 hola ma 37 ° C a hoʻomālamalama ʻia me kahi 10 W blue LED ma ka lumi wela.No ka hoʻopaʻa inoa ʻole ʻana o Parkin, ua hoʻohui ʻia ʻo 10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) me ka probe 3 ma HBSS no 1 hola ma 37°C.Ua like ka cell lysis, click chemistry, reduction and alkylation steps me ka mea i ho'ākāka 'ia ma luna, koe wale nō ka 2 mg o ka lysate i hoʻohui ʻia a me ka biotin PEG3 azide i hoʻohana ʻia i ka pākuʻi kaomi ma kahi o ka biotin azide photodegradable.Ma hope o ka hoʻonuiʻana, holoiʻia nā pēke i 3 manawa me 5 ml o PBS i loaʻa ka 0.2% SDS, 3 manawa me 5 ml PBS i loaʻa ka 1 M urea, a me 3 manawa me 5 ml o PBS.Ma hope iho, hoʻohui ʻia ka 2 µg trypsin i 300 µl 25 mM ABC i loaʻa ka 1 M urea e hoʻokaʻawale i ka protein i ka pō ma 37 ° C.Ua kāpae ʻia ka hopena ma ka hoʻohui ʻana i ka waika formic a hiki i kahi pH o 2-3.Ma hope o ka trypsinization ma nā pahu, ua hoʻoneʻe ʻia ka hopena peptide me ka hoʻohana ʻana i kahi kolamu SOLAµ HRP (Thermo, #60209-001) a maloʻo ʻia i loko o kahi concentrator vacuum Speedvac.Ua hoʻopau hou ʻia nā peptides i ka 0.1% formic acid a me 500 ng o nā peptides i kālailai ʻia me ka Orbitrap Fusion Lumos Tribrid mass spectrometer i lako me ke kumu nano-ESI i hōʻike ʻia ma luna.Ua hoʻokaʻawale ʻia nā peptides ma nā ʻoihana RP-HPLC precolumns (75 μm x 2 cm) (Thermo, no. 164946) a me nā kolamu analytical RP-HPLC (75 μm x 25 cm) (Thermo, no. 164941), hoʻopiha ʻia ʻelua me 2 μm.gradient mai 8% a 35% ACN i 60 mau minuke, a laila hoʻonui i ka linearly i 98% B i 6 mau minuke ma kahi kahe kahe o 300 Nl / min.Ua hōʻiliʻili ʻia ʻo MS spectra (350-1500 m/z) me ka hoʻonā o 60,000, AGC 4 × 105, a me ka manawa hoʻokomo kiʻekiʻe o 50 ms.Ua hoʻokaʻawale ʻia nā ion i koho ʻia e HCD i 3 mau kaʻina me ka ikehu collision maʻamau o 30%, he puka aniani hoʻokaʻawale quadrupole o 1.6 m/z, a me ka hoʻonā ʻana o 15000. ʻO 5 × 104 tandem mass spectrometer AGC pahu hopu a me ka manawa hoʻokele kiʻekiʻe. o 22 ms i hoʻohana ʻia.Hoʻonohonoho ʻia ka hoʻokuʻu ʻia i 45 kekona. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​mea nona ka uku o 1+ a me >7+ no MS/MS. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​mea nona ka uku o 1+ a me >7+ no MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ua hōʻole ʻia nā ion i hāʻawi ʻole ʻia a i ʻole nā ​​​​iona me ka uku o 1+ a me >7+ no 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. Ua hōʻole ʻia nā ʻiona i hōʻike ʻole ʻia a i ʻole nā ​​​​iona me nā koina o 1+ a me >7+ no MS/MS.
