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A na-eji usoro ntinye aha nso nso nke enzymatic dabere na esters na-arụ ọrụ ma ọ bụ radicals phenoxy na-arụ ọrụ n'ọtụtụ ebe iji mapụta proteomes subcellular na ndị na-emekọrịta protein na sel dị ndụ.Otú ọ dị, esters na-arụ ọrụ adịghị arụ ọrụ nke ọma, na-eme ka radius na-edepụta aha dị ukwuu, na phenoxy radicals nke sitere na ọgwụgwọ peroxide nwere ike igbochi ụzọ redox.N'ebe a, anyị na-akọ usoro ntinye aha nso nso nke ndabere fotoactivation (PDPL) nke mepụtara site na ijikọ mkpụrụ ndụ ihe nketa miniSOG photosensitizer protein na protein nke mmasị.N'ịbụ nke ọkụ na-acha anụnụ anụnụ na-achịkwa ma na-achịkwa site n'oge mkpughe, a na-emepụta oxygen singlet wee kpebie n'oge na-adịghị anya iji nweta akara nyocha nke aniline residues histidine.Anyị na-egosipụta nguzosi ike ya dị elu site na nhazi nke organelle-kpọmkwem proteome.Ntụle n'akụkụ n'akụkụ nke PDPL na TurboID na-egosi ihe mkpuchi proteomic zuru oke nke PDPL.Na-esote, anyị tinyere PDPL na ọrịa na-emetụta ọrịa transcriptional coactivator BRD4 na E3 Parkin ligase wee hụ ndị na-emekọrịta ihe na mbụ.Site na nleba anya nke ukwuu, ihe abụọ a na-amaghị, Ssu72 na SNW1, ka achọpụtara maka ọrịa Parkin, onye na-emebi mmebi ya site na ụzọ ubiquitination-proteasome.
Nkọwapụta ziri ezi nke netwọkụ protein na-adabere n'ọtụtụ usoro cellular bụ isi.Ya mere, maapụ spatiotemporal ziri ezi nke mmekọrịta protein ga-enye ihe ndabere mkpụrụ ndụ maka ịkọwapụta ụzọ ndụ, ọrịa ọrịa, na imebi mmekọrịta ndị a maka ebumnuche ọgwụgwọ.Iji mezuo nke a, ụzọ nwere ike ịchọpụta mmekọrịta nwa oge na sel ma ọ bụ anụ ahụ dị ndụ bụ ihe na-achọsi ike nke ukwuu.Affinity Purification Mass Spectrometry (AP-MS) ejirila akụkọ ihe mere eme iji chọpụta ndị mmekọ nke protein mmasị (POI).Site na mmepe nke usoro proteomics quantitative, e mepụtara Bioplex3.0, nchekwa data kacha ukwuu nke netwọkụ protein dabere na AP-MS.Ọ bụ ezie na AP-MS dị ike nke ukwuu, usoro cell lysis na dilution na usoro ọrụ na-adabere na mmekọrịta na-adịghị ike na nke na-adịghị adịte aka ma webata ihe ndị na-eme ka post-lysis dị ka ụzọ abụọ mmekọrịta na-adịghị mma nke na-enweghị nhazi tupu lysis.
Iji lebara okwu ndị a anya, emepụtarala amino acid na-ekwekọghị n'okike (UAA) nwere otu ndị na-emekọrịta ihe yana ikpo okwu enzymatic dị nso (PL) (dịka ọmụmaatụ APEX na BioID)5.Ọ bụ ezie na etinyerela usoro UAA nke ọma n'ọtụtụ ọnọdụ ma na-enye ozi na nrapado protein kpọmkwem, njikarịcha saịtị ntinye UAA ka chọrọ.Nke kachasị mkpa, ọ bụ usoro ịde aha stoichiometric nke na-enweghị ngbanwe mgbanwe nke mmemme ịkpọ aha.N'ụzọ dị iche, usoro enzymatic PL, dị ka usoro BioID, na-ejikọta biotin ligase nke injinia na POI7, nke na-emesị mee biotin ka ọ ghọọ biotinyl-AMP ester intermediate reactive.Enzyme si otú ahụ na-akpali ma wepụta “igwe ojii” biotin na-arụ ọrụ nke na-edepụta ihe fọdụrụ lysine dị nso.Agbanyeghị, BioID chọrọ ihe karịrị awa iri na abụọ iji nweta akara ngosi zuru oke, nke na-egbochi iji ya na mkpebi nwa oge.N'iji evolushọn a na-eduzi dabere na ihe ngosi yist, e mere TurboID dabere na BioID ka ọ na-arụ ọrụ nke ọma, na-enye ohere iji biotin na-ede aha nke ọma n'ime nkeji 10, na-enye ohere ka a mụọ usoro dị ike karị.N'ihi na TurboID na-arụsi ọrụ ike nke ukwuu yana ọkwa biotin endogenous zuru oke maka akara ọkwa dị ala, ịde akara n'azụ na-abụ nsogbu nwere ike mgbe a na-achọ ka mmado nke ukwuu na akara oge site na mgbakwunye nke biotin exogenous.Na mgbakwunye, esters na-arụ ọrụ adịghị arụ ọrụ nke ọma (t1 / 2 ~ 5 min), nke nwere ike iduga na radius dị ukwuu, karịsịa mgbe saturation nke protein ndị agbata obi na biotin 5. N'ụzọ ọzọ, njikọ mkpụrụ ndụ ihe nketa nke engineered ascorbate peroxidase (ie biotin- phenol radicals ma na-enye ohere ntinye aha protein n'ime otu nkeji9,10. A na-eji APEX eme ihe n'ọtụtụ ebe iji chọpụta proteomes subcellular, protein membranes, na cytosolic signaling protein complexes11,12. Otú ọ dị, mkpa maka nnukwu mkpokọta peroxides nwere ike imetụta protein redox ma ọ bụ ụzọ, na-akpaghasị. usoro cellular.
Ya mere, usoro ọhụrụ nke nwere ike iwepụta ụdị mmegide nke akpọrọ-radius suppression nke nwere oke ohere na ogologo oge na-akpaghasịghị ụzọ ekwentị nke ukwuu ga-abụ ihe mgbakwunye dị mkpa na usoro ndị dị adị. N'ime ụdị ndị na-emeghachi omume, oxygen singlet kpaliri mmasị anyị n'ihi obere ndụ ya na obere mgbasa ozi radius (t1/2 <0.6 µs na sel)13. N'ime ụdị ndị na-emeghachi omume, oxygen singlet kpaliri mmasị anyị n'ihi obere ndụ ya na obere mgbasa ozi radius (t1/2 <0.6 µs na sel)13. Среди активных форм наше внимание привлек синглетный кислород из-за его короткого времени жизни и ограниченного радиуса диффузии (t1/2 < 0,6 мкс в клетках)13. N'ime ụdị arụ ọrụ, oxygen singlet dọtara uche anyị n'ihi obere oge ndụ ya na obere mgbasa ozi radius (t1/2 <0.6 µs na sel)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）的有關 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 ограниченного радиуса диффузии (t1/2 < 0,6 мкс в клетках). N'ime ụdị arụ ọrụ, oxygen singlet na-adọta uche anyị n'ihi obere oge ndụ ya na obere mgbasa ozi radius (t1/2 <0.6 μs na sel).A kọwala ikuku oxygen na-enweghị ihe ọ bụla na-eme ka methionine, tyrosine, histidine na tryptophan, na-eme ka ọ bụrụ polar 14,15 maka ntinye aka na amine ma ọ bụ thiol based probes16,17.Ọ bụ ezie na ejirila oxygen singlet kpọọ RNA ngalaba subcellular, a na-ahụbeghị usoro maka iweghachi akara nso nso POI endogenous.N'ebe a, anyị na-eweta ikpo okwu akpọrọ photoactivation-dependent proximity labeling (PDPL), ebe anyị na-eji ọkụ na-acha anụnụ anụnụ na-amụnye POI nke jikọtara ya na miniSOG photosensitizer ma na-akpalite otu ikuku oxygen iji oxidize ihe fọdụrụnụ, na-esote mgbanwe nke amine iji oxidize nyocha kemịkalụ n'ime. sel ndị dị n'etiti..Anyị nwalere otu nyocha kemịkalụ ka iwelie mkpado mkpado yana wepụta saịtị mgbanwe site na iji usoro ọrụ proteomics mepere emepe.Ntụle n'akụkụ n'akụkụ nke PDPL na TurboID na-egosi ihe mkpuchi proteomic zuru oke nke PDPL.Anyị na-etinye usoro a na ihe nrịbama organelle-kpọmkwem nke proteome subcellular na njirimara proteome izugbe nke ndị mmekọ njikọ maka protein epigenetic regulatory protein BRD4 metụtara ọrịa kansa na ọrịa Parkinson metụtara E3 ligase Parkin, bụ nke kwadoro ma netwọkụ protein ama ama na amabeghị. mmekọrịta..Ikike nke PDPL ịmata mkpụrụ E3 na nnukwu protein protein na-anọchite anya ọnọdụ ebe achọrọ nnabata nke ndị na-eke ihe na-apụtaghị ìhè.Achọpụtala ihe mgbochi ogige abụọ amabeghị nke sitere na ubiquitination-proteasome na ọnọdụ.
Usoro ọgwụgwọ Photodynamic (PDT) 19 na chromophore-assisted laser inactivation (CALI) 20, nke ọkụ ọkụ na fotosensitizers na-emepụta oxygen singlet, nwere ike ime ka protein ndị e lekwasịrị anya rụọ ọrụ ma ọ bụ bute ọnwụ cell.Ebe ọ bụ na oxygen singlet bụ ihe na-emeghachi omume nke ukwuu nke nwere usoro mgbasa ozi dị ihe dị ka 70 nm, enwere ike ịchịkwa oxidation na-enweghị oke gburugburu fotosentizer.Dabere na echiche a, anyị kpebiri iji oxygen singlet iji nweta akara nso nke ogige protein na sel dị ndụ.Anyị azụlitela PDPL chemoproteomic obibia iji mezuo ọrụ anọ: (1) iji mee ka ọgbọ nke oxygen singlet na-arụ ọrụ dị ka usoro enzymatic PL;(2) nye akara oge edoziri n'elu mmalite ọkụ;(3) site na ngbanwe (4) Zenarị iji endogenous cofactors (dị ka biotin) iji belata nzụlite, ma ọ bụ jiri nnukwu reagenous exogenous reagents (dị ka peroxides) belata mkpughe cell na nrụgide gburugburu ebe obibi.
Enwere ike ikewa fotosensitizers ụzọ abụọ gụnyere obere fluorophores dị arọ (dịka ọmụmaatụ rose bengal, methylene blue) 22 na mkpụrụ ndụ obere protein nwere mkpụrụ ndụ ihe nketa (dịka miniSOG, KillerRed)23.Iji nweta ihe ngosi modular, anyị mepụtara usoro PDP ọgbọ mbụ site na ịgbakwunye protein fotosentizer (PS) na POI24,25 (Figure 1a).Mgbe ejiri ọkụ na-acha anụnụ anụnụ jiri ọkụ na-acha anụnụ anụnụ, singlet oxygen oxidizes proximal nucleophilic amino acid residues, na-ebute polarity umpolung nke bụ electrophilic ma nwee ike meghachi omume na amine probe nucleophiles16,17.Emebere nyocha ahụ site na iji aka alkyne iji kwe ka kemistri pịa wee wetuo ala maka njirimara LC/MS/MS.
