347 tšepe e sa hloekang e kopantsoeng ka likarolo tsa lik'hemik'hale, Ho tsebahatsoa ha liprotheine tsa chaperone tsa interferon-responsive human leukocyte antigen-A (HLA-A) tse sebelisang cross-linked mass spectrometry (CLMS)

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Li-tube tsa Coil tsa Stainless 347L, Kereiti ea Tšepe: SS347L

Sistimi ea lipontšo tsa interferon e etsa hore ho be le karabelo e matla ea cytokine ho matšoao a mangata a pathogenic le a ka hare ho tikoloho, e leng se etsang hore ho be le li-subsets tsa liprotheine tsa interferon-inducible.Re sebelisitse DSS-mediated cross-link mass spectrometry (CLMS) ho bona litšebelisano tse ncha tsa protheine sebakeng sa liprotheine tse hlahisoang ke interferon.Ho phaella ho liprotheine tse lebeletsoeng tsa li-interferon-inducible, re boetse re khethile li-intermolecular le intramolecular cross-linked adducts tsa canonical interferon-inducible proteins tse kang MX1, USP18, OAS3, le STAT1.Re tsepamisitse maikutlo holim'a netefatso ea orthogonal ea "novel set of interferon-inducible protein networks" e entsoeng ke HLA-A proteins (H2BFS-HLA-A-HMGA1) e sebelisa co-immunoprecipitation le boithuto ba bona bo tsoelang pele ba sebelisa mohlala oa limolek'hule tsa dynamics.Ho etsa mohlala oa conformational dynamics ea protheine complex ho senotse libaka tse 'maloa tsa tšebelisano tse bonts'ang litšebelisano tse khethiloeng ho liphuputso tsa CLMS.Hammoho, re fana ka boithuto ba sefofane sa CLMS ho tsebahatsa li-complexes tse ncha tse hlahisoang ke interferon, 'me re lebelletse ts'ebeliso e pharaletseng ea CLMS ho tsebahatsa matla a macha a ho sebelisana ha protheine sebakeng sa tumor microenvironment.
Pele karabelo e feto-fetohang ea 'mele ea ho itšireletsa mafung e qala, ts'ireletso ea tlhaho ea moamoheli e phahamisa karabelo ea antimicrobial e kentsoeng ke lelapa la li-cytokine tse patiloeng tsa alpha-helical tse bitsoang interferon (IFNs).Mofuta oa I IFN lihlopha tsa IFNα le IFNβ li kenya tšebetsong likarabo tsa cellular, ho kenyelletsa le li-antiviral, proapoptotic, proinflammatory, le li-antiproliferative states.Ho batho, li-subtypes tsa 13 tsa IFNα li tsejoa, kaofela li kopantsoe ka chromosome 91. Hoa makatsa hore ebe ke IFNα2 feela e ithutoang bakeng sa tšebeliso ea meriana.Haufinyane tjena, ho lebisitsoe tlhokomelo e khethehileng ho etsa lipatlisiso ka mefuta e meng ea IFNα.Phuputso ea morao-rao e bontšitse hore IFNα14 ke e 'ngoe ea li-isoform tse sebetsang ka ho fetisisa ho thibela ho pheta-pheta HBV2 le HIV-13,4 ha e bapisoa le subtype ea canonical IFNα2.
Ho netefalitsoe hore mefuta e kentsoeng ea mofuta oa I interferon receptor complexes (IFNAR1 le IFNAR2) e etsa hore ho be le cascade transduction cascade e kentsoeng ke Janus kinases TYK2 le JAK15,6.Tsena Janus kinases phosphorylate signal transducers le transcriptional protheine activators (STAT1 le STAT2) ka masala a tyrosine ho qala SH2 domain-mediated heterodimerization6.Ka mor'a moo, IRF9 e tlama li-heterodimers tsa STAT ho theha motsoako oa trimeric oa IFN-stimulated factor 3 gene (ISGF3), e fetelang khubung le ho etsa hore ho ngoloe liphatsa tsa lefutso tse fetang 2000 tse susumetsang interferon (ISGs)5,6,7,8.
Li-ISG li theha mokokotlo oa sesole sa 'mele, haholo-holo ho arabela tlhaselo ea kokoana-hloko.E le mokhoa oa pele oa tšireletso khahlanong le tšoaetso ea kokoana-hloko, lisele li potlakela ho sebelisa litšebelisano tse ngata tsa liprotheine tsa cellular tse nang le mefuta e mengata ea mesebetsi ea likokoana-hloko.Liprotheine tsena li kenyelletsa li-receptor tse amohelang mohlala, limolek'hule tse tšoaeang, lintlha tse ngotsoeng, le liprotheine tse nang le ts'ebetso e tobileng ea antiviral, hammoho le balaoli ba fosahetseng ba likarabo tsa 'mele oa ho itšireletsa mafung9.Boitsebiso bo bongata mabapi le ts'ebetso ea ISG bo tsoa ho li-skrini tse sebetsang ka ho sebelisa li-skrini tsa overexpression10,11 kapa mekhoa ea ho khutsisa liphatsa tsa lefutso (siRNA, RNAi le CRISPR)12,13 moo li-ISG ka bomong li hlahisoang kapa li thibetsoeng 'me mosebetsi oa bona o lekoa ka likokoana-hloko tse sa tšoaneng.Leha liphuputso tsena li khethile thepa ea antiviral ea li-ISG ka bomong, mekhoa ea limolek'hule ea ISG ka 'ngoe e lula e sa tsejoe haholo.Ka kakaretso ho amoheloa hore liprotheine tse ngata li sebelisana le cytokine e le 'ngoe kapa ho feta ho netefatsa ts'ebetso e felletseng, kahoo li-ISG li sebelisana ka kotloloho kapa litšebelisano tsa tsona li hokahane le liprotheine tsa cellular.Ka mohlala, phuputso ea morao-rao ea photocrosslinked proteomics e bontšitse ATPase VCP/p97 e le molekane ea ka sehloohong oa IFITM3 oa ho sebelisana, eo thibelo ea hae e lebisang mefokolong ea lysosomal sorting, turnover, le cotransport ea IFITM3 e nang le likokoana-hloko tsa 14.Re sebelisa immunoprecipitation, re ile ra tsebahatsa VAPA, protheine e amanang le vesicle, e le molekane oa ho sebelisana le IFITM1 / 2/3 e kopanyang k'holeseterole e amanang le ho hōla ha kokoana-hloko ea k'holeseterole, 'me sena se tiisitsoe ke thuto e' ngoe e sebelisang tomoso tsamaiso ea li-hybrid tse peli.Tšehetso ea Saense 15 , 16 .
Ts'ebetso ea mantlha ea baeloji e amehang khatellong ea ts'oaetso le phetoho e mpe ke tlhahiso ea antigen, e kentsoeng ke limolek'hule tse kholo tsa histocompatibility complex (MHC).Li-peptide (8-12 amino acids nako e telele) ho tloha liprotheine tse arohaneng, tse felisitsoeng pele ho nako kapa tse sa tsitsang li kenngoa ho MHC-I heterodimer (e nang le MHC-I e boima le liketane tse bobebe, tse bitsoang β-2-microglobulin; β2M) 17,18.Li-trimers tse tsitsitseng tsa MHC-I tse hlahisoang li isoa ka holim'a sele, moo li hlahisang li-peptide tsa intracellular ho lisele tsa CD8 + T (lisele tsa cytotoxic T) 17.Lisele tsa T li lemoha le ho senya likokoana-hloko tsena le lisele tse jereng antigen e khethehileng ea tumor.Ka lebaka leo, likokoana-hloko le lisele tsa tumor hangata li hatella ts'ebetso ea tlhahiso ea antigen ho qoba ho lebela sesole sa 'mele.Ho phaella moo, MHC-I e fokotsehile ho 40-90% ea lihlahala tsa batho 'me hangata e amahanngoa le bofokoli bo futsanehileng19.