ʻO nā pae hoʻomākaukau laʻana a hiki i ka hoʻonui ʻana i nā peʻa NeutrAvidin ua like ia me ka loiloi LC-MS/MS i hōʻike ʻia ma luna.Ma kahi o 50 μg o ka lysate i hoʻohana ʻia ma ke ʻano he hoʻokomo no ka hoʻouka ʻana i ka mana a ua hoʻohana ʻia ʻo 2 mg o lysate no ke kaomi ʻana.Ma hope o ka hoʻonui ʻana a me ka holoi ʻana me ka neutravidin, ua hoʻokaʻawale ʻia nā protein i hoʻopaʻa ʻia ma ka hoʻohui ʻana i 50 μl o ka paʻa o Laemmli i nā pahu resin agarose a hoʻolapalapa ʻia ma 95 ° C. no 5 mau minuke.Ua kālailai ʻia ka hoʻokomo ʻana i ka ukana a me ka bead i hoʻonui ʻia e SDS-PAGE a hoʻoneʻe ʻia i nā membranes PVDF (Millipore, #ISEQ00010) e nā ala Western blot maʻamau.Hoʻopaʻa ʻia nā membrane me ka waiu skim 5% (Sangon, #A600669) i loko o ka TBS i loaʻa ka 0.1% ma waena o-20 (TBST) a hoʻomoʻi ʻia me nā antibodies mua a me ka lua.Ua hoʻoheheʻe ʻia nā antibodies mua i ka 1:1000 i ka waiū skim 5% i TBST a hoʻomoʻa ʻia i ka pō ma 4 ° C.Ua hoʻohana ʻia nā antibodies lua i ka ratio o 1: 5000 a hoʻomoʻa ʻia no 1 hola ma ka lumi wela.ʻIke ʻia nā membrane e ka chemiluminescence me ka hoʻohana ʻana i ka ʻōnaehana kiʻi Chemidoc MP.Hōʻike ʻia nā kiʻi ʻoki ʻole a pau o nā blots a me nā gels i ke kiʻi ma ke ʻano he ʻikepili maka.
ʻO nā antibodies mua i hoʻohana ʻia i kēia haʻawina ʻo ka rabbit anti-SFPQ monoclonal antibody (CST, no. 71992), rabbit anti-FUS monoclonal antibody (CST, no. 67840), rabbit anti-NSUN2 polyclonal antibody (Proteintech, no. 20854-1- AP), rabbit anti-mSin3A polyclonal antibody (Abcam, #ab3479), anti-tag monoclonal antibody (TransGen, #HT201-02), anti-β-actin monoclonal antibody (TransGen, #HC201-01), rabbit antibody -CDK2 monoclonal antibody (ABclonal, #A0094), rabbit monoclonal antibody i CTBP1 (ABclonal, #A11600), rabbit polyclonal antibody i DUT (ABclonal, #A2901), rabbit polyclonal antibody i PSMC4 (ABclonal, #A2505), rabbit anti- DNAJB1 polyclonal antibody (ABclonal, # A5504).Ua hoʻohana ʻia kēia mau antibodies ma kahi dilution 1:1000 i 5% waiu skim i TBST.ʻO nā antibodies lua i hoʻohana ʻia i kēia haʻawina ʻo ia ka anti-rabbit IgG (TransGen, #HS101-01), anti-mouse IgG (TransGen, #HS201-01) ma kahi dilution 1:5000.
No ka noiʻi hou ʻana inā pili ʻo BRD4 me SFPQ, ua hoʻopaʻa ʻia nā ʻāpana HEK293T a me BRD4-miniSOG e overexpressing HEK293T i nā kīʻaha 10 cm.Ua holoiʻia nā pūnaewele me ka PBS anu a lysed i ka 1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) me EDTA-free protease inhibitor no 30 mau minuke ma 4 ° C.Ma hope o kēlā, ua hōʻiliʻili ʻia nā lysates i 1.5 ml centrifuge tubes a centrifuged ma 15,871 xg no 10 min ma 4 ° C.Ua hōʻiliʻili ʻia ka supernatant a hoʻomoʻi ʻia me 5 µg o anti-V5 labeled mouse monoclonal antibody (CST, #80076) i ka pō ma 4°C.Holoi ma kahi o 50 µl o nā kinipona A/G magnetic beads (MCE, #HY-K0202) ʻelua me ka PBS he 0.5% Tween-20.A laila ua hoʻokomoʻia nā lysates pūnaewele me nā pahu magnetic no 4 mau hola ma 4 ° C me ka hoʻololiʻana mai lalo a luna.A laila holoi ʻia nā pahu i ʻehā manawa me 1 ml o ka PBST buffer a hoʻolapalapa ʻia ma 95 ° C no 5 min.Hoʻopili ʻia nā laʻana ma nā gel SDS-PAGE a hoʻoneʻe ʻia i nā membranes PVDF me ka hoʻohana ʻana i nā ala Western blot maʻamau.Hoʻopaʻa ʻia nā membrane i 5% waiu skim i TBST a hoʻokomo pū ʻia me nā antibodies mua a me ka lua.Ua hoʻohana ʻia ʻo Primary Antibody Rabbit anti-SFPQ monoclonal antibody (CST, #71992) ma ka ratio o 1:1000 i 5% waiu skim i TBST a hoʻomoʻa ʻia i ka pō ma 4°C.Ua hoʻohana ʻia ʻo Anti-rabbit IgG ma kahi ratio o 1:5000 a hoʻomoʻa ʻia no 1 hola ma ke ana wela.ʻIke ʻia nā membrane e ka chemiluminescence me ka hoʻohana ʻana i ka ʻōnaehana kiʻi Chemidoc MP.