Nleta atụmatụ nke ịde aha ogige protein nke miniSOG na-elekọta ya.Mgbe ekpughere ya na ọkụ na-acha anụnụ anụnụ, mkpụrụ ndụ na-egosipụta miniSOG-POI na-emepụta oxygen singlet, nke na-agbanwe protein ndị na-emekọrịta ihe ma ọ bụghị protein na-enweghị njikọ.A na-ejide ngwaahịa dị n'etiti nke photooxidation site na akara relay nke nyocha kemịkalụ amine iji mepụta nkwado ndabere.Otu alkynyl na nyocha kemịkalụ na-enye ohere pịa kemịkalụ maka ịba ụba site na ịdọrọ ala na-esote LC-MS/MS quntitation.b Usoro ihe omimi nke amine nyocha 1-4.c Nnọchite anya fluorescent gel nyocha nke mitochondrial localized miniSOG-mediated proteomic markers na-eji nyocha 1-4 na nha nha dabere na densitometry gel.A tụlere ogo mgbama na azụ azụ nke nyocha kemịkalụ site na iji nnwale njikwa na-adịghị mma ewepu ọkụ na-acha anụnụ anụnụ ma ọ bụ iji mkpụrụ ndụ HEK293T na-enweghị okwu miniSOG.n = 2 ihe nlere nke nwere onwe ya.Ntụpọ ọ bụla na-anọchite anya oyiri ndu.d Nchọpụta ndị nnọchite anya na ọnụọgụ nke PDPL site na iji nyocha kachasị mma 3 na ọnụnọ ma ọ bụ enweghị akụrụngwa PDPL egosipụtara dị ka c.n = 3 ihe nlere anya nwere onwe ya.Ntụpọ ọ bụla na-anọchite anya oyiri ndu.Akara etiti na afụ ọnụ na-anọchi anya ihe pụtara na ± ọkọlọtọ.CBB: Coomassie Brilliant Blue.e Confocal onyogho nke otu oxygen na-acha uhie uhie Si-DMA unyi.Ogwe ọnụ ọgụgụ: 10µm.A na-emegharị ihe onyonyo gel na nyocha nlegharị anya n'onwe ha ma ọ dịkarịa ala ugboro abụọ na nsonaazụ yiri ya.
Anyị buru ụzọ nwale ike nke ndị tozuru okè photosensitizers miniSOG26 na KillerRed23, guzosie ike egosipụta na HEK293T, mediate propargylamine labeling nke proteome dị ka kemịkalụ nyocha (Mgbakwunye Fig. 1a).Gel fluorescence analysis gosiri na a na-enweta akara proteome dum site na iji miniSOG na irradiation ọkụ na-acha anụnụ anụnụ, ebe ọ nweghị ngwaahịa akara ngosi a na-ahụ anya na KillerRed.Iji meziwanye ogo mgbama gaa n'azụ, anyị nwalere usoro nyocha kemịkalụ nwere aniline (1 na 3), propylamine (2), ma ọ bụ benzylamine (4).Anyị chọpụtara na mkpụrụ ndụ HEK293T n'onwe ha nwere akara ngosi dị elu ma e jiri ya tụnyere enweghị ìhè na-acha anụnụ anụnụ, ikekwe n'ihi riboflavin photosensitizer endogenous, flavin mononucleotide (FMN) 27. Nyocha kemịkalụ nke Aniline 1 na 3 nyere nkọwapụta ka mma, yana HEK293T na-egosipụta nke ọma miniSOG na mitochondria na-egosipụta> mmụba okpukpu 8 na mgbama maka nyocha 3, ebe nyocha 2 ejiri mee akara akara RNA CAP-seq naanị na-egosipụta ~ 2.5- Mpịakọta mgbaama na-abawanye, ikekwe n'ihi mmasị mmeghachi omume dị iche iche n'etiti RNA na protein (Fig 1b, c). Nyocha kemịkalụ nke Aniline 1 na 3 nyere nkọwapụta ka mma, yana HEK293T na-egosipụta nke ọma miniSOG na mitochondria na-egosipụta> mmụba okpukpu 8 na mgbama maka nyocha 3, ebe nyocha 2 ejiri mee akara akara RNA CAP-seq naanị na-egosipụta ~ 2.5- Mpịakọta mgbaama na-abawanye, ikekwe n'ihi mmasị mmeghachi omume dị iche iche n'etiti RNA na protein (Fig 1b, c).Nyocha kemịkalụ nke Aniline 1 na 3 gosipụtara nkọwapụta ka mma: HEK293T, nke na-egosipụta nke ọma miniSOG na mitochondria, na-egosi ihe karịrị okpukpu 8 na mgbama maka nyocha 3, ebe nyocha 2, ejiri na usoro akara akara CAP-seq RNA, naanị. na-egosi ~ 2.5 ugboro mgbama mmụba, eleghị anya n'ihi dị iche iche reactivity mmasị n'etiti RNA na protein (Fig. 1b, c).苯胺 的 1 和 3 和 具有 3 和 具有 具有 具有 具有 粒体 My 在 Minisus, 探针 3 倍 信号, 倍, 用, 用 用 Rna 标记 探针 2 显示 显示 2 显示 显示 2 显示 显示 2 显示 显示 2 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）.苯胺 的 1 的 3 和 3 具有 3 具有 具有 Minisog, 探针 3 的 而 而 而 而 rna 标记 col-eq 仅 2 ~ ~ ~ 2.5 -倍信号增加，可能是由于RNANyocha kemịkalụ nke Aniline 1 na 3 nwere nkọwa dị mma karịa, HEK293T gosipụtara miniSOG n'ụzọ kwụ ọtọ na mitochondria, na nyocha 3 nwere mmụba nke okpukpu 8 karịa, ebe nyocha 2 maka akara akara CAP-seq RNA gosipụtara naanị ~ 2.5 - mmụba.na mgbaama, eleghị anya n'ihi mmasị mmeghachi omume dị iche iche n'etiti RNA na protein (Fig 1b, c).Na mgbakwunye, a nwalere nyocha 3 isomers na hydrazine probes (nyocha 5, 6, 7), na-akwado njikarịcha nke nyocha 3 (Mgbakwunye 1b, c).N'otu aka ahụ, nyocha nke in-gel fluorescence na-ekpughe ihe nyocha ndị ọzọ kachasị mma: irradiation wavelength (460 nm), nyocha nyocha nke kemịkalụ (1 mM), na oge irradiation (20 min) (Mgbakwunye 2a-c).Ịhapụ akụkụ ọ bụla ma ọ bụ nzọụkwụ ọ bụla na usoro PDPL mere ka mgbama dị ịrịba ama gbanwee n'azụ (Fig 1d).N'ụzọ doro anya, a na-ebelata akara ngosi protein nke ọma na ọnụnọ sodium azide ma ọ bụ trolox, nke a maara na-emenyụ ikuku oxygen na-adịghị.Ọnụnọ nke D2O, nke a maara na ọ na-eme ka ikuku oxygen guzosie ike, na-eme ka akara ngosi akara.Iji nyochaa ntinye aka nke ụdị oxygen ndị ọzọ na-emeghachi omume na ịde aha, mannitol na vitamin C agbakwunyere iji guzobe hydroxyl na superoxide radical scavengers, n'otu n'otu, 18, 29, mana achọtaghị ha iji belata labeling.Mgbakwunye H2O2, ma ọ bụghị ọkụ, arụpụtaghị akara (Mgbakwunye Fig. 3a).Fluorescence singlet oxygen imaging na Si-DMA probes kwadoro ọnụnọ nke ikuku oxygen dị na waya HEK293T-miniSOG, mana ọ bụghị na waya HEK293T mbụ.Tụkwasị na nke ahụ, mitoSOX Red enweghị ike ịchọpụta mmepụta superoxide mgbe ìhè gasịrị (Fig 1e na Fig. 3b) 30. Data ndị a na-egosi na ọ bụ nanị otu oxygen na-emeghachi omume bụ ụdị ikuku oxygen na-eme ka ọ bụrụ akara proteomic na-esote.A na-enyocha cytotoxicity nke PDPL gụnyere ọkụ ọkụ na-acha anụnụ anụnụ na nyocha kemịkalụ, ọ dịghịkwa ihe dị ịrịba ama cytotoxicity a hụrụ (Mgbakwunye Fig. 4a).
Iji mụọ usoro nrịbama ma mee ka njirimara proteomic nke ogige protein site na iji LC-MS/MS, anyị kwesịrị ibu ụzọ chọpụta nke amino acid emezigharịrị yana ọnụọgụ delta nke akara nyocha.A kọwo na methionine, histidine, tryptophan na tyrosine na-emezi ya site na singlet oxygen14,15.Anyị na-ejikọta usoro ọrụ TOP-ABPP31 na ọchụchọ enweghị mmasị na-emeghe nke FragPipe computing ikpo okwu na-enye dabere na MSFragger32.Ka emechara mgbanwe oxygen singlet na akara nyocha kemịkalụ, pịa kemịkalụ ka emere site na iji akara mbelata biotin nwere njikọ nwere ike ihie, na-esote neutravidin stretching na mgbaze trypsin.peptide emezigharịrị, ka ejikọta ya na resin, ka ewepụchara foto maka nyocha LC-MS/MS (Ọnọdụ 2a na Data mgbakwunye 1).Ọnụ ọgụgụ dị ukwuu nke mgbanwe mere na proteome nwere ihe karịrị 50 peptide map (PSM) e depụtara (Fig. 2b).N'ụzọ dị ịtụnanya, anyị hụrụ naanị mgbanwe nke histidine, ikekwe n'ihi mmeghachi omume dị elu nke oxidized histidine n'ihe nyocha aniline karịa amino acid ndị ọzọ.Dị ka usoro e bipụtara nke histidine oxidation site na singlet oxygen, 21,33 usoro nhazi nke delta-mass nke +229 Da kwekọrọ na ntinye nke nyocha 3 na 2-oxo-histidine mgbe oxidation abụọ gasịrị, ebe + 247 Da bụ ngwaahịa hydrolysis. nke +229 Da (Mgbakwunye Fig. 5).Ntụle nke ụdịdị dị iche iche MS2 gosipụtara ntụkwasị obi dị elu nke njirimara nke ọtụtụ n'ime y na b ion, gụnyere njirimara nke ion gbanwetụrụ (y na b) (Fig. 2c).Ntụle ihe omimi nke usoro mpaghara nke PDPL-gbanwetụrụ histidines gosipụtara mmasị na-adịghị mma maka obere hydrophobic residues na ± 1 ọnọdụ (Mgbakwunye 4b).Ná nkezi, 1.4 histidines ka a chọpụtara n'otu protein, na saịtị ndị a nrịbama ka e kpebisiri ike site ihe mgbaze inweta n'elu ebe (SASA) na ikwu ihe mgbaze nnweta (RSA) analysis (Mgbakwunye Fig. 4c, d).