Liphatsa tsa lefutso tse amehang ho arabeleng likokoana-hloko li tlameha ho fetoha kapele pakeng tsa boemo ba ho phomola le boemo ba ho ngoloa ka mafolofolo.Ka hona, liprotheine tse 'maloa tsa lisele tsa cellular li nahanoa hore li ameha karabelong ea tlhokahalo e phahameng ea IFN ka nako e khutšoanyane, ho kenyelletsa le ho lokisoa le ho fetoloa ha chromatin ea 20,21.Liphuputso tse ngata li tsepamisitse maikutlo ho khethoeng ha balekane ba protheine ea ISG ka boteng ba IFN.Lithuto tse 'maloa tsa proteomic le transcriptomic ka har'a litsamaiso tsa lisele tsa mohlala li hlakisitse phello ea IFN sebakeng sa cellular.Leha ho le joalo, ho sa tsotellehe kutloisiso e ntseng e hōla ea matla a bakoang ke li-interferon, re ntse re tseba hanyenyane ka ho kenya letsoho ha li-ISG.Ha ho nahanoa ka ho rarahana le matla a itšetlehileng ka nako ea pontšo ea interferon, ho hlaha lipotso tse peli: (i) na hoa khoneha ho tsitsisa le ho tšoasa li-multiprotein complexes tse amehang ka pontšo e potlakileng, 'me (ii) na litšebelisano tsee li ka kenngoa sebakeng sa 3D?
Ho rarolla mathata ana, re kentse tšebetsong disuccinimide suberate-mediated chemical cross-linking (DSS) hammoho le mass spectrometry (CLMS) ho ithuta marang-rang a tšebelisano ea protheine ea IFNα le matla a eona.DSS e eketsa maqhama a covalent lipakeng tsa masala a proximal a liprotheine le/kapa protheine complexes in vivo.Tlhahlobo e latelang ea MS e senola libaka tse ikhethileng tse hokahanyang tse bonts'ang sebaka sa libaka tse ka har'a protheine e itseng, tse bitsoang li-internal linkages, kapa li-subunits tse ka har'a protheine complexes, tse bitsoang interrelationships.Ka ho sebelisa mokhoa ona, re fumane li-protein-protein complexes tse 'maloa tse ncha hammoho le marang-rang a interferon-induced multiprotein interaction.Ka ho tsoela pele ho leka karoloana ea litšebelisano tsena tse ncha, re bontša hore H2BFS (H2B histone-type FS; eo hamorao e bitsoang H2B) le MDN1 li sebetsa e le balekane ba tlamang bakeng sa HLA-A.
Lisele tsa Flo-1 ke e 'ngoe ea mefuta e tsebahalang haholo ea in vitro ea esophageal adenocarcinoma kaha li etsisa likarolo tsa bohlokoa tsa lihlahala tsa 'metso22,23.Leha ho le joalo, hase lihlahala tsohle tse nang le immunogenic, le ho fumana hore na lisele tsa Flo-1 li arabela kalafo ea interferon, re ile ra tšoara lisele tsa Flo-1 ka 10 ng / ml IFNα bakeng sa lihora tsa 72.Lisele tsa Flo-1 li bontšitse ho kenngoa pele ho pSTAT1 le IRF1, ho qala lihora tsa 2 ka mor'a phekolo le ho tsoela pele ka lihora tsa 72, ka ho fokotseha ho itšetlehileng ka nako ea litekanyetso tsa IRF1 (Setšoantšo sa 1A).Li-ISG (MX1, IFITM1, OAS1/2, le ISG15) li fumanoe li susumelitsoe ka matla ka mor'a lihora tsa 6, li etsisa likarabo tsa khale tsa bohareng le tsa morao ho IFNα (Setšoantšo sa 1A).Hammoho, lintlha tsena li fana ka maikutlo a hore mohlala ona oa selefouno o ka sebelisoa ho ithuta likarabo tsa interferon.
Likarabo tse fapaneng tsa polelo ea protheine liseleng tsa Flo-1 kamora kalafo ea IFNα.(A) Polelo ea protheine ka lisele tsa Flo-1 tse tšoaroang ka 10 ng / ml IFNα bakeng sa 2, 6, 24, 48 le lihora tsa 72 li ile tsa hlahlojoa ke immunoblot ho sebelisa li-antibodies tsa ISG tse bontšitsoeng.(B) Coomassie blue stained SDS-PAGE gels ea all cell extracts kamora ho hokahana le DSS bakeng sa linako tse bontšitsoeng le likhakanyo.(C) Moemeli oa immunoblot o hlahlobiloe ka antibody ea p53 (DO-1) ho tloha lisampole tse tšoanang ho hlahloba tekanyo ea li-protein cross-linking.
Ho hapa sebopeho sa tšebelisano ea protheine ea in situ, re sebelisitse DSS, sesebelisoa se kopanyang se sebelisoang haholo ka lebaka la ho kenella ha lera le phahameng le nako e khuts'oane ea karabelo.Nako e khuts'oane ea karabelo e thusa ho thibela ho thehoa ha likarolo tse kholo tsa liprotheine tse kopantsoeng, ka hona ho boloka botsitso ba crosslinker.Ho tseba boemo bo nepahetseng ba DSS le ho qoba ho hokahana ho feta tekano, re ile ra qala ho pepesa lisele ho 5, 2.5, le 1 mM DSS bakeng sa metsotso e 5, 10, 5 le 30, ka ho latellana, le ho sekaseka li-lysates ka Coomassie-stained SDS-PAGE. (data ha e bonts'e).Li-cell lysate li bonahala li hokahane haholo maemong a tlase haholo le ka nako e khuts'oane.Ka hona, DSS e ne e lekanyelitsoe ho 1, 0.5, le 0.1 mmM ho feta metsotso e 5 (Setšoantšo sa 1B).Tsela e nepahetseng ea ho tšela e ile ea hlokomeloa ka 0.5 mM DSS bakeng sa metsotso ea 5, 'me maemo ana a khethiloe bakeng sa lisele tse tšoaroang ka IFNα.Ho feta moo, Setšoantšo sa 1C se bonts'a blot ea Bophirimela e entsoeng ho sebelisoa lesole la 'mele la p53 (DO-1) ho lekola boemo ba protheine cross-linking.
Lisele tsa Flo-1 li ne li tšoaroa ka 10 ng / ml IFNα bakeng sa lihora tsa 24 pele li eketsa crosslinker.Lisele tse amanang le lifapano li ile tsa qetella li lysed ka proteolysis ea mehato e 'meli le liprotheine li ile tsa sebetsoa ke FASP (Fig. 2) 24,25.Li-peptide tse nang le li-tryptic tse amanang le sefapano li ile tsa hlahlojoa ke boima ba spectrometry (setšoantšo sa 2).The MS/MS spectra e ntan'o bapisoa le tatellano ea liprotheine 'me e lekantsoe le MaxQuant26,27.Li-peptide tse amanang le sefapano li ile tsa khetholloa ho tloha ponong e fumanoeng ka ho sebelisa lenaneo la SIM-XL, 'me metsoako ea motho ka mong e ne e kopantsoe le marang-rang a rarahaneng a sebelisa xQuest28 le SIM-XL29 liphaephe tsa software tse bulehileng tsa k'homphieutha (setšoantšo sa 2).SIM-XL e khetholla litšebelisano tsa protheine-protein, liketane tsa ka hare le liketane tsa motho ka mong ka metsoako e bonolo kapa e rarahaneng ea liprotheine 'me e fana ka litokomane bakeng sa ho bona litšebelisano tsa liprotheine.Ntle le moo, e beha sesupo se seng le se seng e le lintlha tsa ID ho latela boleng ba spectrum ba MS/MS29.Litšebelisano tse 'maloa tsa protheine-protein le li-complexes tse' maloa tse tšepahalang li 'nile tsa fumanoa,' me ho 'nile ha etsoa lipatlisiso tse ncha ho sebelisa li-co-immunoprecipitation le liphetoho tsa conformational tsa li-complexes tse sebelisang mohlala oa molecular dynamics (MD) (Fig. 2) 30, 31.
Kakaretso ea moralo oa mokhoa oa CLMS.Lisele tsa Flo-1 li ne li tšoaroa ka 10 ng / ml IFNα bakeng sa lihora tsa 24 tse lateloa ke in situ protein cross-linking sebelisa DSS e lateloa ke cell lysis le trypsinization.Mehlala e amanang le sefapano e ile ea hlahlobjoa ho sebelisoa spectrometer ea boima ba Orbitrap 'me ea etsoa sampuli e eketsehileng bakeng sa ho aroloa ha li-precursors tsa peptide nakong ea LC-MS/MS.Li-peptide tse peli tse hokahaneng li ile tsa khetholloa ho tsoa ho sebatli se fumanoeng ho sebelisoa Spectrum Recognition Machine ea lenaneo la Crosslinked Peptides (SIM-XL), 'me metsoako eohle e kopantsoe ho ba marang-rang a rarahaneng a sebelisa liphaephe tsa computational.Hlakola litšebelisano tse tlase tse thehiloeng ho lintlha tse fosahetseng (FDR).Litšebelisano tse 'maloa tse ncha tse phahameng tsa protheine-protein li ile tsa tiisoa ka ho eketsehileng ho sebelisa co-immunoprecipitation,' me liphetoho tse lumellanang le li-complexes li ile tsa hlahlojoa ho sebelisoa mohlala oa molecular dynamics (MD).