Loaʻa nā hale āpau i hoʻohana ʻia no ka Solvent Accessible Surface Area (SASA) kānana mai ka Protein Data Bank (PDB)52 a i ʻole ka AlphaFold Protein Structure Database53.Ua helu ʻia ʻo SASA piha no kēlā me kēia koena me ka hoʻohana ʻana i ka polokalamu FreeSASA.Ua hoʻohana wale ʻia ka ʻikepili SASA piha a maopopo ʻole no ka histidine a me kona mau hoalauna e loaʻa ai ka SASA mean no kēlā me kēia hale.Ua helu ʻia ka ʻae ʻana o ka solvent (RSA) no kēlā me kēia histidine ma ka puʻunaue ʻana i ka waiwai SASA ponoʻī e ka empirical ʻoi loa o ke koena o ka ʻili i loaʻa i ka solvent.Hoʻokaʻawale ʻia nā histidines a pau i hūnā ʻia inā ʻoi aku ka nui o ka RSA ma lalo o 20%, inā ʻaʻole i ʻike ʻia56.
Ua ʻimi ʻia nā faila i loaʻa ma ke ʻano DDA me ka hoʻohana ʻana i ka Proteome Discoverer (v2.5) a i ʻole MSfragger (Fragpipe v15.0) i loko o ka SwissProt verified protein database i loaʻa nā mea haumia maʻamau.Pono nā peptides i ka trypsin piha me ʻelua mau wahi cleavage i nalowale, ka methylation carbamoyl ma ke ʻano he hoʻololi paʻa a me ka oxidation methionine ma ke ʻano he hoʻololi ikaika.Ua hoʻonohonoho ʻia ka precursor a me ka ʻāpana kaumaha i ka 10 ppm a me 0.02 Da (MS2 Orbitrap), i kēlā me kēia. Ua wehe ʻia nā mea hoʻohaumia, a ua kānana ʻia nā protein no ka loaʻa ʻana o ka helu ʻike wahaheʻe o <1%. Ua wehe ʻia nā mea hoʻohaumia, a ua kānana ʻia nā protein no ka loaʻa ʻana o ka helu ʻike wahaheʻe o <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэффициент ложного %. Ua wehe ʻia nā mea hoʻohaumia a ua kānana ʻia nā protein e hāʻawi i ka helu ʻike wahaheʻe o <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 обнаружения. Ua wehe ʻia nā mea hoʻohaumia a ua kānana ʻia nā protein e loaʻa i kahi helu kūpono wahaheʻe o <1%.No ka hoʻohālikelike ʻana me ka ʻole o ka hoʻohana ʻana i nā lepili, ua hoʻohana ʻia ka maʻiʻo protein maʻamau mai ʻekolu mau hana hou.Ua hana ʻia ka nānā ʻana o ka protein subcellular localization me ka hoʻohana ʻana i ka loiloi Gene Ontology (GO) mai DAVID Bioinformatics Resources, MitoCarta 3.0 a me nā ʻikepili i hōʻuluʻulu ʻia a paʻi ʻia e ka hui ʻo Alice Ting.Loaʻa ka palapala ʻāina pele mai Perseus (v1.6.15.0). Ua ho'āʻo ʻia nā hoʻololi ʻana i ka nui o ka protein no ke ʻano helu helu me ka hoʻohana ʻana i kahi t-ʻaoʻao ʻelua ʻaoʻao, a ua ʻike ʻia nā hits protein me ka loli nui> 2 (koe ke ʻōlelo ʻia) a me ka waiwai p <0.05. Ua ho'āʻo ʻia nā hoʻololi ʻana i ka nui o ka protein no ke ʻano helu helu me ka hoʻohana ʻana i kahi t-ʻaoʻao ʻelua ʻaoʻao, a ua ʻike ʻia nā hits protein me ka loli nui> 2 (koe ke ʻōlelo ʻia) a me ka waiwai p <0.05. Изменения кратности содержания белка были проверены на статистическую значимость с использованием двустороннего t-критерия, и совпадения белков были идентифицированы с изменением содержания> 2 (если не указано иное) и значением p <0,05. Ua ho'āʻo ʻia nā hoʻololi ʻana o ka protein no ka nui o ka helu helu me ka hoʻohana ʻana i kahi hoʻokolohua t-huelo ʻelua, a ua ʻike ʻia nā pāʻani protein me ka loli maʻiʻo> 2 (koe ke ʻike ʻole ʻia) a me ka waiwai ap <0.05.使用 双边 t 检验 "丰度蛋白质 蛋白质 的 统计统计, 丰度 变化 mp3 2 0.0使用 双边 t 检验 "倍测测 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. Ua hoʻāʻo ʻia ke ʻano o ka helu helu o nā loli i loko o ka protein ma ka hoʻohana ʻana i ka t-huelo ʻelua, a ua hoʻoholo ʻia nā pāʻani protein no nā loli ʻikepili> 2 (koe ke ʻole i hōʻike ʻia) a me nā p-values ​​<0.05.Ua hana ʻia ka ʻikepili pili protein me ka hoʻohana ʻana i ka ʻikepili GO me ka waihona String.