Usoro ọrụ na-enweghị isi maka ịmụ nhọrọ ihe fọdụrụ site na iji ikpo okwu FragPipe nke MSFragger kwadoro.A na-eji ndị na-ahụ maka ihie ụzọ na Pịa kemịkalụ iji kwe ka ikpochapụ peptides gbanwetụrụ site na resin streptavidin.A malitere nchọta mepere emepe iji chọpụta ọtụtụ mgbanwe, yana ihe fọdụrụ dị mkpa.b Nyefee oke mgbanwe na-eme na proteome dum.Peptide maapụ PSM.c MS2 spectral nkọwa nke saịtị histidine ejiri nyocha 3. Dị ka ihe atụ nnọchite anya, mmeghachi omume covalent nwere nyocha 3 gbakwunyere +229.0938 Da na amino acid emezigharịrị.d Mgbanwe nyocha ejiri nwalee maka akara PDPL.PRDX3 (H155A, H225A) na PRDX1 (H10A, H81A, H169A) ka ejiri ụdị plasmid nke anụ ọhịa butere ya maka nchọpụta mgbochi ọkọlọtọ.e meghachiri peptide sịntetik na miniSOG dị ọcha n'ihu nyocha 3 na ngwaahịa kwekọrọ na Δm +247 na +229 ka edepụtara na ụdị LC-MS.f In vitro protein-na-protein mmekọrịta nke ejiri miniSOG-6xHis-tag na mgbochi 6xMgbochi ya.Antibiotin (streptavidin-HRP) na mgbochi òké Western blot analysis nke miniSOG-6xHis/anti-6xMgbochi mgbochi ya nke akara 3, dabere na oge ikpughe na ìhè.A na-egosipụta akara maka protein n'otu n'otu n'ụdị nha nha nha nha: LC antibody light chain, HC antibody heavy chain.Emegharịrị nnwale ndị a n'onwe ha ma ọ dịkarịa ala ugboro abụọ nwere nsonaazụ yiri ya.
Maka nkwenye biochemical nke saịtị ịde aha, PRDX3 na PRDX1 nke a chọpụtara site na mass spectrometry gbanwere site na histidine gaa na alanine ma jiri ya tụnyere ụdị anụ ọhịa na nyocha mbufe.Nsonaazụ PDPL gosiri na ngbanwe ahụ belatara nke ukwuu akara aha (Fig 2d).Ka ọ dị ugbu a, a na-ahazi usoro peptide ndị a chọpụtara na nchọta mepere emepe ma meghachi omume na vitro na miniSOG dị ọcha n'ihu nyocha 3 na ìhè na-acha anụnụ anụnụ, na-emepụta ngwaahịa na ngbanwe nke + 247 na + 229 Da mgbe LC-MS chọpụtara (Fig). .2e).).Iji nwalee ma enwere ike ịkpọ protein proximal na-emekọrịta ihe na vitro na nzaghachi miniSOG photoactivation, anyị haziri nyocha ịdị nso nke artificial site na mmekọrịta dị n'etiti protein miniSOG-6xHis na mgbochi mgbochi ya monoclonal in vitro (Nyocha 2f).N'ime nyocha a, anyị tụrụ anya na a ga-eji miniSOG akara akara nso nso nke mgbochi mmadụ dị arọ na nke dị mfe.N'ezie, mgbochi òké (ịghọta na agbụ dị arọ na nke dị mfe nke mgbochi 6xHis-labeled antibody) na streptavidin Western blots gosipụtara biotinylation siri ike nke ụdọ dị arọ na ọkụ.N'ụzọ doro anya, anyị hụrụ miniSOG autobiotinylation n'ihi mkpado 6xYa na njikọ njikọ n'etiti ọkụ na agbụ dị arọ, nke nwere ike jikọta ya na oghere a kọwara na mbụ n'etiti lysine na 2-oxo-histidine proximal nzaghachi.N'ikpeazụ, anyị kwubiri na PDPL na-agbanwe histidine n'ụzọ dabere nso.
Ihe mgbaru ọsọ anyị na-esote bụ ịkọwapụta proteome subcellular iji nwalee kpọmkwem nke ịde aha ọnọdụ.Ya mere, anyị kwuputara nke ọma miniSOG na nucleus, mitochondrial matrix, ma ọ bụ mpụta ER akpụkpọ anụ nke sel HEK293T (Fig. 3a).Nyocha nke gel fluorescence kpughere ọtụtụ band ndị akpọrọ aha n'ebe atọ dị n'okpuru cell yana usoro akara aha dị iche iche (Fig 3b).Nnyocha nyocha nke fluorescence gosipụtara nkọwa dị elu nke PDPL (Fig 3c).A na-esochi usoro ọrụ PDPL site na mmeghachi omume pịa ya na akwa rhodamine iji kọwaa proteomes subcellular site na iji microscopy fluorescence, na akara PDPL ejiri DAPI, mitochondrial trackers, ma ọ bụ ER trackers, na-akwado ntụkwasị obi dị elu nke PDPL.Maka ebe organelle atọ, ntụnyere n'akụkụ n'akụkụ PDPL na TurboID site na iji avidin western blot gosiri na a na-akpọ PDPL nke ọma ma e jiri ya tụnyere njikwa ha.N'okpuru ọnọdụ PDPL, ọtụtụ eriri ndị akpọrọ aha pụtara, na-egosi ọtụtụ protein ndị akpọrọ PDPL (Mgbakwunye 6a-d).
ihe nnochite anya nke miniSOG-mediated organelle-specific proteome labeling.miniSOG lekwasịrị anya matriks mitochondrial site na njikọta na N-terminal 23 amino acids nke mmadụ COX4 (mito-miniSOG), nucleus site na njikọ na H2B (nucleus-miniSOG), na Sec61β site na akụkụ cytoplasmic nke akpụkpọ ahụ ER (ER-miniSOG). ).Ngosipụta gụnyere onyonyo gel, onyonyo confocal, na spectrometry mass.b Ihe nnochite anya gel onyogho nke profaịlụ PDPL nke organelle puru iche.CBB Coomassie na-acha anụnụ anụnụ.c onyogho confocal nke mkpụrụ ndụ HEK293T na-egosipụta nke ọma miniSOG na mpaghara subcellular dị iche iche nke ihe mgbochi akpọrọ V5 (red) achọpụtara.A na-eji akara subcellular maka mitochondria na ER (green).Usoro ọrụ PDPL gụnyere nchọpụta nke miniSOG (edo edo) nke akpọrọ proteomes subcellular na-eji Cy3-azide click chemistry.Ogwe ọnụ ọgụgụ: 10µm.d Atụmatụ mgbawa ugwu nke proteomes akara PDPL n'ime akụkụ akụkụ ahụ dị iche iche akọwapụtara site na ọnụọgụ enweghị akara (n = 3 nnwale nke ndu ndu onwe ya).Ejiri t-ule nwata akwụkwọ nwere ọdụ abụọ na ala mgbawa ugwu.A na-eji ụdị anụ ọhịa HEK293T mee ihe nchịkwa na-adịghị mma. A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> 2-fold ion intensity intensity). A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> 2-fold ion intensity intensity). Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в интенсивности ионом). A na-akọwapụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> ọdịiche okpukpu abụọ na ike 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). A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> ọdịiche okpukpu abụọ na ike ionic).Protein emetụtara dị mkpa maka HEK293T-miniSOG mana ọ bụghị ihe dị mkpa maka HEK293T ka egosiri na akwụkwọ ndụ akwụkwọ ndụ.e Ntụle nke nkọwapụta datasets proteomic sitere na nnwale d.Ngụkọta ọnụ ọgụgụ protein dị ịrịba ama na akụkụ akụkụ ọ bụla (ntụpọ uhie na akwụkwọ ndụ akwụkwọ ndụ) ka akara n'elu.Histogram na-egosi protein ndị etinyere na organelles dabere na MitoCarta 3.0, GO analysis na A. Ting et al.ndị mmadụ.Ntọala dị iche iche maka mitochondria, nuclei, na ER.Emegharịrị nnwale ndị a n'onwe ha ma ọ dịkarịa ala ugboro abụọ nwere nsonaazụ yiri ya.A na-enye data raw n'ụdị faịlụ data raw.
N'ịgba ume site na gel na nsonaazụ onyonyo, a na-eji ọnụọgụ na-enweghị akara akara iji tụọ proteome amatara na akụkụ ọ bụla (Data mgbakwunye 2).Ejiri HEK293T na-enweghị mbugharị ka ejiri mee ihe nchịkwa na-adịghị mma iji wepụ ihe nrịbama azụ. Nyochaa nkata mgbawa ugwu gosipụtara protein ndị bara ụba nke ukwuu (p <0.05 na> 2-fold ion intensity) yana protein dị otuton dị naanị na ahịrị miniSOG na-egosipụta (Fig 3d red na green ntụpọ). Nyochaa nkata mgbawa ugwu gosipụtara protein ndị bara ụba nke ukwuu (p <0.05 na> 2-fold ion intensity) yana protein dị otuton dị naanị na ahịrị miniSOG na-egosipụta (Fig 3d red na green ntụpọ). 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). Nyocha nke mgbawa ugwu gosipụtara protein ndị bara ụba nke ukwuu (p <0.05 na> 2-fold ion intensity) yana otu protein dị naanị na miniSOG na-egosipụta ahịrị (Fig 3d, ntụpọ uhie na akwụkwọ ndụ akwụkwọ ndụ).分析 显示 <<0.05 和> 2 倍 仅 仅 minisog 表达 中 中 中 中 中 中 (图 3d 红色).分析 显示 (p <0.05 和> 2 离子 存 Minisog 表达 在于 的 Mini 单一 (图 3rd 红色 ...)))))))) 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). Nyochaa nkata mgbawa ugwu kpughere protein bara ụba nke ukwuu (p<0.05 na> 2x ionic ike) yana otu protein dị naanị na ahịrị okwu miniSOG ( ntụpọ uhie na akwụkwọ ndụ akwụkwọ ndụ na Fig. 3d).N'ịkọkọta data ndị a, anyị chọpụtara 1364, 461, na 911 ngwangwa nuklia, mitochondrial, na ER n'elu membrane protein, n'otu n'otu.Iji nyochaa izi ezi nke organelle-localized PDPL, anyị jiri MitoCarta 3.0, Gene Ontology (GO) analysis, na A. Ting et al.A na-eji data set8 mee ihe maka mitochondria, nucleus, na ER iji nwalee nkọwa nke organelle nke protein ndị achọpụtara, kwekọrọ na izi ezi nke 73.4, 78.5, na 73.0% (Fig 3e).Nkọwapụta nke PDPL na-akwado na PDPL bụ ngwá ọrụ dị mma maka ịchọpụta proteome nke organelle.N'ụzọ doro anya, nyocha nke subochondrial nke protein mitochondrial achọpụtara gosiri na a na-ekesa proteome tọrọ atọ na matrix na akpụkpọ anụ dị n'ime (226 na 106, n'otu n'otu), na-aza 91.7% (362) nke ngụkọta ọnụ ọgụgụ protein mitochondrial.A kwadoro ọkwa dị elu nke PDPL (Foto mgbakwunye 7a).N'otu aka ahụ, nyocha nke subnuclear gosiri na a na-ekesa proteome e weghaara na nucleus, nucleoplasm, na nucleolus (Fig. 7b).Ntụle proteomic nke nuklia na peptide localization nke nuklia (3xNLS) gosipụtara izi ezi yiri nke ahụ na nrụpụta H2B (Foto mgbakwunye 7c-h).Iji chọpụta kpọmkwem ihe nrịbama PDPL, a họọrọ laminin nuklia dị ka ọnyà POI7 nke nwere ezi uche karịa.PDPL kwuru na protein 36 bara ụba nke ukwuu, nke protein 12 (30.0% gụnyere lamin A) bụ protein na-emekọrịta nke ọma nke ejiri mara ọdụ data String, yana pasent dị elu karịa usoro BioID (protein 122) 28 nke 28. , 22.9 %) 7. Usoro anyị chọpụtara na protein ole na ole, ikekwe n'ihi oke akara akara, bụ nke mere ka o kwe omume site na otu ikuku oxygen na-arụsi ọrụ ike.Nnyocha GO gosiri na protein ndị ahụ achọpụtara dị na nucleoplasm (26), akpụkpọ anụ nuklia (10), akpụkpọ anụ nuklia (9), na pores nuklia (5).N'ịchịkọta, protein ndị a na-emepụta nuklia na-enweta 80% nke protein ndị bara ọgaranya, na-egosipụtakwa nkọwa nke PDPL (Fig. 8a-d).