Kakaretso ea ~ 30,500 le ~ 28,500 li-peptide li ile tsa fumanoa li sebelisa MaxQuant ka lisampole tse sa tsitsang le tse tsosolositsoeng tsa IFNα, ka ho latellana (Supplementary Table S1, Fig. 3A).Kabo ea bolelele ba peptide maemong ana ka bobeli e bontšitse karolo e phahameng ea li-peptide tse kholoanyane, e bonts'ang boteng ba li-peptide tse amanang le sefapano (setšoantšo sa 3B, C).Ho phaella moo, karolo e kholoanyane ea li-peptide tse khōloanyane li ne li le teng ka har'a mefuta ea 40-55 ka lisampole tse tšoaroang ke IFNα (setšoantšo sa 3C).'Mapa oa protheine khahlano le matla a log2 o bonts'itse hore liprotheine tsa khale tse hlohlellelitsoeng ke interferon ke tsona tse ngata haholo ha li bapisoa le lisampole tse sa phekolehang, ho kenyeletsoa MX1, IFIT1/3, OAS2/3, DDX58, le HLA-F (Figure 3D).Tlhahlobo ea litsela tsa liprotheine tse fetang makhetlo a mararo a matlafalitsoeng ho arabela kalafo ea IFNα ho sebelisa database ea tsela ea Reactome e bontšitse hore MHC-I-mediated antigen presentation and processing e ne e le tsela e ka sehloohong ka ho fetisisa (Setšoantšo sa 3E).Tumellanong le litlaleho tsa pejana, likarabo tsa antiviral tse kentsoeng ke OAS le ISG15 hammoho le IFNα / β le pontšo ea cytokine li ne li le har'a litsela tse entsoeng.Ho feta moo, li-cross-link tsa protheine ea lysine le serine-specific li ile tsa khetholloa ho tsoa ho li-spectra tse fumanoeng qalong tsa MS/MS ho sebelisoa SIM-XL.Boithuto ba morao-rao bo tlalehile li-ISG tse 104 tse nkileng livaerase tse 20 ho tsoa lihlopheng tse 9 tsa vaerase ka tlhahlobo ea meta ea lithuto tsa ISG ka bomong ho mefuta e 5 ea lisele9.Leha ho le joalo, ho hlōla mefokolo ea computational ea ho hlahloba li-datasets tse kholo, re ile ra qala ka dataset e nyenyane ho hlahloba litšebelisano tse ka khonehang pakeng tsa lethathamo la liphatsa tsa lefutso tsa IRDS tse tlalehiloeng ke Padaria et al., tseo boholo ba tsona e leng li-ISG.
Ho tsebahatsa liprotheine tse amanang le sefapano ka mokhoa o fapaneng ho arabela IFNα (lintlha tse fumanoang ho MaxQuant).(A) Setšoantšo sa Venn se emelang palo ea li-peptide tse tloaelehileng le tse khethehileng tse khetholloang ho IFNα14 lisampole tse tšoaroang le tse sa phekoloang tsa Flo-1.Kabo ea bolelele ba Peptide ea lisampole tse sa tšoaroeng (B) le IFNα tse tšoaroang (C) tse fapaneng.(D) 'Mapa oa mocheso o emelang log2 (LFQ intensity) pakeng tsa lisele tsa Flo-1 tse sa phekoloeng le IFNα14.Karolo e ka letsohong le letšehali e bonts'a liprotheine tse sebetsang ka ho fetisisa ka pel'a IFNα.(E) Histogram e emelang litsela tse kholo tsa 20 tsa ntlafatso ka mor'a phekolo ea IFNα.Database ea tsela ea Reactome e hlahlobile liphetoho tse fetang makhetlo a mane ho liprotheine tse arabelang ka IFNα.
Tšusumetso ea Interferon-mediated ISG e ngotsoe hantle, empa boemong ba limolek'hule ha e utloisisoe hantle hore na liprotheine tsena li fella joang ka mesebetsi e mengata ea likokoana-hloko.Re batlisitse tšebelisano ea protheine ka kholiseho e phahameng lipakeng tsa li-ISG tse tsebahalang.Hoa thahasellisa hore re bone marang-rang a kenyeletsang MX1, USP18, ROBO1, OAS3, le liprotheine tsa STAT1 tse etsang motsoako o moholo ho arabela phekolo ea IFNα (Setšoantšo sa 4, Letlapa la S2) 32,33,34.Habohlokoa ka ho fetisisa, litšebelisano tsena li ile tsa fumanoa likarolong tsohle tse tharo tse tšoaroang ka IFNα 'me ha lia ka tsa fumanoa ka mehlala e sa phekoleheng, ho fana ka maikutlo a hore li thehiloe ka ho khetheha ho arabela phekolo ea IFNα.Hoa tsebahala hore STAT1 e laola ka ho ngoloa polelo ea li-ISG tsena, empa tšebelisano ea eona le li-ISG maemong a protheine ha e so ithutoe.Sebopeho sa kristale sa STAT1 se bontšitse hore sebaka sa eona sa helical (CCD) ha se amehe ho sebelisana le DNA kapa protomers nakong ea ho thehoa ha dimers35.Li-α-helices tsena li etsa sebopeho sa helical helix se fanang ka sebaka se seholo sa hydrophilic bakeng sa ho sebelisana ho etsahala 35.Lintlha tsa rona tsa CLMS, re hlokometse hore boholo ba litšebelisano le STAT1 li etsahetse sebakeng sa SH2 pele ho CCD, sebaka sa linker, kapa mohatla oa C-terminal (masala 700-708) (Setšoantšo sa 4A).Phuputso e fetileng e tlalehile hore USP18 e tlama ho CCD le DNA-binding domain (DBD) ea STAT2 'me e hiriloe ho subunit ea mofuta oa I interferon receptor IFNAR2 ho buisana le thibelo ea mofuta oa I interferon signaling 24 .Lintlha tsa rona li boetse li bonts'a hore USP18 catalytic domain e sebelisana le STAT1 DBD (Figure 4A, D), e fana ka maikutlo a hore STAT1 le STAT2 ka bobeli li ka bapala karolo ea ho hohela USP18 ho IFNAR2.
I-protein-protein ISG network e khetholloa ka liseleng tse amanang le sefapano tse tšoaroang ka IFNα.(A) Morero oa ho sebelisana oa 2D o bontšang litšebelisano tsa protheine-protein (tse hlahisoang lenaneong la SIM-XL), ka mela e emelang litšebelisano tsa li-intermolecular (crosslink cutoff set to 3.5).Libaka tsa boitsebiso bo fapaneng li tšoauoa ka mebala ea tsona32: MX1 domain, Dynamin_N (73–249), Dynamin_M (259–547), le GED (569–660).OAS3 libaka: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), le OAS1_C (903-108).Domain ROBO1, Ig_3 (67–151), I-set (170–258), I-set (262–347), Ig_3 (350–432), Ig_3 (454–529), fn3 (562–646), fn3 (678–758) le fn3 (777–864).Mabaka a STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), le STAT1_TAZ2bind (715–739).(B) Sebali sa selikalikoe sa liprotheine tse amanang le sefapano (MX1, UBP18, OAS3, ROBO1, le STAT1) ka litšebelisano le litšebelisano tse ngotsoeng ka 'mala o moputsoa le o mofubelu, ka ho latellana.Moeli oa li-cross-link o ne o behiloe ho 3.5.Lits'oants'o tsa matheba li bonts'a libaka tsa tšebelisano tsa STAT1 le MX1 (C), USP18 (D), ROBO1 (E), le OAS3 (F), hammoho le libaka tsa tšebelisano tsa K kapa S lipakeng tsa li-peptide tse peli.Setšoantšong, moeli oa lintlha tsa cross-link o behiloe ho 3.0.+STAT1 (pdb id: 1bf533) le OAS3 (pdb id: 4s3n34).) lenaneo.