ʻEkolu hoʻopiʻi olaola i hana ʻia me nā hopena like.Ua hana ʻia ka ʻikepili helu me GraphPad Prism (polokalamu GraphPad) a ua hana ʻia nā ʻāpana lua pele me Perseus (v1.6.15.0).No ka hoʻohālikelike ʻana i nā pūʻulu ʻelua, ua hoʻoholo ʻia nā p-values ​​me ka hoʻohana ʻana i ka t-test a Student's two-tailed.ʻO nā protein singleton wale nō i ʻike ʻia ma ka liʻiliʻi ʻelua i loko o ka hui hoʻokolohua i hoʻokomo ʻia i loko o nā pā lua pele, a ua hoʻololi ʻia nā waiwai i nalowale i ka pūʻulu mana me Perseus mai kahi mahele maʻamau i hiki ke helu ʻia ka p-value.Hōʻike nā pahu kuhi i ka mean ± ka hoʻokaʻawale maʻamau.I loko o nā kānana proteomic no ka nānā ʻana i ka helu helu, ua mālama ʻia ka nui o nā protein i ʻike ʻia ma ka liʻiliʻi o ʻelua mau kope biological.ʻAʻole i hoʻohana ʻia nā ʻano helu helu no ka hoʻoholo mua ʻana i ka nui hāpana.ʻAʻole kūʻokoʻa nā hoʻokolohua.ʻAʻole makapō nā mea noiʻi i nā hana i ka wā o ka hoʻokolohua a me ka loiloi o nā hopena.
No ka ʻike hou aku e pili ana i ka hoʻolālā haʻawina, e ʻike i ka abstract Nature Research Report i pili i kēia ʻatikala.
Ua waiho ʻia ka ʻikepili spectrometry nui i loaʻa i kēia noiʻi i ka ProteomeXchange Consortium ma o ka waihona hoa iProX57 ma lalo o ka waihona ID PXD034811 (datadata PDPL-MS).Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.Hāʻawi kēia ʻatikala i ka ʻikepili kumu.
ʻO Gingras, AC, Abe, KT & Raught, B. Ka ʻike ʻana i ke kaiāulu: me ka hoʻohana ʻana i ka biotinylation pili pili kokoke e ʻike i nā paʻakikī protein a me nā organelles palapala. ʻO Gingras, AC, Abe, KT & Raught, B. Ka ʻike ʻana i ke kaiāulu: me ka hoʻohana ʻana i ka biotinylation pili pili kokoke e ʻike i nā paʻakikī protein a me nā organelles palapala.ʻO Gingras, AS, Abe, KT a me Raut, B. Kamaʻāina me ka puni: me ka hoʻohana ʻana i ka biotinylation pili pili kokoke e ʻike i nā paʻakikī protein a me nā organelles palapala. Gingras, AC, Abe, KT & Raught, B. Gingras, AC, Abe, KT & Raught, B. Ka hoʻomaopopo ʻana i ke kaiāulu: hoʻohana i ka hilinaʻi o ke kaiāulu i ke ola olaola.ʻO Gingras, AS, Abe, KT a me Raut, B. Ka hoʻomaopopo ʻana i ka pili kokoke: ke ʻano o nā paʻakikī protein a me ka palapala ʻana i nā organelles me ka hoʻohana ʻana i ka biotinylation pili kokoke.ʻO kēia manawa.ʻO koʻu manaʻo.Kemika.biology 48, 44–54 (2019).
Geri, JB et al.Ka palapala ʻāina i ka microenvironment ma ka hoʻoili ʻana i ka ikehu Dexter i nā cell immune.ʻEpekema 367, 1091–1097 (2020).
Hertling, EL et al.ʻIke ʻia nā ʻupena pākēneka ʻelua i ka hoʻoponopono hou ʻana o ka cell-specific remodeling o ke kamaʻilio kanaka.Nā Pūnaewele 184, 3022–3040.e3028 (2021).