N'ịbụ onye guzobeworo ike nke PDPL ime akara nso nso na organelles, anyị nwalere ma enwere ike iji PDPL nyochaa ndị mmekọ POI.Karịsịa, anyị chọrọ ịkọwapụta nyocha nke PDPL nke protein cytosolic, bụ ndị a na-ewere dị ka ebumnuche siri ike karịa ndị ibe ha nwere mpaghara akpụkpọ ahụ n'ihi ọdịdị ha siri ike.Bromodomain na extraterminal (BET) protein BRD4 dọtara uche anyị maka isi ọrụ ya na ọrịa dị iche iche 35, 36.Mgbagwoju anya nke BRD4 hibere bụ ihe nkwado ederede na ebumnuche ọgwụgwọ dị mkpa.Site n'ịchịkwa okwu nke c-myc na ihe odide Wnt5a, a na-eche na BRD4 bụ isi ihe na-achọpụta ọrịa leukemia myeloid ukwu (AML), multiple myeloma, lymphoma Burkitt, ọrịa cancer colon na ọrịa mkpali37,38.Na mgbakwunye, ụfọdụ nje na-elekwasị anya BRD4 iji mezie ndenye nje na cellular, dị ka papillomavirus, HIV, na SARS-CoV-236,39.
Iji mapụta mmekọrịta BRD4 site na iji PDPL, anyị jikọtara miniSOG na obere N- ma ọ bụ C-terminal isoform nke BRD4.Nsonaazụ proteomic gosipụtara ogo dị elu nke ndakọrịta n'etiti ihe nrụpụta abụọ ahụ (Fig 9a mgbakwunye).Ihe nchebe nuklia nke ejikọtara na miniSOG-H2B na-ekpuchi 77.6% nke protein ndị na-emekọrịta ihe na BRD4 (Mgbakwunye 9b).Mgbe ahụ, a na-eji oge dị iche iche nke ọkụ (2, 5, 10, 20 min) mee ihe iji dozie radius akara (Fig. 4a na data mgbakwunye 3).Anyị kwubiri na na obere oge foto, PDPL ga-ebu ụzọ kpọọ ndị mmekọ na-ejikọta ọnụ, ebe ogologo oge ga-agụnye protein ndị achọpụtara n'oge oge fotoactivation dị mkpụmkpụ yana ebumnuche na-apụtaghị ìhè na ụlọ ịde aha.N'ezie, anyị chọtara nkwụsịtụ siri ike n'etiti oge dị n'akụkụ (84.6% maka 2 na 5 min; 87.7% maka 5 na 10 min; 98.7% maka 10 na 20 min) (Fig. 4b na Fig. 9c).N'ime otu nnwale niile, anyị hụrụ ọ bụghị naanị BRD4 ịde aha onwe, mana ọtụtụ ebumnuche ama ama dịka MED1, CHD8, BICRA, NIPBL, SMC1A, na HMGB1 akọwapụtara na nchekwa data eriri.Ike ionic nke ebumnuche ndị a dabara na oge mkpughe (Fig 4c na Fig. 9d mgbakwunye).Nyocha GO nke protein ndị a chọpụtara na nkeji nkeji 2 gosiri na a na-edozi protein ndị a chọpụtara na nucleus ma tinye aka na nhazi chromatin na ọrụ RNA polymerase.A na-eme ka ọrụ molekụla nke protein dịkwuo mma na chromatin na-ejikọta ma ọ bụ nchịkọta ederede, na-adabere na ọrụ BRD4 (Fig. 4d).Ntụle mkparịta ụka protein na-enye eriri nchekwa data gosipụtara ọkwa mbụ nke mmekọrịta na-apụtaghị ìhè n'etiti BRD4 na HDAC ezinụlọ na-emekọrịta ihe dị ka SIN3A, NCOR2, BCOR, na SAP130 (Fig. 4e na Mgbakwunye Fig. 9e), kwekọrọ na BRD4 na HDAC na-ejikọta acetylated histones. ..Tụkwasị na nke ahụ, ndị nnọchiteanya nke LC-MS/MS chọpụtara, gụnyere Sin3A, NSUN2, Fus, na SFPQ, kwadoro site na Western blotting (Fig. 4f).N'oge na-adịbeghị anya, a kọwapụtara obere isoform nke BRD4 ka ọ mepụta nuclei nwere ihe nkewa mmiri-mmiri mmiri (LLPS).Ndị na-edozi RNA na-ejikọta Fus na SFPQ na-edozi LLPS nke usoro cellular dị iche iche ma chọpụta ebe a dị ka protein na-ejikọta BRD4 edekọghị.Ekwenyere mmekọrịta dị n'etiti BRD4 na SFPQ site na nyocha nke co-immunoprecipitation (co-IP) (Nyocha 4g), na-atụ aro usoro ọzọ maka BRD4-mediated liquid-mmire nkewa nke kwesịrị nyocha ọzọ.Ejikọtara ọnụ, nsonaazụ ndị a na-egosi na PDPL bụ ezigbo ikpo okwu maka ịchọpụta mmekọrịta BRD4 ama ama yana protein na-ejikọ ama ama.
ihe nnochite anya nke miniSOG-mediated BRD4 nso nso, oge mkpughe: 2, 5, 10, na 20 min.b Mkpokọta protein achọpụtara n'oge ọkụ dị iche iche.Nkwalite protein nke achọpụtara na HEK293T-miniSOG-BRD4 dị ịrịba ama n'ụzọ mkpokọta tụnyere ụdị anụ ọhịa HEK293T.c Ion ike mgbe ị na-agụta onye nnọchi anya na-enweghị aha amaara protein protein na-ejikọta BRD4 n'oge oge mkpughe akọwapụtara.n = 3 ihe nlere anya nwere onwe ya.A na-egosi data dị ka ± ọkọlọtọ ndahie.d Gene ontological analysis (GO) nke protein achọpụtara n'ime otu nkeji 2.Edepụtara okwu GO iri mbụ.Afụfụ na-acha ka ụdị okwu GO siri dị, na nha afụ dabara na ọnụọgụ protein dị na nke ọ bụla.e eriri nyocha nke protein na-emekọrịta ihe na BRD4.okirikiri odo bụ gluu ozugbo na okirikiri isi awọ bụ oyi akwa nke gluu na-apụtachaghị ìhè.Ahịrị uhie na-anọchi anya mmekọrịta ekpebisiri ike na nnwale yana ahịrị anụnụ anụnụ na-anọchi anya mmekọrịta ndị e buru n'amụma.f Ihe nnochite anya BRD4 agbakwunyere ebumnobi achọpụtara na LC-MS/MS bụ nke Western blotting kwadoro.g Nnwale mgbochi mgbochi ọrịa na-akwado mmekọrịta dị n'etiti SFPQ na BRD4.Emegharịrị nnwale ndị a n'onwe ha ma ọ dịkarịa ala ugboro abụọ nwere nsonaazụ yiri ya.A na-enye data raw n'ụdị faịlụ data raw.
Na mgbakwunye na ịchọpụta ebumnuche ndị metụtara POI na-edebanyeghị aha, anyị na-eche na PDPL ga-adabara maka ịchọpụta mkpụrụ maka enzymes, nke ga-achọ njirimara nke protein na-ejikọ ọnụ na nnukwu ogige iji kọwaa ihe ndị na-edebanyeghị aha.Parkin (nke PARK2 debere ya) bụ ligase E3 na mmụgharị na ogige mara amara na-ebute ọrịa Parkinson na-akpata autosomal (AR-JP)42.Na mgbakwunye, a kọwawo ogige ntụrụndụ dị ka ihe dị mkpa maka mitophagy (mitochondrial autophagy) na iwepụ ụdị oxygen na-emeghachi omume.Agbanyeghị, n'agbanyeghị na achọpụtala ọtụtụ ihe ndị na-eme ogige ntụrụndụ, ọrụ ogige ntụrụndụ na ọrịa a ka edobeghị anya.Iji kọwapụta mkpụrụ okwu ya na-enweghị njirimara, nwalere PDPL site na ịgbakwunye miniSOG na N- ma ọ bụ C-terminus nke ogige.A na-agwọ mkpụrụ ndụ na carbonyl cyanide proton transporter m-chlorophenylhydrazone (CCCP) iji mee ka ogige rụọ ọrụ site na ụzọ PINK1-Parkin.Tụnyere nsonaazụ BRD4 PDPL anyị, ngwakọta N-terminus parkin kpughere ọtụtụ protein ebumnuche, n'agbanyeghị na ọ kpuchiri akụkụ buru ibu nke C-terminus (177 n'ime 210) (Nyocha 5a,b na Data mgbakwunye 4).nsonaazụ ya kwekọrọ na akụkọ na mkpado N-terminal nwere ike ime ka Parkin44 rụọ ọrụ n'ụzọ na-adịghị mma.N'ụzọ dị ịtụnanya, enwere naanị protein 18 jikọtara ọnụ na data anyị nwere nsonaazụ AP-MS ebipụtara maka Parkin43, ikekwe n'ihi ọdịiche dị n'etiti ahịrị cell na proteomics workflows.Na mgbakwunye na protein anọ mara amara (ARDM1, HSPA8, PSMD14, na PSMC3) nke ejiri ụzọ abụọ mara (Fig 5c)43.Iji kwadokwuo nsonaazụ LC-MS/MS, a na-eji ọgwụgwọ PDPL na ihichapụ ọdịda ọdịda anyanwụ na-esote ya tụnyere nsonaazụ nke nne na nna HEK293T yana ahịrị ogige N-terminal kwụsiri ike.Ezubere iche CDK2, DUT, CTBP1, na PSMC4 nke a na-amabughị na-eji ihe eji ama ama, DNAJB1 (Fig 5d).
Ibé ugwu mgbawa nke protein na-emekọrịta ihe n'ime sel HEK293T nwere miniSOG egosipụtara nke ọma na N- ma ọ bụ C-terminus nke ogige (n = 3 nnwale nke ndu ndu onwe ya).Ejiri t-ule nwata akwụkwọ nwere ọdụ abụọ na ala mgbawa ugwu.A na-eji HEK293T dị ka njikwa na-adịghị mma. A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> 2-fold ion intensity intensity). A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> 2-fold ion intensity intensity). Значительно измененные белки выделены красным цветом (p <0,05 и > 2-кратная разница в интенсивности ионом). A na-akọwapụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> ọdịiche okpukpu abụọ na ike 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). A na-egosipụta protein ndị gbanwere nke ọma na ọbara ọbara (p <0.05 na> ọdịiche okpukpu abụọ na ike ionic).Protein emetụtara dị mkpa maka HEK293T-miniSOG mana ọ bụghị ihe dị mkpa maka HEK293T ka egosiri na akwụkwọ ndụ akwụkwọ ndụ.b Ihe osise Venn na-egosi protein dị n'etiti N-terminal na C-terminal.Mkpado N-terminal nwere ike ime ka ogige ahụ rụọ ọrụ nke ọma wee rụpụta protein ndị a na-amata karịa.c Venn eserese na-egosi protein dị n'etiti PDPL na AP-MS.Edepụtara ndị mmekọ ama ama, gụnyere 4 n'ime protein 18 jikọtara ọnụ yana 11 nke protein 159 akọwapụtara na PDPL.d Ebumnobi ndị nnọchi anya nke LC-MS/MS chọpụtara bụ nke Western blotting kwadoro.e Ssu72 na SNW1 ka achọpụtara dị ka ihe ndị na-edoghị anya n'akwụkwọ.A na-atụgharị plasmids protein ndị a na-akpado FLAG na HEK293T na HEK293T-Parkin-miniSOG na-esote ọgwụgwọ CCCP n'oge dị iche iche.Mbibi ahụ pụtakwara nke ọma na ahịrị oke okwu Parkin.f N'iji proteasome inhibitor MG132, a kwadoro na usoro mmebi nke Ssu72 na SNW1 na-edozi site na proteasome-ubiquitination.Emegharịrị nnwale ndị a n'onwe ha ma ọ dịkarịa ala ugboro abụọ nwere nsonaazụ yiri ya.A na-enye data raw n'ụdị faịlụ data raw.