Li-isoform tse peli tsa USP18 li hlalositsoe ho batho, protheine e bolelele bo felletseng e fumanehang ka har'a khubu, le isoform ntle le sebaka sa N-terminal, USP18-sf, e ajoang ka ho lekana ho cytoplasm le nucleus 36.Ho feta moo, ho ile ha boleloa esale pele hore N-terminus ha e na sebopeho ebile ha e hloke ts'ebetso ea isopeptidase kapa ISG1537 e tlamang.Boholo ba litšebelisano tse khethiloeng thutong ea rona li ne li le N-terminus ea protheine, e fana ka maikutlo a hore litšebelisano tsena li kenyelletsa USP18 e tletseng bolelele (Figure 4A, D) 'me kahoo mohlomong e etsahala khubung.Ho feta moo, lintlha tsa rona li boetse li bontša hore N-terminus e ikhethile bakeng sa ho sebelisana ha protheine le protheine.Sebaka se tlamang sa IFNAR2 se teng pakeng tsa masala a 312-368, 'me ka ho khetheha, ha ho na liprotheine tse rarahaneng tse tlamang sebakeng sena (setšoantšo sa 4A) 37,38.Lintlha tsena tse nkiloeng hammoho li bontša hore sebaka se tlamang sa IFNAR2 se sebelisoa feela ke protheine ea receptor.Ho phaella moo, ke OAS3 le ROBO1 feela tse fumanoeng li amahanngoa le libaka tse holimo ho N-terminus le IFNAR2 sebaka se tlamang (Setšoantšo sa 4A).
ROBO1 ke ea immunoglobulin (Ig) superfamily ea transmembrane signaling molecules 'me e na le libaka tse hlano tsa Ig le libaka tse tharo tsa fibronectin (Fn) sebakeng sa extracellular.Libaka tsena tsa extracellular li lateloa ke sebaka sa membrane-proximal le helix e le 'ngoe ea transmembrane helix 39. Sebaka se sa tsitsang sa intracellular se sebakeng sa C-terminus 'me se na le li-sequence motifs tse bolokiloeng tse kopanyang protheine e tlamang39.Sebaka se tlohang ho amino acid ~ 1100 ho isa ho 1600 hangata se na le pherekano.Re fumane hore MX1 e sebelisana le ROBO1 ka Ig, Fn, le libaka tsa intracellular, ha litšebelisano tse ngata le STAT1 li etsahala pakeng tsa CCD, sebaka sa linker, le C-terminus ea ROBO1 (Fig. 4A, E).Ka lehlakoreng le leng, litšebelisano le libaka tsa li-linker tsa DI, DIII, le OAS3 li ile tsa ajoa ho pholletsa le protheine ea ROBO1 (Fig. 4A).
Lelapa la liprotheine la oligoadenylate synthase (OAS) le amohela le ho tlama RNA (dsRNA) e nang le methapo e 'meli ea intracellular, e etsa liphetoho tsa conformational,' me e kopanya 2',5'-linked oligoadenylates (2-5 As) 40.Ho fumanoe hore har'a li-OAS tse tharo, OAS3 e bonts'a kamano e phahameng ka ho fetisisa bakeng sa dsRNA mme e kopanya bonyane ba 2-5 As, e ka kenyang RNase L 'me kahoo e fokotsa ho ikatisa ha kokoana-hloko 41.Lelapa la OAS le na le libaka tsa polymerase beta (pol-β) tse kang nucleotide transferase.Patlisiso e fetileng e bonts'itse hore ts'ebetso ea catalytic ea sebaka sa C-terminal (DIII) se its'etleha ho dsRNA-binding domain (DI), e hlokahalang bakeng sa ts'ebetso ea OAS342.Re hlokometse hore libaka tsa DI le DII tsa OAS3 li sebelisana le CCD le sebaka se senyenyane sa mateano pakeng tsa SH2 le STAT1 TAD (Setšoantšo sa 4A, F).Ho koahela libaka tse fapaneng tsa crosslinking holim'a sebopeho sa protheine ho senotse ho sebelisana pakeng tsa β-sheet le DBD STAT1 loop le pokotho e bulehileng kapa sekoti se entsoeng ke masala 60-75 sebakeng sa DI sa OAS3 (Fig. 4G).Boikutlo ba liprotheine bo rarahaneng bo boetse bo bontšitse hore ha ho le e 'ngoe ea litšebelisano le OAS3 e sitisang matla a DNA a tlamang sebaka sa eona sa DI (Fig. S1A).Ho phaella moo, sebaka sa N-terminal sa GTPase MX1 se sebelisana haholo le libaka tsa DI le DIII tsa OAS3 (Fig. 4A).Re boetse re hlokometse tšebelisano pakeng tsa OAS1 le MX1 ho pheta-pheta tse tharo tse tšoaroang ke IFNα, moo sebaka se le seng sa OAS1 (le sona se sebetsang hantle) se sebelisaneng le libaka tsohle tse tharo tsa MX1 (Setšoantšo sa S2A, B).
Liprotheine tsa MX ke karolo ea lelapa le leholo la li-GTPase tse kang dynein tse nang le sebaka sa N-terminal GTPase se tlamang le ho hydrolyzes GTP, sebaka se mahareng se ikemetseng ho kopana, le zipper ea C-terminal leucine e sebetsang joaloka GTPase (LZ). ).domain effector domain25,43.MX1 e tlamella likaroloana tsa lipolymerase tsa vaerase ho thibela ho ngoloa ha kokoana-hloko ea gene43.Skrine e tlalehiloeng ea tomoso ea li-hybrid tse peli e bonts'itse hore PIAS1 e amanang le MX1 e thibela ts'ebetso ea liphatsa tsa lefutso tsa STAT1 ka ho thibela ts'ebetso e tlamang DNA hape e na le ts'ebetso ea ligase ea SUMO E344,45.Mona, re bontša hore MX1 e tlama ho STAT1 (Setšoantšo sa 4C, D), leha ho le joalo hore na tšebelisano ena e ama ts'ebetso ea liphatsa tsa lefutso tsa STAT1-mediated ho arabela IFNα e hloka thuto e eketsehileng.Ho phaella moo, re boetse re fumane hore MX1 e sebelisana le IFIT3 le DDX60 ka makhetlo a mararo a phetoa-pheta-pheta (Fig. S2C).
DDX60 ke IFN-induced cytoplasmic helicase eo pele ho tlalehiloeng hore e na le karolo ho senyeha ho ikemetseng ha RNA46 ea kokoana-hloko ea RNA46.E sebelisana le RIG-I mme e kenya tšebetsong pontšo ea eona ka mokhoa o khethehileng oa ligand 46. DDX60 e na le sebaka sa DEXD/H-Box sa helicase le sebaka sa C-terminal helicase se tlamang likokoana-hloko tsa RNA le DNA47.Boholo ba litšebelisano tsa eona le MX1 le IFIT3 li etsahala ka har'a libaka tse telele tsa N- le C-terminal ntle le libaka tsa canonical kapa motifs (Fig. S2E, F).Leha ho le joalo, MX1 e boetse e amahanngoa le sebaka sa helicase sa DEXD / H-Box (Fig. S2E).Liprotheine tsa lelapa la IFIT li na le likopi tsa tandem tsa mofuta o ikhethileng oa helix-turn-helix o bitsoang tetrapeptide repeat (TPR).IFIT3 e fumanoe e le modulator e ntle ea pontšo ea RIG-I 'me kahoo e le karolo ea motsoako oa MAVS.Ha re kopane, lintlha tsa rona li fana ka maikutlo a hore IFIT3 le DDX60 li sebelisana haholo-holo sebakeng se pakeng tsa TPR 3-6 ea IFIT3 'me e ka bapala karolo ea RIG-I / MAVS pontšo (Fig. S2F).
Ka lebaka la hore ho hlahloba proteome eohle ho matla haholo, re ile ra hlahloba boitsebiso bohle ba batho ba UniProt bakeng sa boteng ba e 'ngoe ea pheta-pheta e tšoaroang ke IFNα.Setšoantšong sena, re fumane marang-rang a mangata a ts'epahalang haholo a HLA-A.Tlhahlobo ea litsela tsa protheine e khetholloang ke MS / MS spectra e bontšitse hore MHC-I-based antigen processing le tlhahiso ke tsela e ka sehloohong e bakoang ke interferon (Fig. 3D).Ka hona, re tsepamisitse maikutlo ho ithuteng litšebelisano tsa protheine tsa limolek'hule tsa MHC-I tse nang le kholiseho e phahameng ho mehlala eohle e amanang le sefapano.HLA e na le libaka tsa α1, α2 le α3 le liketane tse bobebe, 'me microglobulin β2 (β2m) ke protheine ea kamehla ea chaperone49.Ha e se e bokane ho endoplasmic reticulum, HLA ha e tsitsa ha ho se na peptide ligands50.Mokotla o tlamang peptide o entsoe ke libaka tse nang le polymorphic tse ngata haholo le tse sa hlophisoang tsa α1 le α2 ka sebopeho se seng sa peptide le sebaka se batlang se le tlase sa polymorphic α351.Ka pel'a IFNα, re fumane li-complexes tse peli tsa HLA-A: e 'ngoe e sebelisana le HMGA1 le H2B (Setšoantšo sa 5, Letlapa la S3)' me e 'ngoe e sebelisana le MDN1, LRCH4 le H2B (Setšoantšo sa 6).
IFNα e kenya marang-rang a HLA-A a sebelisanang le H2B (H2BFS) le HMGA1.(A) 2D plot (e hlahisitsoeng ka software ea SIM-XL) e bontšang mefuta e sa tšoaneng ea litšebelisano mofuteng oa H2B-HLA-A-HMGA1: interlink (blue), interlink (red) le single link (e ntšo)..Libaka tsa boitsebiso bo fapaneng li ngotsoe ka mebala32: H2B (histone; 2–102) le MHC-I (MHC_1; 25–203, sehlopha sa C1; 210–290 le MHC_I_C; 337–364).Moeli oa li-cross-link o ne o behiloe ho 3.5.Lits'oants'o tsa matheba li bonts'a libaka tsa tšebelisano tsa HLA-A le H2B (B) le HMGA1 (C), hammoho le libaka tsa tšebelisano tsa K kapa S lipakeng tsa li-peptide tse peli.Setšoantšong, moeli oa lintlha tsa cross-link o behiloe ho 3.0.(D) Likamano pakeng tsa liprotheine tse bontšitsoeng ka har'a mehaho ea liprotheine tsa H2B, HLA-A, le HMGA1 lenaneong la PyMOL.Mehaho ena e entsoe ka mohlala ho sebelisa seva sa Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) 'me libopeho tsa li-template tsa liprotheine tsa H2B, HLA-A le HMGA1 e ne e le 1kx552, 1kj349 le 2eze55, ka ho latellana.
IFNα e kenya marang-rang a HLA-A a sebelisanang le H2B (H2BFS), MDN1 le LRCH4.(A) Li-crosslink tsa Intramolecular (red) le intermolecular (blue) tse hlahisitsoeng 'mapeng o kopanetsoeng oa 2D (o entsoeng ka software ea SIM-XL) e nang le MDN1 e emetsoeng e le selikalikoe.Moeli oa li-cross-link o ne o behiloe ho 3.5.Libaka tsa boitsebiso bo fapaneng ke mebala e mebala32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, sehlopha sa C1; 210–290 le MHC_I_C; 337–364) le LRCH4 (LRR_8 (68–126), LRR_8 (137-194) le CH (535-641)).(B) Likamano pakeng tsa liprotheine tse bontšitsoeng ka har'a mehaho ea liprotheine tsa H2B, HLA-A, LRCH4, le MDN1 lenaneong la PyMOL.Mehaho ena e entsoe ka mohlala ho sebelisa seva ea Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) e nang le li-template structures 1kx552, 1kj349, 6hlu62 le 6i2665 bakeng sa liprotheine tsa H2B, HLA-A, LRCH4 le MDN1, ka ho latellana.Mehaho e bontšang libaka tsa khokahanyo tsa K kapa S bakeng sa HLA-A e nang le H2B (C), LRCH4 (D), le MDN1 (E).Bakeng sa meralo, moeli oa lintlha tsa cross-link o ne o behiloe ho 3.0.
Ntle le ho boloka botšepehi ba genome, histone H2B e boetse e ameha taolong ea ho ngoloa.Protheine ea H2B e na le sebaka se bohareng sa histone (HFD) se entsoeng ke li-α-helice tse tharo tse arohaneng ka li-loops le mohatla oa C-terminal 41,52.Boholo ba ho sebelisana le H2B ho etsahala ho α1 helix, e fanang ka trimerization le HFD heterodimer (Setšoantšo sa 5A, B).Leha li-lysin li kenya letsoho ho tlamang DNA, li-lysin tse ling le tsona ke libaka tse ling tsa acetylation kapa methylation.Ka mohlala, masala a K43, K46, le K57 ho tloha H2B ha a amehe ho tlamahaneng ha DNA ka ho toba, empa ke lipehelo tsa liphetoho tse fapaneng tsa morao-rao53.Ka mokhoa o ts'oanang, masala a K44, K47, le K57 ho H2B a ka 'na a phetha karolo e' ngoe ka pel'a IFNα, ho kenyelletsa le ho sebelisana le liprotheine tse ling (Fig. 5A, B).Ho feta moo, extrachromosomal histone H2B e kenya tšebetsong karabelo ea 'mele ea ho itšireletsa mafung ka mefuta e fapaneng ea lisele, e sebetsa joalo ka sensor ea cytosolic ho bona likaroloana tse peli tsa DNA (dsDNA) tse tsoang ho li-agent tse tšoaetsanoang kapa lisele tse senyehileng54.Ka pel'a likokoana-hloko tsa DNA, ho fokotseha ha H2B ho thibela tlhahiso ea IFN-β le STAT154 phosphorylation.H2B e boetse e tsejoa ka ho kena le ho tsoa khubung kapele ho feta li-core histones54.Litšebelisano tsa H2B le MDN1 le LRCH4 li ile tsa boela tsa hlokomeloa ka lisampole tse khethiloeng tse sa phekoloang.Re fumane hore HLA-A e sebelisana le H2B lisampoleng tsohle tse tharo tse phekoloang ke IFNα le sampuling e le 'ngoe e sa phekoleheng e phetoang.Lintlha tsena li bonts'a karolo ea H2B ts'ebetsong e 'ngoe ea fisioloji ntle le taolo ea mongolo.
HMGA1 (sehlopha se phahameng sa ho tsamaea sa AT-Hook 1), nucleoprotein e nyenyane e ruileng ka li-amino acid tse khothalletsang maloetse, e 'nile ea tsejoa ka ho kopana le HLA-A.E na le mohatla o nang le asiti ea C-terminal le li-DBD tse tharo tse ikhethileng tse bitsoang li-hook tsa AT hobane li tlamella groove e nyenyane ea sebaka sa AT-rich ho dsDNA55,56.Tlamahano ena e etsa hore DNA e kobehe kapa e otlolohe, e leng ho lumellang lintlha tse ngotsoeng ka molao hore li fihlele tatellano ea eona e lumellanang.Mohatla oa C-terminal ho lumeloa hore o ameha litšebelisanong tsa protheine le protheine le ho hira lintlha tse ngotsoeng, kaha liphetoho tsa C-terminal deletion ha li khone ho qala transcription57.Ho feta moo, sebaka sena se na le libaka tse 'maloa tse bolokiloeng tsa phosphorylation tse tsejoang ka li-substrates tsa kinases 58.Re hlokometse litšebelisano tsa HLA-A le H2B le HMGA1 ka ntle ho sebaka sa C-terminal, ho fana ka maikutlo a hore sebaka sa C-terminal se sebelisoa haholo-holo bakeng sa ho tlama ntho e ngotsoeng (Fig. 5A, C).Liprotheine tsa HMGA li qothisana lehlokoa le histone H1 bakeng sa ho tlamella ho adaptara DNA, ka hona ho eketsa phihlello57.Ka mokhoa o ts'oanang, ho bonahala eka HMGA e sebelisana le histone H2B hammoho le linker DNA tlholisanong le histone H1.HMGB1 e etsa hore ho be le polelo ea HLA-A, -B, le -C ka liseleng tsa dendritic, e lebisang ho ts'ebetsong ea bona59, empa tšebelisano pakeng tsa HMG le HLA ha e e-s'o tlalehoe pele.Re fumane hore HMGA1 e sebelisana le libaka tsa α1 le α3 tsa HLA-A, tse nang le litšebelisano tse ngata ka ntle ho 3 DBD ea eona (Setšoantšo sa 5A, C).Matsohong a rona, HLA-A e ile ea fumanoa e le sebakeng sa khubu (data e sa bonts'itsoeng), mme ha ho nahanoa hore H2B le HMGA1 le tsona li teng khubung, tšebelisano ena e kanna ea etsahala khubung.Li-adducts tse khethehileng tse lekantsoeng lipakeng tsa H2B, HLA-A, le HMGA1 li bonts'itsoe ho Setšoantšo sa 5D.