N'ụzọ dị ịrịba ama, protein ndị PDPL chọpụtara ga-agụnye protein na-ejikọta ogige na ihe ha.Iji chọpụta mkpụrụ ndụ ogige na-edeghị aha, anyị họrọla protein asaa achọpụtara (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 na SNW1) na plasmids ebugharị iji kpughee mkpụrụ ndụ ihe nketa ndị a na HEK293T nkịtị wee kwupụta nke ọma miniSOG-Parkin's HEK2CCCPT sochiri ya.E belatara ọkwa nke protein Ssu72 na SNW1 n'ahịrị miniSOG-Pakin kwụsiri ike (Fig 5e).Ọgwụgwọ na CCCP maka awa 12 butere mbibi kachasị ukwuu nke mkpụrụ abụọ ahụ.Iji nyochaa ma a na-achịkwa mmebi nke Ssu72 na SNW1 site na proteasome-ubiquitination, a na-agbakwunye proteasome inhibitor MG132 iji gbochie ọrụ proteasome, na n'eziokwu anyị chọpụtara na a na-egbochi usoro mmebi ha (Fig 5f).A kwadoro ebumnuche ndị ọzọ na-abụghị nke mkpụrụ okwu dị ka ndị na-emekọrịta ihe na Parkin na-eji Western blotting (Mgbakwunye Fig. 10), nke gosipụtara nsonaazụ na-agbanwe agbanwe na LC-MS/MS.N'ikpeazụ, njikọ nke PDPL workflow na nkwenye nnyefe protein lekwasịrị anya na-enye ohere iji chọpụta ihe ndị E3 ligase na-edeghị aha.
Anyị ewepụtala ikpo okwu akara nso nso nke na-enye gị ohere ịchọpụta oghere na oge mmekọrịta POI.Ikpo okwu dabere na protein miniSOG photosensitizer, nke dị naanị ihe dịka 12 kDa, ihe na-erughị ọkara nha nke enzyme APEX2 tozuru oke (27 kDa) yana otu ụzọ n'ụzọ atọ nha TurboID (35 kDa).Obere nha kwesịrị ịgbasa ngwa ngwa maka ịmụ obere mmekọrịta protein.Nchọgharị ọzọ nke fotosensitizers ndị ọzọ, ma protein agbakwunyere mkpụrụ ndụ ihe nketa ma ọ bụ obere ụmụ irighiri ihe, dị mkpa iji mee ka mkpụrụ nke ikuku oxygen dị n'otu n'otu wee gbasaa nghọta nke ụzọ a.Maka ụdị miniSOG dị ugbu a, enwere ike nweta mkpebi nwa oge dị elu site na iji ọkụ na-acha anụnụ anụnụ iji mee ka akara nso nso.Na mgbakwunye, ogologo oge mkpughe weputara “igwe ojii” ka ukwuu nke oxygen singlet, na-ebute mgbanwe nke ihe fọdụrụnụ nke histidine dị anya, radius na-ebuwanye ibu, na ikike imezigharị mkpebi oghere nke PDPL.Anyị nwalekwara nyocha kemịkalụ asaa iji bulie ogo mgbama na-azụ wee nyochaa usoro molekụla n'azụ ụzọ a.Usoro ọrụ TOP-ABPP jikọtara ya na ọchụchọ na-enweghị mmasị na-emeghe gosipụtara na mgbanwe mere naanị na histidines na ọ dịghị microenvironment na-agbanwe agbanwe ka a hụrụ maka mmụba nke mgbanwe histidine, ma e wezụga maka mmasị na-agafeghị oke maka histidines na mpaghara loop.
A na-ejikwa PDPL kọwaa proteome subcellular nwere nkọwapụta proteome na mkpuchi ma ọ dịkarịa ala tụnyere akara ndị ọzọ nso na ụzọ nyocha kemịkalụ akọwapụtara nke akụkụ ahụ.A na-ejikwa ihe nrịbama nso nso mee ihe nke ọma iji mara elu, lysosomal, na proteomes metụtara secretome46,47.Anyị kwenyere na PDPL ga-adaba na akụkụ ndị a nke subcellular.Na mgbakwunye, anyị gbara PDPL aka mgba site na ịchọpụta ebumnuche maka njikọ protein cytosolic nke dị mgbagwoju anya karịa protein na-ekekọta akpụkpọ ahụ n'ihi njirimara ike ha na itinye aka na mmekọrịta oge.A na-etinye PDPL na protein abụọ, transcriptional coactivator BRD4 na ligase E3 Parkin metụtara ọrịa.A họọrọ protein abụọ a ọ bụghị naanị maka ọrụ ndu ha bụ isi, kamakwa maka mkpa ahụike ha na ikike ọgwụgwọ ha.Maka POI abụọ a, ndị mmekọ mmekọ ama ama yana ebumnuche edebanyeghị aha ka achọpụtara.N'ụzọ doro anya, protin SFPQ jikọtara akụkụ nke ngalaba IP kwadoro, nke nwere ike igosi usoro ọhụrụ nke BRD4 (obere isoform) na-achịkwa LLPS.N'otu oge ahụ, anyị kwenyere na njirimara nke mkpụrụ ndụ Parkin bụ ihe atụ nke achọrọ njirimara nke adhesives na-apụtaghị ìhè.Anyị chọpụtara mkpụrụ osisi abụọ a na-amaghị ama ma gosipụta mmebi ha n'akụkụ ụzọ ubiquitination-proteasome.N'oge na-adịbeghị anya, e mepụtala usoro ọnyà nke dabeere na usoro iji chọpụta ihe ndị na-emepụta hydrolase site na iji enzymes ọnyà.Ọ bụ ezie na nke a bụ usoro dị ike nke ukwuu, ọ naghị adabara nyocha nke ihe ndị na-etinye aka na nhazi nke nnukwu ogige ma na-achọ ka e guzobe njikọ covalent n'etiti enzyme na mkpụrụ.Anyị na-atụ anya na enwere ike ịgbatị PDPL iji mụọ ogige protein ndị ọzọ na ezinụlọ enzyme, dị ka ezinụlọ deubiquitinase na metalloprotease.
Ụdị miniSOG ọhụrụ, nke a na-akpọ SOPP3, ewepụtala site na mmepụta ikuku oxygen dị mma.Anyị tụlere miniSOG na SOPP3 wee hụ ịrụ ọrụ nrịbama ka mma, ọ bụ ezie na nrịbama-na-mkpọtụ agbanweghị (Foto mgbakwunye. 11).Anyị chere na njikarịcha nke SOPP3 (dịka ọmụmaatụ, site na evolushọn a na-eduzi) ga-eduga na protein fotosentizer na-arụ ọrụ nke ọma nke na-achọ oge ọkụ dị mkpụmkpụ ma si otú a na-enye ohere ka ejide usoro cellular ndị ọzọ siri ike.N'ụzọ doro anya, ụdị PDPL dị ugbu a bụ naanị na gburugburu cellular ka ọ na-achọ ọkụ na-acha anụnụ anụnụ na enweghị ike ịbanye n'ime anụ ahụ dị omimi.Akụkụ a na-egbochi iji ya na ọmụmụ ihe nlereanya anụmanụ.Otú ọ dị, njikọ nke optogenetics na PDPL nwere ike inye ohere maka nchọpụta anụmanụ, karịsịa na ụbụrụ.Na mgbakwunye, fotosensitizers infrared ndị ọzọ arụpụtara na-ewepụkwa mmachi a.A na-eme nyocha ugbu a na mpaghara a.
E nwetara ahịrị cell HEK293T site na ATCC (CRL-3216).Ahịrị cell nwalere ihe na-adịghị mma maka ọrịa mycoplasma ma mebe ya na DMEM (Thermo, #C11995500BT) agbakwunyere na serum bovine nwa ebu n'afọ 10% (FBS, Vistech, #SE100-B) na 1% penicillin/streptomycin (Hyclone, #SV30010).toro na.
3-Aminophenylene (ihe nlele 3) na (4-ethynylphenyl) methanamine (ihe nlele 4) zụtara na Bidepharm.A zụtara Propylamine (nchọpụta 2) n'aka Energy-chemicals.N- (2-Aminophenyl) pent-4-ynamide (nchọpụta 1) ka emebere dịka ụzọ ebipụtara.
Tebụl mgbakwunye 1 depụtara usoro mkpụrụ ndụ ihe nketa ejiri mee ihe n'ọmụmụ ihe a.E mechiri usoro miniSOG na KillerRed site na plasmid onyinye sitere na P. Zou (Mahadum Peking).Usoro nlebara anya nke mitochondrial matriks sitere na 23 N-terminal amino acids nke COX4 wee tinye n'ime vectors egosiputara site na iji mgbakọ Gibson (Beyotime, #D7010S).Iji lekwasị anya na akpụkpọ ahụ na nucleus nke reticulum endoplasmic, SEC61B DNA mmadụ (NM_006808.3) (NEB, #M0491L) kwadoro site na PCR site na ụlọ akwụkwọ cDNA nke mkpụrụ ndụ HEK293T, na H2B DNA (nke D. Lin nyere, Shenzhen Bay Laboratory) na cloned , dị ka e kwuru n'elu.Ọ gwụla ma egosiputara n'ụzọ ọzọ, mkpụrụ ndụ ihe nketa protein ndị ọzọ ejiri maka mbufe na iwu ahịrị sel kwụsiri ike bụ PCR sitere na ọbaakwụkwọ HEK293T cell cDNA.A na-eji G3S (GGGS) na G4S (GGGGS) mee ihe dị ka njikọ n'etiti protein bait na miniSOG.Agbakwunyere mkpado epitope V5 (GKPIPNPLLGLDST) na nrụkọ ọnụ ndị a.Maka nkwupụta n'ime anụ mamma na iji guzobe ahịrị cell kwụsiri ike, ihe nrụpụta miniSOG jikọtara ya na vector lentiviral pLX304.Maka okwu nje bacteria, miniSOG ka etinyere n'ime pET21a vector nke akpọrọ 6xHis na C-terminus.
A na-amịpụta mkpụrụ ndụ HEK293T na sel 2.0 x 105 kwa nke ọma na efere isii nke ọma ma nyefee 24 awa mgbe e mesịrị na recombinant lentiviral plasmids (2.4 μg pLX304) na plasmids nkwakọ ngwaahịa viral (1.5 μg psPAX2 na 1.2 μG) na Limd pMD. , #C0533), ihe dịka 80% ngwakọta.Mgbe nnyefe nke abalị gasịrị, a gbanwere usoro ahụ wee tinye ya maka awa 24 ọzọ.Emere mkpokọta nje a mgbe awa 24, 48 na 72 gachara.Tupu ọrịa nke ahịrị cell ezubere iche, a na-enyocha usoro nje site na nzacha 0.8 μm (Merck, #millex-GP) yana polybrene (Solarbio, #H8761) agbakwunyere na ntinye nke 8 μg/ml.Mgbe awa 24 gachara, a na-ahapụ sel ahụ ka ọ gbakee site n'ịgbanwe usoro.A na-ahọrọ mkpụrụ ndụ site na iji 5 μg/ml blasticidin (Solarbio, #3513-03-9) maka akụkụ atọ mbụ dị ka nhọrọ siri ike dị ala.Mgbe ahụ jiri 20 μg/ml dị ka usoro siri ike maka akụkụ atọ na-esote.