Litšebelisano tse ngata tsa HLA-A le liprotheine tse ling li etsahala ka har'a libaka tsa eona tsa α1 le α2 le sebaka se senyehileng sa C-terminal (Setšoantšo sa 6).Ho e 'ngoe ea mehlala ena, re fumane hore HLA-A e sebelisana le mohatla o sa sebetseng oa N-terminal oa LRCH4 (Setšoantšo sa 6A,D).LRCH4 e laola ts'ebetso ea TLR4 le LPS cytokine induction, ka hona e fetola karabelo ea tlhaho ea 'mele ea ho itšireletsa mafung60,61.Ke protheine ea membrane e nang le li-leucine-rich repeats (LRRs) le calmodulin (CH) homology motif ho ectodomain ea eona, e lateloa ke sebaka sa transmembrane (TMD) 60, 62.Libaka tsa CH li 'nile tsa tlalehoa hore li na le litšebelisano tsa protheine-protein 60.Karolo e ka bang 300 ea li-amino acid pakeng tsa LRR le CH e fumaneha habonolo empa e sa sebetse.Ho itšetlehile ka mosebetsi oa libaka tse sa tsitsang e le bakena-lipakeng ba marang-rang a liprotheine le lipalangoang tsa vesicular 63, re fumane hore boholo ba likamano tsa protheine li etsahala libakeng tse sa tsitsang.Litšebelisano le MDN1 li ne li ajoa ho pholletsa le bolelele ba protheine, ho kenyelletsa le LRR1, LRR6, CH domains, le libaka tse sa tloaelehang, ha H2B e ne e tlamahane haholo le sebaka sa CH (Fig. 6A, B).Ka ho hlakileng, ha ho le e 'ngoe ea litšebelisano e neng e kenyelletsa TMJ, e fana ka maikutlo a ho khetheha ha mokhoa oa CLMS (Setšoantšo sa 6A, B).
MDN1 e boetse e khethiloe e le karolo ea marang-rang a protheine ea HLA-A (Setšoantšo sa 6A).Ke ea lelapa la AAA la liprotheine (ATPases tse amanang le mesebetsi e fapaneng).Ena ke sebaka se ts'oanang sa N-terminal AAA se hlophisang selikalikoe sa hexameric mme se tlosa ntho ea kopano ho tloha 60S 64 ribosomal subunit.e bonahala e tšoana le dynein64,65,66.Ho phaella moo, sebaka se ruileng sa Asp / Glu se lateloa ke sebaka sa MIDAS (sebaka se itšetlehileng ka ion ea tšepe).Ka lebaka la boholo bo boholo ba MDN1 (hoo e ka bang 5600 amino acid) le homology ea eona e fokolang e nang le liprotheine tse ithutoang hantle, ha ho tsejoe hakaalo ka sebōpeho le mosebetsi oa eona bathong.Re hloaile HLA-A, H2B, le LRCH4 e le balekane ba tlamang ba MDN1 mme ra senola maikutlo a bona joalo ka liprotheine complexes ho PyMol (Fig. 6A,B).Liprotheine tsena tse tharo li sebelisana le sebaka sa AAA, sebaka sa dynein-like linker, mohlomong le sebaka sa MIDAS MDN1.Tlalehong e fetileng, tlhoekiso ea kamano ea liprotheine tsa bait e bontšitse MDN1 e le protheine e amanang le histone H2B67.Ho phaella moo, phuputso ea morao tjena e boetse e tlalehile tšebelisano pakeng tsa MDN le HLA-B liseleng tsa HCT116 tse sebelisang li-mass spectrometry tse hloekisitsoeng, tse tšehetsang seo re se fumaneng68.Ho tsebahatsoa ha motsoako ona ho lisampole tse tšoaroang ke IFNα ho fana ka maikutlo a karolo ea MDN1 ka pontšo ea interferon.
Hobane liphatsa tsa lefutso tsa HLA li na le polymorphic haholo, re ntšitse tatellano e balehang ka 'mapa oa HLA-A, -B, le -C ho tsoa ho data ea RNA ea tatellano ea lisele tsa Flo-1 (boitsebiso bo sa bonts'itsoeng).Tatelano ea peptide e tsamaellanang le ho bala ka tatellano e senotse phapang e kholo lipakeng tsa HLA-A, -B, le -C libakeng tseo li-peptide tse amanang le sefapano li neng li le teng ho HLA-A (Figure S3).Ho feta moo, ha rea ​​ka ra bona khokahano ea protheine le protheine ea limolek'hule tsa HLA-B/C tse nang le liprotheine tsa H2B/HMGA1/MDN1/LRCH4.Sena se fana ka maikutlo a hore tšebelisano ea protheine e fumanehang lipakeng tsa HLA-A, MDN1, LRCH1 le HMGA1 e ikhethile ka HLA-A.Ntle le moo, tlhahlobo ea proteomic ea lisampole tse sa kopanngoeng (Letlapa la S4) li bonts'itse hore HLA-A e na le tšireletso e phahameng ea tatelano ha e bapisoa le HLA-B kapa HLA-C.Li-peptide tse khethiloeng bakeng sa HLA-A li ne li le matla haholo ho lisampole tse tšoaroang ke IFNα le tse sa phekoloang.
Ho etsa bonnete ba hore litšebelisano tse boletsoeng mona ha li bakoe ke likhokahano tse sa tobang tsa liprotheine tse peli tse haufi-ufi, re netefalitse lintlha tse peli tse ncha tse sebelisanang tsa HLA-A ka ho etsa liteko tsa co-immunoprecipitation.Litšebelisano tsa HLA-A le MDN1 le H2B tse sa feleng li ile tsa fumanoa liseleng tsa Flo-1 tse tšoaroang ka bobeli le tse sa tšoaroeng ka IFNα (Setšoantšo sa 7, Setšoantšo sa S4).Re netefalitse hore HLA-A e hapiloe ke H2B ho li-immunoprecipitates le hore mokhatlo ona o ne o bakoa ke phekolo ea IFNα kaha HLA-A e ne e le sieo ka lisampole tsa immunoprecipitate ho tloha lisele tse sa phekoloang (Figure 7A).Leha ho le joalo, lintlha tsa rona li fana ka maikutlo a hore IFNα e laola ka tsela e fapaneng HLA-A e tlamang H2B le MDN1.IFNα e etsa hore ho be le kamano pakeng tsa H2B le HLA-A, empa e fokotsa kamano ea eona le MDN1.Re fumane hore MDN1 e ne e amahanngoa le HLA-A ho laola, 'me ho eketsoa ha IFNα ho fokotsa tšebelisano ena e ikemetseng ea MDN1 ho kenngoa ke IFNα (Setšoantšo sa 7B, C).Ho phaella moo, HLA-A immunoprecipitation e nkile H2B liseleng tsa A549 (Fig. S4), e fana ka maikutlo a hore tšebelisano ena e ikemetse ho mofuta oa sele.Ha li kopane, liphetho tsena li tšehetsa litšebelisano tsa interferon-mediated tsa HLA-A le H2B le MDN1.
HLA-A e hloekisa H2B le MDN1 hammoho.Endogenous H2B (A) le MDN1 (B) li-immunoblots tsa endogenous li ne li se na immunoprecipitated ho tloha liseleng tsa Flo-1 tse tšoaroang ke IFNα 'me tsa hlahlojoa bakeng sa li-antibodies tse bontšitsoeng.Mouse le mmutla IgG li ne li sebelisoa e le taolo e mpe.(C) Litekanyo tse lekanang (tse kenang) tsa li-antigen tse fapaneng li tšoantšetsoa ke li-immunoblots tse hlahlojoang khahlanong le li-antibodies tse bontšitsoeng, β-actin e ne e sebelisoa e le taolo ea ho laela.
Thepa ea sebopeho sa e 'ngoe ea marang-rang a marang-rang a tšeptjoang haholo a nang le li-interferon, H2B-HLA-A-HMGA1, a ile a hlahlojoa.Re sebelisitse mohlala oa limolek'hule tsa dynamics e le mokhoa o mong oa ho utloisisa matla a conformational a liprotheine tse amehang mofuteng ona (Setšoantšo sa 8).Lits'oants'o tse tsoang ho data ea CLMS li fana ka maikutlo a monyetla oa ho ba le likhokahano tse fapaneng tsa liprotheine tsa H2B, HLA-A, le HMGA1.Ka hona, li-complexes tse latelang tse ka 'nang tsa e-ba teng li ile tsa etsoa ka mokhoa oa ho qhibiliha: H2B-HLA-A, HMGA1-HLA-A, le H2B-HLA-A-HMGA1.Sesepa sa pele sa protheine-protein docking se sebelisang MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) sephutheloana se ile sa fana ka tlhahiso ea hore ho ka ba le conformations e fapaneng pakeng tsa liprotheine tsena (Fig. 8A).Pono ea protheine ea docking e senotse litšebelisano tse 'maloa le li-conformations tse ka bang teng (Setšoantšo sa 5A, 8).Kahoo, phetoho e le 'ngoe e ka bang teng e bontšoa ho Setšoantšo sa 8A (se nang le li-cross-link tse ngotsoeng)' me se ile sa hlahlojoa ho ea pele ho sebelisoa pipeline ea mohlala ea MD.Ho phaella moo, ho tlama H2B kapa HMGA1 ho HLA-A ho totobatsa kamano e phahameng ea H2B bakeng sa HLA-A (Setšoantšo sa 8A).
Matla a lumellanang le marang-rang a ka khonehang pakeng tsa li-complexes tsa H2B (H2BFS)-HLA-A, HMGA1-HLA-A, le H2B-HLA-A-HMGA1.(A) Karolo e ka ho le letšehali ke 'mapa oa 2D (o hlahisitsoeng ka SIM-XL software) ea intramolecular (red) le intermolecular (blue) crosslinks (crosslink cutoff set to 3.5).Ho feta moo, masala a hokahantsoeng a tšoailoeng a ngotsoe meahong ea liprotheine tsa H2B, HLA-A, le HMGA1.Li-conformations tse amanang le liprotheine tsena li ile tsa ntšoa ho sebelisoa lipeipi tsa docking tse kenngoeng ka har'a sephutheloana sa MOE.Karolo e ka tlase e ka letsohong le letšehali e bontša mefuta e fapaneng e ka bang teng ea likhomphutha tsa H2B-HLA-A le HMGA1-HLA-A tse nang le li-protein tse tlamang tse fapaneng (GBVI/WSA dG; kcal/mol).(B) Phapang e tloaelehileng (RMSD) ea maemo a athomo (ntle le liathomo tsa hydrogen) bakeng sa sebopeho se seng le se seng sa protheine.(C) Litšebelisano tsa liprotheine tsa protheine ea hydrogen bond tse tsoang ho li-complexes tse sa tšoaneng tse etsisang ho nahana ka litšebelisano tse khethehileng tsa nako ≥ 10 ns.Sebaka sa "h-bond donor-acceptor cutoff" se ne se behiloe ho 3.5 Å, 'me sekheo sa "donor-H-acceptor cutoff" se behiloe ho ≥ 160 ° -180 °.(D) Masala a tšoailoeng a etsang litšebelisano tsa protheine ea HLA-A le balekane ba bona ba fapaneng, ho tloha ho ≥ 20 ns, e nkiloeng ho dummy HLA-A-H2B le HLA-A-HMGA1 complexes.Mehaho ea protheine e emela sebopeho se tloaelehileng sa 100 ns MDS.(E) Litšebelisano lipakeng tsa li-complexes tsa HLA-A-H2B le HLA-A-HMGA1 ha li bapisoa le litšebelisano tse lateloang ke papiso ea H2B-HLA ho feta 100 ns ho ipapisitse le sebaka sa tšebelisano sa K kapa S lipakeng tsa li-peptide tse peli.Mathata /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Boleng ba moeli oa tlhahlobo ea li-cross-link bo ne bo behiloe ho 3.0, 'me litšebelisano tse khethehileng tse tsoang ho MDS tse nkang ≥ 10 ns li ile tsa nkoa.Mehaho ea protheine e ne e bonoa ho sebelisoa BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) le liphutheloana tsa Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
Ho tsitsisa ha limolek'hule tsa HLA-A ha nako e ntse e ea (ho kheloha ho tloaelehileng; RMSD kapa ho kheloha ho tloaelehileng; RMSF) e bontšitse hore boteng ba liprotheine tsa H2B kapa HMGA1 mefuteng e tsitsitseng HLA-A (Figure 8B, Figure S5).Protheine ea HMGA1 e tlama ka thata sebakeng sa B2M sa HLA-A, e etsa hore ho be le botsitso ba HLA-A amino acid ho HLA-A-HMGA1 kapa H2B-HLA-A-HMGA1 complex (Figure 8B, Figure S5).haholo-holo, masala a HLA ~ 60-90 le ~ 180-210 a fumanoe a sa fetohe habonolo ka pel'a H2B (FIG. 8B).H2B le HMGA1 li bonts'itse tlamahano e betere ho HLA-A ka har'a komporo ea H2B-HLA-A-HMGA1 ha e bapisoa le HLA-A e tlamang H2B kapa HMGA1 feela (Setšoantšo sa 8C,D; Letlapa la S5).Masala a amehang ho hydrogen bonding (MD modeled high occupancy ≥ 10 ns) a lumellana le libaka tsa ho sebelisana le CLMS (masala a K kapa a S) a rarahaneng, a fana ka maikutlo a hore litšebelisano tse khethiloeng ke CLMS li tšepahala haholo.Ho tšepahala (setšoantšo sa 8E).Ho CLMS le MD modelling, masala a HLA-A pakeng tsa hoo e ka bang 190-210 le hoo e ka bang 200-220 amino acid a fumanoe a tlama H2B le HMGA1, ka ho latellana (FIG. 8E).
Litšebelisano tsa protheine le liprotheine li theha marang-rang a matla a sebopeho a hokahanyang puisano ea intracellular ho arabela tšusumetso e itseng.Hobane mekhoa e mengata ea liprotheine e lemoha liphetoho boemong bo tsitsitseng ba protheine, matla a ho sebelisana le protheine a hloka lisebelisoa tse eketsehileng tsa ho tšoara li-interfaces tse tlamang, 'me CLMS ke e' ngoe ea lisebelisoa tse joalo.Sistimi ea lipontšo tsa interferon ke marang-rang a cytokine a lumellang lisele ho arabela mefuteng e mengata ea tikoloho le matšoao a ka hare a pathological, ho fella ka ho kenngoa ha li-subsets tsa liprotheine tsa interferon-inducible.Re sebelisitse CLMS ho fumana hore na litšebelisano tse ncha tsa protheine le protheine li ka tsejoa har'a sehlopha sa liprotheine tse hlahisoang ke interferon.Tlhahlobo ea liprotheine tsa lefats'e ka bophara ka mokhoa oa lisele tsa Flo-1 tse arabelang li-interferon li ile tsa sebelisoa ho tšoara liprotheine tse rarahaneng.Ho ntšoa ha li-peptide tsa tryptic ho tsoa liseleng tse sa amaneng le tse hokahaneng ho lumella ho bala peptide, ho ntlafatsa tsela, le ho ajoa ha bolelele ba peptide ka matla a hlalositsoeng a LFQ.Liprotheine tsa Canonical interferon-inducible li ile tsa tsejoa e le taolo e ntle ea ka hare, ha li-adducts tse ncha tsa intermolecular le intramolecular cross-linked tsa canonical interferon-inducible proteins tse kang MX1, UP18, OAS3 le STAT1 li ile tsa hlokomeloa.Likarolo tse fapaneng tsa sebopeho le likamano libakeng tsa ts'ebetso li 'nile tsa batlisisoa.