A na-akụ mkpụrụ ndụ mkpụrụ ndụ n'ime ụlọ olulu mmiri iri na abụọ (Ibidi, #81201) na njupụta nke ihe dịka sel 20,000 kwa olulu mmiri.Iji meziwanye adhesion nke mkpụrụ ndụ HEK293T, tinye 50 µg/ml fibronectin (Corning, #356008) diluted na phosphate buffered saline (PBS, Sangon, #B640435) na 37°C.A na-edozi ụlọ ndị ahụ maka awa 1 wee wepụ ya na PBS.Mgbe awa 24 gachara, ejiri PBS sachaa mkpụrụ ndụ otu ugboro, tinye ya na nyocha 1 mM 3 na ngwọta nnu ziri ezi nke Hanks (HBSS, Gibco, #14025092) maka 1 h na 37 Celsius C, wee tinye ya na ọkụ na-acha anụnụ anụnụ (460 nm). ).) na-agbapụta maka 10 min na ụlọ okpomọkụ.Mgbe nke ahụ gasịrị, a na-asacha mkpụrụ ndụ ahụ ugboro abụọ na PBS ma dozie ya na 4% formaldehyde na PBS (Sangon, # E672002) maka nkeji iri na ise na okpomọkụ.E wepụrụ oke formaldehyde n'ime sel edoziri site na iji PBS saa ugboro atọ.Ejiri 0.5% Triton X-100 (Sangon, #A600198) mebie cell na PBS wee jiri PBS sachaa ugboro atọ.Wepụ ụlọ ahụ ma gbakwunye na nlele ọ bụla 25 µl nke ngwakọta mmeghachi omume nke nwere 50 µM Cy3-azide (Aladdin, #C196720), 2 mM CuSO4 (Sangon, # A603008), 1 mM BTTAA (Confluore, #BDJ-4) na 0.5 mg / ml sodium ascorbate (Aladdin, no. S105024) na incubated maka 30 nkeji na ụlọ okpomọkụ.Mgbe mmeghachi omume snap gasịrị, a na-asacha mkpụrụ ndụ ugboro isii na PBS nwere 0.05% Tween-20 (Sangon, #A600560) (PBST) wee gbochie ya na 5% BSA (Abcone, #B24726) na PBST maka nkeji 30 n'ime ụlọ okpomọkụ.
Maka collocalization immunostaining, a na-ejikọta mkpụrụ ndụ na ọgwụ nje ndị bụ isi dị ka ọnọdụ egosipụtara: òké mgbochi V5 mkpado mAb (1: 500, CST, #80076), oke bekee anti-Hsp60 mAb (1: 1000), ABclonal, #A0564), mgbochi oke bekee polyclonal anti-calnexin antibody (1:500, Abcam, #ab22595) ma ọ bụ oke bekee mgbochi lamin A/C monoclonal mgbochi (1:500; CST, #2032) na 4 Celsius C n'abali.Mgbe ejiri PBST sachaa ugboro 3, a na-etinye mkpụrụ ndụ ihe mgbochi nke abụọ: ewu anti-rabbit Alexa Fluor 488 (Thermo, #A11034) diluted 1:1000, ewu mgbochi òké Alexa Fluor 594 (CST, #8889) diluted 1:1000.dilution Dilu na ụlọ okpomọkụ maka nkeji 30.A na-eji PBST sachaa mkpụrụ ndụ ugboro atọ ma were DAPI (Thermo, #D1306) mee ya na PBS maka nkeji iri na okpomọkụ.Mgbe 3 sachara na PBS, a na-emechi sel na 50% glycerol (Sangon, #A600232) na PBS maka eserese.Enwetara onyonyo nke immunofluorescent site na iji microscope ZEISS LSM 900 Airyscan2 confocal na ngwanrọ ZNE 3.5.
Maka onyonyo fluorescent nke otu oxygen, ejiri Hanks HEPES buffer sachara mkpụrụ ndụ ugboro abụọ tupu agbakwunye 100 nM Si-DMA na nchekwa Hanks HEPES (DOJINDO, #MT05).Mgbe ekpughere ya na ìhè, a na-etinye mkpụrụ ndụ ahụ na CO2 incubator na 37 ° C maka nkeji 45.Ejiri ihe nchekwa Hanks 'HEPES sachaa mkpụrụ ndụ ugboro abụọ wee were Hoechst kpuchie ya na nchekwa Hanks' HEPES maka nkeji iri na ọnụ ụlọ ma were anya ya na-eji ZEISS LSM 900 confocal microscope., #M36008) na nchekwa HBSS nwere calcium na magnesium.Mgbe ekpughere ìhè ma ọ bụ doxorubicin (MCE, #HY-15142A), a na-etinye mkpụrụ ndụ na CO2 incubator na 37 ° C. maka nkeji 10, jiri ihe nchekwa HBSS sachaa ugboro abụọ, ma tinye ya na Hoechst na nchekwa HBSS na ụlọ okpomọkụ.nkeji.A na-eji Doxorubicin dị ka njikwa nyocha dị mma ebe a na-agwọ mkpụrụ ndụ na 20 μM doxorubicin na HBSS nwere 1% BSA maka nkeji 30.Enwetara onyonyo nke immunofluorescent site na iji microscope confocal Zeiss LSM 900.
Akụnyere mkpụrụ ndụ HEK293T na-ekwupụta nke ọma mito-miniSOG na njupụta nke ihe dịka 30% na efere 15 cm.Mgbe awa 48 gachara, mgbe ~ 80% rutere, a na-asacha mkpụrụ ndụ ahụ otu ugboro na PBS, tinye ya na 1 mM Probe 3 na nchekwa HBSS ọhụrụ maka 1 hour na 37 ° C wee jiri ọkụ na-acha anụnụ anụnụ mee ihe maka nkeji 10 n'ime ụlọ. okpomọkụ..Mgbe nke ahụ gasịrị, a na-asacha mkpụrụ ndụ ahụ ugboro abụọ na PBS, kpochapu ma weghachite ya na ihe nchekwa PBS oyi nke nwere EDTA na-enweghị ihe mgbochi protease (MCE, #HY-K0011).A na-asachapụ mkpụrụ ndụ site n'ịkpọ ọnụ ọnụ maka nkeji 1 (1 sekọnd na 1 sekọnd na njupụta 35%).A na-eme ka ngwakọta a na-esi na ya pụta na 15,871 xg maka 10 min na 4 ° C iji wepụ ihe mkpofu, ma gbanwee ntinye uche dị elu na 4 mg / mL site na iji ngwa nyocha protein BCA (Beyotime, #P0009).Gwakọta 1 ml nke lysate dị n'elu na 0.1 mM photodegradable biotin azide (Confluore, #BBBD-14), 1 mM TCEP (Sangon, #A600974), 0.1 mM TBTA ligand (Aladdin, #T162437), na 1 mMMtor CuSO4 na ala. ntụgharị maka awa 1 na ụlọ okpomọkụ.Mgbe mmeghachi omume ọnyà gasịrị, gbakwunye ngwakọta na ngwọta agwakọtara (MeOH: CHCl3: H2O = 4 ml: 1 ml: 3 ml) na vial iko 10 ml.A gwakọtara ihe atụ ndị ahụ na centrifuged na 4500 g maka nkeji 10 na ụlọ okpomọkụ.A na-atụfu ihe ngwọta nke ala na nke elu, a na-asacha mmiri ahụ ugboro abụọ na 1 ml nke methanol na centrifuged na 15871 × g maka 5 min na 4 ° C.Tinye 1 ml nke 8 M urea (Aladdin, no. U111902) na 25 mM ammonium bicarbonate (ABC, Aladdin, ọ dịghị. A110539) iji gbazee mmiri ozuzo.Emegharịrị ihe nlele na 10 mM dithiothreitol (Sangon, # A100281 na 25 mM ABC) maka nkeji 40 na 55 Celsius C na-esote 15 mM ọhụrụ iodoacetamide (Sangon, # A600539) na ụlọ okpomọkụ n'ime ọchịchịrị.Alkylation n'ime nkeji 30..Agbakwunyere 5 mM dithiothreitol iji kwụsị mmeghachi omume ahụ.Kwadebe ihe dịka 100 µl NeutrAvidin agarose beads (Thermo, #29202) maka nlele ọ bụla site na iji 1 ml PBS saa ugboro 3.A na-agbaze ihe ngwọta proteome dị n'elu na 5 ml PBS ma tinye ya na NeutrAvidin agarose beads nke a sachara maka awa 4 n'ime ụlọ okpomọkụ.A na-asacha beads ahụ ugboro atọ na 5 ml PBS nwere 0.2% SDS (Sangon, #A600485), ugboro 3 na 5 ml PBS nwere urea 1M, na ugboro 3 nwere 5 ml ddH2O.A na-egbute beads ahụ site na centrifugation wee maliteghachi na 200 μl nke 25 mM ABC nwere 1 M urea, 1 mM CaCl 2 (Macklin, #C805228) na 20 ng/μl trypsin (Promega, #V5280).Trypsinize n'abali na 37 Celsius na ntụgharị.A kwụsịrị mmeghachi omume ahụ site n'ịgbakwunye formic acid (Thermo, # A117-50) ruo mgbe pH ruru 2-3.A na-asacha beads ugboro 3 na 1 ml nke PBS nwere 0.2% SDS, ugboro 3 na 1 ml nke PBS nwere 1 M urea, na mgbe ahụ, ugboro atọ na 1 ml nke mmiri mmiri.A tọhapụrụ peptides emezigharịrị site na lysis ọkụ (365 nm) maka nkeji 90 site na iji 200 μl nke 70% MeOH.Mgbe centrifugation gasịrị, a na-anakọta supernatant.Ejiri 100 μl nke 70% MeOH sachaa bead ndị ahụ otu ugboro ma jikọta ihe ndị dị elu.Akpọkọrọ ihe nlele n'ime oghere Speedvac ma chekwaa na -20 Celsius ruo mgbe nyocha.
Iji chọpụta ma chọpụta peptides gbanwetụrụ otu ikuku oxygen, atụgharịrị n'ụdị 0.1% formic acid na 1 μg nke peptides nyochara site na iji Orbitrap Fusion Lumos Tribrid mass spectrometer nke nwere nano ESI sitere na Tune na Xcalibur sitere na ngwanrọ na-ere ahịa 4.3.Ekewapụrụ ihe nlele na kọlụm capillary 75 µm × 15 cm n'ime ya nwere ihe 3 µm C18 (ReproSil-pur, #r13.b9.) wee jikọọ na usoro EASY-nLC 1200 UHPLC (Thermo).E kewapụrụ peptides site na chromatography nkeji nkeji 95 linear site na 8% ihe mgbaze B ruo 50% mgbaze B (A = 0.1% formic acid na mmiri, B = 0.1% formic acid na 80% acetonitrile), wee mụbaa linearly ruo 98% B min. na 6 min na ọsọ ọsọ nke 300 nl / min.Orbitrap Fusion Lumos na-anakọta data ọzọ n'etiti nyocha MS zuru oke na nyocha MS2 dabere na data ahụ.Edobere voltaji na-agbapụta ka ọ bụrụ 2.1kV yana okpomọkụ nke ion transport capillary bụ 320C.Achịkọtara MS spectra (350-2000 m/z) na mkpebi nke 120,000, AGC 4 × 105, yana oge ntinye kacha nke 150 ms.A na-ekewa 10 ndị a na-ebu ụzọ gbabawanye n'ụba na nyocha zuru ezu nke ọ bụla site na iji HCD nwere ike nkukota ahazi nke 30%, windo ikewapụ anọ nke 1.6 m/z, yana ntọala mkpebi nke 30,000.Ebumnuche AGC maka tandem mass spectrometry na-eji 5 × 104 yana oge ntinye kacha 150 ms.Ewepụghị ike ka atọrọ ka 30 sekọnd. Ajụjụ ion ekenyeghị ma ọ bụ ndị nwere ụgwọ 1+ na >7+ maka MS/MS. Ajụjụ ion ekenyeghị ma ọ bụ ndị nwere ụgwọ 1+ na >7+ maka MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ajụjụ ion ma ọ bụ ion na-enyeghị nke nwere ụgwọ 1+ na >7+ maka 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. Ajụjụ ion ma ọ bụ ion na-akọwapụtaghị nke nwere ụgwọ 1+ na >7+ maka MS/MS.
A na-ahazi data raw ahụ site na iji ikpo okwu FragPipe dabere na MSFragger.Ekpebiri nhụsianya oke na amino acid kwekọrọ site na iji algọridim ọchụchọ mepere emepe nwere nnabata nnabata nke -150 ruo 500 Da.Achọpụtara peptides emezigharịrị site na iji mgbanwe histidine nwere nnukwu uru nke +229.0964 na +247.1069 Da na PD (Proteome Discoverer 2.5, Thermo).
Edebere mkpụrụ ndụ ndị na-egosipụta nke ọma mkpụrụ ndụ miniSOG agwakọtara na efere 6 cm.Mgbe ha rutere ~ 80% confluence, a na-asacha mkpụrụ ndụ otu ugboro na HBSS (Gibco, #14025092), wee tinye ya na nyocha kemịkalụ na HBSS maka elekere 1 na 37 Celsius ma were ọkụ na-acha anụnụ anụnụ mee ka ìhè.10W LED maka nkeji 20 na ụlọ okpomọkụ.Iji chọpụta ụdị ụdị oxygen reactive na-etinye aka na 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 ka etinyere na sel dị ka ihe mgbakwunye.Mgbe a sachara ya na PBS oyi, a na-ehichapụ mkpụrụ ndụ, na-anakọta na 1.5 ml centrifuge tubes, na sonicated na ọnụ maka 1 min na 200 μl nke PBS na 1x protease inhibitor na-enweghị EDTA (1 s na 1 s enweghị, njupụta 35%).A na-eme ka ngwakọta a na-esi na ya pụta na 15,871 × g maka 10 min na 4 Celsius C ma gbanwee ntinye uche dị elu na 1 mg/mL site na iji ngwa nyocha protein BCA.Ihe dị ka 50 µl nke lysate dị n'elu ka ejiri 0.1 mM rhodamine azide (Aladdin, no. T131368), 1 mM TCEP, 0.1 mM TBTA ligand, na 1 mM CuSO4 maka 1 awa na okpomọkụ ụlọ na ntụgharị site na ala ruo n'elu.Mgbe mmeghachi omume pịa, a na-eme mmiri ozuzo na acetone site n'ịgbakwunye 250 μl nke acetone chilled tupu oge eruo, na-emepụta na -20 ° C maka 20 min na centrifuging na 6010 × g maka 10 min na 4 ° C.Na-anakọta pellet ahụ wee sie na 50 µl nke 1x Laemmli nke nchekwa maka nkeji 10 na 95 Celsius.A na-enyocha ihe nlele na SDS-PAGE ogologo gels wee hụ ya site na iji Bio-rad ChemiDoc MP Touch imaging system na Image Lab Touch software.
A na-eme nkwupụta na ịdị ọcha nke recombinant miniSOG-6xProtein ya dị ka akọwara na mbụ.Na nkenke, E. coli BL21 (DE3) sel (TransGen, # CD701-02) gbanwere na pET21a-miniSOG-6xHis na okwu protein sitere na 0.5 mM IPTG (Sangon, #A600168).Mgbe cell lysis gasịrị, a na-asachapụ protein site na iji Ni-NTA agarose beads (MCE, no. 70666), dialyzed megide PBS, ma chekwaa na -80 ° C.
Maka nyocha ihe mgbochi nke dabere na vitro label proximity assay, gwakọta 100 μM dị ọcha miniSOG, 1 mM probe 3, na 1 μg anti-label mouse monoclonal antibody (TransGen, #HT501-01) na PBS na ngụkọta mmeghachi omume nke 50 μl..A na-eji ọkụ ọkụ na-acha anụnụ anụnụ mee ihe ngwakọta mmeghachi omume maka 0, 2, 5, 10, na 20 min na ụlọ okpomọkụ.Ejikọtara ngwakọta ahụ na 0.1 mM biotin-PEG3-azide (Aladdin, #B122225), 1 mM TCEP, 0.1 mM TBTA ligand, na 1 mM CuSO4 maka 1 h n'ime ụlọ okpomọkụ na ihe mgbago elu.Mgbe mmeghachi omume snap gasịrị, tinye ihe nchekwa 4x Laemmli ozugbo na ngwakọta wee sie ya na 95 Celsius maka nkeji 10.A na-enyocha ihe nlele na SDS-PAGE gels wee nyochaa site na ọdịda ọdịda anyanwụ na streptavidin-HRP (1: 1000, Solarbio, #SE068).
A na-eji peptide synthetic nke nwere histidine nwere C-terminal amidation (LHDALDAK-CONH2) iji nyochaa akara peptide dị nso na akara in vitro.Na nyocha a, 100 μM dị ọcha miniSOG, 10 mM probe 3 na 2 μg / ml synthetic peptide na-agwakọta na PBS na ngụkọta mmeghachi omume nke 50 μl.A na-eji ọkụ na-acha anụnụ anụnụ na-acha anụnụ anụnụ mee ka ngwakọta mmeghachi omume na-egbuke egbuke maka 1 hour na ụlọ okpomọkụ.A tụlere otu microliter nke sample site na iji usoro LC-MS (Mmiri, SYNAPT XS Ions Mobility Time-of-Flight mass spectrometer na MassLynx spectrum analysis software).
A na-akụ mkpụrụ ndụ HEK293T nke na-egosipụta mkpụrụ ndụ miniSOG fusion na efere 10 cm maka ahịrị nwere mpaghara akụkụ akụkụ dị iche iche (Mito, ER, Nucleus) na efere 15 cm maka ahịrị Parkin-miniSOG na BRD4-miniSOG.Mgbe ha rutere ~90% confluence, a na-asacha mkpụrụ ndụ ahụ otu ugboro na HBSS, wee tinye ya na nyocha 3 na HBSS maka elekere 1 na 37 Celsius ma jiri ọkụ na-acha anụnụ anụnụ 10 W mee ka ọkụ na ụlọ okpomọkụ.Maka ịkpọ aha Parkin na-abụghị kọntaktị, 10 µM proton carbonyl cyanide carrier m-chlorophenylhydrazone CCCP (Solarbio, #C6700) nwere nyocha 3 na HBSS ka agbakwunyere maka elekere 1 na 37°C.The cell lysis, click kemistrị, mbelata na alkylation nzọụkwụ bụ otu ihe ahụ a kọwara n'elu, ma e wezụga na 2 mg nke lysate na-agbakwunyere na biotin PEG3 azide na-eji na click mmeghachi omume kama photodegradable biotin azide.Mgbe emechara ya, a na-asacha beads ugboro 3 na 5 ml nke PBS nwere 0.2% SDS, ugboro 3 na 5 ml nke PBS nwere 1 M urea, na ugboro 3 na 5 ml nke PBS.Mgbe nke ahụ gasịrị, 2 µg trypsin gbakwụnye na 300 µl 25 mM ABC nwere 1 M urea iji kewaa protein n'otu abalị na 37 ° C.A kwụsịrị mmeghachi omume ahụ site n'ịgbakwunye formic acid ruo mgbe pH nke 2-3 ruru.Mgbe trypsinization na beads, a na-ehichapụ ihe ngwọta peptide site na iji kọlụm SOLAµ HRP (Thermo, #60209-001) wee kpoo ya na ngwa ngwa ngwa ngwa Speedvac.A gbazere peptides na 0.1% formic acid na 500 ng nke peptides ka a na-enyocha site na iji Orbitrap Fusion Lumos Tribrid mass spectrometer nke nwere isi nano-ESI akọwara n'elu.E kewapụrụ Peptides na kọlụm RP-HPLC azụmahịa (75 μm x 2 cm) (Thermo, no. 164946) na kọlụm RP-HPLC nyocha (75 μm x 25 cm) (Thermo, ọ. 164941), ha abụọ jupụtara na 2 μm.gradient site na 8% ruo 35% ACN na nkeji 60, wee mụbaa linearly ruo 98% B n'ime nkeji 6 na ọsọ ọsọ nke 300 Nl/min.Achịkọtara MS spectra (350-1500 m/z) na mkpebi nke 60,000, AGC 4 × 105, yana oge ntinye kacha nke 50 ms.HCD kewara ion ahọpụtara n'usoro n'ime usoro 3 s nke nwere ike nkuku nkịtị nke 30%, windo ikewapụrụ anọ nke 1.6 m/z, na mkpebi nke 15000. A 5 × 104 tandem mass spectrometer AGC lekwasịrị anya na oge ịgba ọgwụ kacha nke 22 ms ejiri.Edobere mwepu ike na sekọnd 45. Ajụjụ ion ekenyeghị ma ọ bụ ndị nwere ụgwọ 1+ na >7+ maka MS/MS. Ajụjụ ion ekenyeghị ma ọ bụ ndị nwere ụgwọ 1+ na >7+ maka MS/MS. Неназначенные ионы или ионы с зарядом 1+ и >7+ были отклонены для МС/МС. Ajụjụ ion ma ọ bụ ion na-enyeghị nke nwere ụgwọ 1+ na >7+ maka 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. Ajụjụ ion ma ọ bụ ion na-akọwapụtaghị nke nwere ụgwọ 1+ na >7+ maka MS/MS.
Usoro nkwadebe ihe nlele ruo na ịba ụba nke NeutrAvidin beads bụ otu ihe ahụ na nyocha LC-MS/MS akọwara n'elu.A na-eji ihe dịka 50 μg nke lysate mee ihe dị ka ntinye maka njikwa ntinye na 2 mg nke lysate na-eji maka mmeghachi omume pịa.Mgbe emechara ya na sachaa ya na neutravidin, a na-ekpochapụ protein ndị ahụ site n'ịgbakwunye 50 μl nke ihe nchekwa Laemmli na beads resin agarose na esi na 95° C. maka nkeji ise.SDS-PAGE nyochara ntinye ibu njikwa na ihe nlere anya emejuru bead wee bufee ya na membran PVDF (Millipore, #ISEQ00010) site na usoro Western blot ọkọlọtọ.Eji 5% mmiri ara ehi skim (Sangon, #A600669) kpuchie akpụkpọ ahụ ahụ na TBS nke nwere 0.1% tween-20 (TBST) ma tinye ya na ọgwụ mgbochi nke isi na nke abụọ.A gbanyere ọgwụ mgbochi nke mbụ 1:1000 na mmiri ara ehi 5% na TBST wee tinye ya n'otu abalị na 4 Celsius.A na-eji ọgwụ mgbochi nke abụọ mee ihe na nha 1:5000 ma tinye ya maka awa 1 n'ime ụlọ okpomọkụ.Chemiluminescence na-ahụ anya akpụkpọ ahụ ahụ site na iji usoro onyonyo Chemidoc MP.A na-egosi nyocha niile nke blots na gels na ọnụ ọgụgụ a dị ka data raw.
Ọgwụ mgbochi mbụ ejiri mee ihe n'ọmụmụ ihe a gụnyere oke bekee anti-SFPQ monoclonal antibody (CST, no. 71992), rabbit anti-FUS monoclonal antibody (CST, no. 67840), oke bekee mgbochi NSUN2 polyclonal antibody (Proteintech, mba 20854-1- AP), mgbochi mgbochi mSin3A polyclonal mgbochi (Abcam, #ab3479), mgbochi mkpado mgbochi monoclonal òké (TransGen, #HT201-02), òké mgbochi β-actin monoclonal mgbochi (TransGen, #HC201-01), oke bekee mgbochi -CDK2 monoclonal mgbochi (ABclonal, #A0094), oke bekee monoclonal mgbochi CTBP1 (ABclonal, #A11600), oke bekee polyclonal mgbochi DUT (ABclonal, #A2901), oke bekee polyclonal mgbochi PSMC4 (ABclonal, #A2505), oke bekee mgbochi- DNAJB1 polyclonal mgbochi (ABclonal, # A5504).A na-eji ọgwụ mgbochi ndị a mee ihe na 1: 1000 dilution na mmiri ara ehi 5% na TBST.Ọgwụ mgbochi nke abụọ ejiri na ọmụmụ a gụnyere mgbochi oke bekee IgG (TransGen, #HS101-01), mgbochi òké IgG (TransGen, #HS201-01) na 1: 5000 dilution.
Iji nwetakwuo nyocha ma BRD4 na-emekọrịta ihe na SFPQ, sel HEK293T kwụsiri ike na BRD4-miniSOG na-egosipụta HEK293T na efere 10 cm.Ejiri PBS oyi saa cell cell ma gbanye ya na 1 ml Pierce IP lysis buffer (Thermo Fisher, #87787) ya na ihe mgbochi protease na-enweghị EDTA maka nkeji 30 na 4 Celsius.Mgbe nke ahụ gasịrị, a na-anakọta lysates na tubes centrifuge 1.5 ml na centrifuge na 15,871 xg maka 10 min na 4 ° C.A na-egbute ihe dị elu ma were 5 μg nke mgbochi V5 akpọrọ mouse monoclonal antibody (CST, #80076) n'otu abalị na 4°C.Saa ihe dị ka 50 µl nke protein A/G magnetik beads (MCE, #HY-K0202) ugboro abụọ na PBS nwere 0.5% Tween-20.Mgbe ahụ, a na-ejikọta lysates cell na bead magnet maka awa 4 na 4 Celsius na ntụgharị site na ala ruo n'elu.Mgbe ahụ, a na-asacha beads ahụ ugboro anọ na 1 ml nke ihe nchekwa PBST ma sie ya na 95 ° C maka 5 min.A tụlere ihe nlele na gels SDS-PAGE wee bufee ya na membran PVDF site na iji usoro ntụpọ Western.E gbochiri akpụkpọ ahụ n'ime mmiri ara ehi skị 5% na TBST wee were ọgwụ mgbochi mbụ na nke abụọ tinye ya n'usoro.Ejiri ọgwụ mgbochi mgbochi SFPQ monoclonal nke mbụ (CST, #71992) mee ihe na nha 1:1000 na mmiri ara ehi 5% na TBST wee tinye ya n'otu abalị na 4°C.A na-eji mgbochi-oke bekee IgG mee ihe na nha 1:5000 ma tinye ya maka awa 1 n'ime ụlọ okpomọkụ.Chemiluminescence na-ahụ anya akpụkpọ ahụ ahụ site na iji usoro onyonyo Chemidoc MP.
Arụmọrụ niile ejiri mee nyocha ebe mgbaze nweta n'elu ala (SASA) nwetara site na Protein Data Bank (PDB)52 ma ọ bụ nchekwa data AlphaFold Protein Structure53.A gbakọrọ SASA zuru oke maka ihe fọdụrụ site na iji mmemme FreeSASA.Naanị data SASA zuru oke na enweghị mgbagha maka aha histidine na ndị agbata obi ya ka ejiri nweta SASA pụtara maka nhazi ọ bụla.A na-agbakọ ohere ịnweta ihe mgbaze (RSA) maka histidine ọ bụla site n'ịkewaa uru SASA zuru oke site na mpaghara ebe ihe mgbaze nwere ike ịnweta.Ekewaziri histidines niile dị ka ezoro ezo ma ọ bụrụ na RSA pụtara n'okpuru 20%, ma ọ bụghị ekpughere56.
Achọtara faịlụ raw enwetara na ọnọdụ DDA site na iji Proteome Discoverer (v2.5) ma ọ bụ MSfragger (Fragpipe v15.0) na nchekwa data protein ziri ezi nke SwissProt nke nwere mmetọ nkịtị.The peptides chọrọ trypsin zuru ezu na saịtị abụọ na-efu efu, carbamoyl methylation dị ka mgbanwe agbanweela yana methionine oxidation dị ka mgbanwe dị ike.Edebere ndidi na iberi dị arọ na 10 ppm na 0.02 Da (MS2 Orbitrap), n'otu n'otu. E wepụrụ ihe ndị na-emerụ emerụ, na-enyocha protein iji nweta ọnụọgụ nchọpụta ụgha nke <1%. E wepụrụ ihe ndị na-emerụ emerụ, na-enyocha protein iji nweta ọnụọgụ nchọpụta ụgha nke <1%. Попадания загрязняющих веществ были удалены, а белки отфильтрованы, чтобы получить коэфнцие . E wepụrụ ihe ndị na-emerụ emerụ ma kpochaa protein iji nye ọnụọgụ nchọpụta ụgha nke <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. E wepụrụ ihe ndị na-emerụ emerụ ma kpochaa protein iji nweta ọnụego ziri ezi ụgha nke <1%.Maka nyocha ọnụọgụgụ na-ejighi akara, a na-eji ọdịnaya protein emezigharị site na nkwugharị ndu atọ.Emere nyocha mpaghara subcellular protein site na iji nyocha Gene Ontology (GO) sitere na DAVID Bioinformatics Resources, MitoCarta 3.0 na ọdụ data ndị otu Alice Ting chịkọtara ma bipụta ya.E nwetara map ugwu mgbawa site na Perseus (v1.6.15.0). A nwalere mgbanwe n'ụba protein maka mkpa ndekọ ọnụ ọgụgụ site na iji t-ule akụkụ abụọ, a chọpụtakwara protein hits na mgbanwe dị ukwuu> 2 (ọ gwụla ma ekwuru ya) na p value <0.05. A nwalere mgbanwe n'ụba protein maka mkpa ndekọ ọnụ ọgụgụ site na iji t-ule akụkụ abụọ, a chọpụtakwara protein hits na mgbanwe dị ukwuu> 2 (ọ gwụla ma ekwuru ya) na p value <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. A nwalere mgbanwe mpịakọta protein maka mkpa ndekọ ọnụ ọgụgụ site na iji t-ule ọdụdụ abụọ, na egwuregwu protein ka achọpụtara na mgbanwe ọdịnaya> 2 (ọ gwụla ma amataghị ya) yana uru ap <0.05.双边 T 检验 测试 确定 确定 确定 确定 确定 的 的 的 的 丰度 丰度 丰度 变化 另 有 另 p 值 0.05.双边 t 检验 测试 统计 统计 并 命 命 的 的 的 丰度 丰度 丰度 有 有 有 有 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. A nwalere ihe ndekọ ọnụ ọgụgụ nke mgbanwe mpịaji na ọdịnaya protein site na iji t-ule ọdụdụ abụọ, na egwuregwu protein kpebiri maka mgbanwe ọdịnaya> 2 (ọ gwụla ma egosighi ya) na p-ụkpụrụ <0.05.Emere nyocha mmekọrịta protein site na iji nyocha GO yana ọdụ data String.
Emere ihe ngbanwe nke ndu atọ nwere nsonaazụ yiri ya.Ejiri GraphPad Prism (ngwa ngwa GraphPad) mee nyocha ndekọ ọnụ ọgụgụ na Perseus (v1.6.15.0).Iji tụnyere otu abụọ ahụ, a na-ekpebi p-uru site na iji t-ule nwata akwụkwọ nwere ọdụ abụọ.Naanị protein ndị a chọpụtara ma ọ dịkarịa ala ugboro abụọ n'ime otu nnwale ahụ ka etinyere n'ime atụmatụ mgbawa ugwu ahụ, a na-eji Perseus dochie ụkpụrụ ndị na-efu efu na otu njikwa site na nkesa nkịtị ka e wee gbakọọ p-uru.Ogwe mperi na-anọchite anya ndahie ọkọlọtọ ±.Na nyocha proteomic maka nyocha ndekọ ọnụ ọgụgụ, ụbara protein ndị pụtara na opekata mpe abụọ n'ụdị ndu ka edobere.Ejighị ụzọ ọnụ ọgụgụ buru ụzọ chọpụta nha nlele.Nnwale ndị a abụghị enweghị usoro.Ndị nchọpụta ahụ kpuru ìsì na ọrụ ndị ahụ n'oge nnwale na nyocha nke nsonaazụ ya.
Maka ozi ndị ọzọ gbasara imepụta ọmụmụ ihe, lee akụkọ nyocha nke okike ejikọtara na edemede a.
Edebere data spectrometry nke a na-enweta na ọmụmụ a na ProteomeXchange Consortium site na ebe nchekwa ndị mmekọ iProX57 n'okpuru dataset ID PXD034811 (PDPL-MS dataset).A na-enye data raw n'ụdị faịlụ data raw.Edemede a na-enye data izizi.
Gingras, AC, Abe, KT & Raught, B. Ịmata agbata obi: iji biotinylation na-adabere na nso nso iji mara ogige protein na maapụ organelles. Gingras, AC, Abe, KT & Raught, B. Ịmata agbata obi: iji biotinylation na-adabere na nso nso iji mara ogige protein na maapụ organelles.Gingras, AS, Abe, KT na Raut, B. Mmata na gburugburu: iji biotinylation dabere na nso nso iji mara ogige protein na akụkụ ahụ maapụ. Gingras, AC, Abe, KT & Raught, B. 了解邻里：使用邻近依赖性生物素化来表征蛋白白质复合物制复得物列。 Gingras, AC, Abe, KT & Raught, B. Ịghọta agbataobi: jiri ndabere agbata obi na ndụ ndu.Gingras, AS, Abe, KT na Raut, B. Ịghọta ịdị nso: njirimara nke ogige protein na nkewa nke akụkụ ahụ site na iji biotinylation na-adabere na nso nso.Ugbu a.Echiche m.Chemical.bayoloji 48, 44–54 (2019).
Geri, JB et al.Ịdepụta ihe microenvironment site na ịnyefe ike Dexter na sel ndị na-alụso ọrịa ọgụ.Sayensị 367, 1091–1097 (2020).
Hertling, EL et al.Netwọk ọnụ ọgụgụ proteome abụọ na-achọpụta mgbanwe mkpụrụ ndụ akọwapụtara nke mmekọrịta mmadụ na ibe ya.Mkpụrụ ndụ 184, 3022–3040.e3028 (2021).
Oge nzipu: Sep-15-2022