Tšebelisano pakeng tsa HLA-A, MDN1 le H2B e ile ea fumanoa ka immunoblotting liseleng tsa Flo-1 le A549 tse tšoaroang le ho sa phekoloe ka IFNα.Liphetho tsa rona li totobatsa hore HLA-A e kopana le H2B ka mokhoa o itšetlehileng ka IFNα.Mosebetsi oa rona o emetse mokhoa o khahlisang oa tlhahlobo e tsoelang pele ea ho kopanngoa ha libaka tsena tse peli.Hape ho ka ba monate ho atolosa mokhoa oa CLMS ho phanele ea mela ea lisele ho tsebahatsa litšebelisano tsa protheine tse ikemetseng tsa mofuta oa lisele tsa interferon.Qetellong, re sebelisitse mohlala oa MD e le mokhoa o mong oa ho utloisisa matla a conformational a liprotheine tse amehang mofuteng oa H2BFS-HLA-A-HMGA1, o neng o latela lipuisano tsa intramolecular le intermolecular cross-talk.Lits'oants'o tse tsoang ho data ea CLMS li fana ka maikutlo a hore ho ka ba le likhokahano tse fapaneng tsa liprotheine tsa H2BFS, HLA-A, le HMGA1.Likhokahano tse fapaneng tse ka bang teng lipakeng tsa liprotheine tsena tsa docking li senotse litšebelisano tse 'maloa tse ts'oanang le tse bonoang ho dataset ea CLMS.E 'ngoe ea matla a mantlha a mokhoa oa rona ke hore o lumella ho tsebahatsa habonolo ha liphatsa tsa lefutso tse nang le polymorphic tse ngata joalo ka HLA, ka hona ho tla ba monate ho ithuta ka tšebelisano ea liprotheine tse ikhethileng tsa HLA haplotype tseo ho seng bonolo ho li ithuta.Ha re kopane, lintlha tsa rona li bontša hore CLMS e ka sebelisoa ho atolosa kutloisiso ea rona ea marang-rang a lipontšo tsa interferon le ho fana ka motheo oa ho ithuta mekhoa e rarahaneng ea li-intercellular ka har'a tumor microenvironment.
Lisele tsa Flo-1 li ile tsa fumanoa ho tloha ATCC 'me tsa bolokoa ho DMEM (Gibco) e tlatsitsoeng ka 1% penicillin / streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) mme e bolokiloe ho 37 ° C le 5% CO2.Ho qhomisa.Lisele li ne li hōlile ho 70-80% confluence pele li tšoaroa ka IFNα14 (e entsoeng ke Edinburgh Protein Production Facility).Lik'hemik'hale tse ling kaofela le li-reagents li ile tsa rekoa ho Sigma Aldrich ntle le haeba ho boletsoe ka tsela e 'ngoe.
Lisele tsa Flo-1 li ne li hlahisoa ka lipoleiti tsa 6 'me letsatsing le hlahlamang lisele li ne li tšoaroa ka 10 ng / ml IFNα14 bakeng sa lihora tsa 24 ho ea ho hoo e ka bang 80% confluence.Lisele li ne li hlatsuoa ka makhetlo a mararo ka PBS 'me li kopantsoe le DSS e sa tsoa lokisoa (Thermo Fisher Scientific) (e qhibilihisoa ka DMSO) ho PBS bakeng sa 5 min ho 37 ° C. ho ea ho khatello ea ho qetela ea 0.5 mM.DSS crosslinking reaction e ile ea nkeloa sebaka ke PBS mme DSS e setseng e ile ea tima ka ho eketsa 20 mM Tris (pH 8.0) ho PBS bakeng sa 15 min ho 37 ° C.Lisele li ne li bokelloa ka ho phunya ebe li bokelloa ka li-tubes tse tlamang tse tlase (Axygen).
Pellet ea sele e ne e tšeloa ka 300 µl ea urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) bakeng sa metsotso e 30 mocheso oa kamore ka ho sisinyeha nako le nako.Mehato eohle ea centrifugation e entsoe ka 14,000 xg ho 8°C.Centrifuge the lysate metsotso e 10 'me u fetise supernatant ho tube e ncha.Likaroloana tse setseng tse hlakileng li ile tsa qhibiliha ka 150 μl ea bobeli ba lysis buffer (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) ka metsotso e 30 kapa ho feta ho fihlela ho fumanoa tharollo ea metsi a homogeneous.Lysate e ne e le centrifuged ka metsotso e 20 'me supernatant e kopantsoe le lysate e fumanoeng mohato o fetileng.Maemo a protheine a ile a hlahlojoa ho sebelisoa tlhahlobo ea Micro BCA (Thermo Fisher Scientific) ho latela litaelo tsa moetsi bakeng sa mekhoa ea microplate.Mehlala e ile ea hoamisoa ka potlako ka har'a naetrojene e metsi 'me ea bolokoa ho -80 ° C.
Hoo e ka bang 100 μg ea protheine e qhibilihang ka har'a marang-rang e ile ea sebetsoa ho sebelisoa protocol e fetotsoeng ea ho lokisa sampole (FASP) joalo ka ha ho hlalositsoe ke Wisniewski et al.69 Ka bokhuts'oanyane, protheine e hokahane le 200 µl ea urea buffer (8 M urea ho 0.1 M Tris, pH 8.5), e teteaneng ebile e halohile.Mehato eohle ea centrifugation e entsoe ka 14,000 xg ho 25°C.Halofo ea pele ea protheine ea lysate e amanang le sefapano e ile ea fetisetsoa ho sesebelisoa sa filthara sa 10 kDa Microcon centrifugal se nang le lera la Ultracel-10 (Merck), se lateloang ke centrifugation sefaneng ka metsotso e 25.Ebe u eketsa halofo ea bobeli ea protheine ho sefa ebe u pheta mehato e tšoanang.Ho hlaphoheloa ha protheine ho entsoe ka ho kenyelletsa 100 μl ea 17 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) ka har'a buffer ea urea.Pholiso e ile ea hlohlelletsoa ka thermomixer ka 600 rpm bakeng sa metsotso e 30 ho 37 ° C.Ntle le moo, kholomo e ne e kentsoe bohareng mme protheine e fokotsehileng e hokahantsoeng e ile ea etsoa alkylated ho sebelisoa 100 μl ea 50 mM iodoacetamide ho urea buffer.Karabelo ea alkylation e ne e etsoa ka mocheso oa kamore ka metsotso e 20 lefifing.Fetola kholomo, hlatsoa marako a kholomo ka makhetlo a 3 ka 100 µl urea buffer, ebe u centrifuge.Opereishene e tšoanang e ile ea etsoa ka makhetlo a 3 ho sebelisoa 100 μl ea 100 mM ammonium bicarbonate.Pele ho trypsinization, nka sebaka sa tube ea pokello ka e ncha.Kenya tšilo ea lijo e nang le 50 mM ammonium bicarbonate le 1 µl trypsin e hlapolotsoeng ka har'a buffer ea trypsin (Promega).Karo-karolelano ea trypsin le protheine e ile ea bolokoa hoo e ka bang 1:33, 'me liphetoho tsa tšilo ea lijo li ile tsa kenngoa bosiung ba 37° C. ka phaposing e mongobo.Peptide e kopantsoeng e ile ea ntšoa sefahlaneng ka centrifugation ka metsotso e 25.Ho hlaphoheloa ha peptide ho ile ha ntlafatsoa ka ho kenyelletsa 50 μl ea 0.5 M NaCl ho sefe, 'me e lateloe ke centrifugation metsotso e 25.
Likholomo tsa C18 Micro Spin (Harvard Apparatus) li ne li sebelisetsoa ho tlosa letsoai li-peptide tsa tryptic tse amanang le sefapano ho latela melaoana e hlalositsoeng ke Bouchal et al.70 ka liphetoho tse nyane.Ka bokhutšoanyane, litšiea tsa C18 spin li ile tsa sebelisoa ka litlhapo tse tharo tsa 0.1% formic acid (FA) ho acetonitrile (AcN) (Merck) le lihlatso tse peli tsa 0.1% FA.Kholomo e ile ea kolobisoa ka 0.1% FA ka metsotso e 15.Laela lisampole ka har'a li-spin columns 'me u hlatsoe makhetlo a 3 ka 0.1% FA.Li-peptide tse hlakotsoeng li ile tsa hlakisoa ka tatellano ka gradient ea mohato ka mohato ho sebelisoa 50%, 80% le 100% AcN ho 0.1% FA.Mehlala e ile ea omisoa ka har'a sekontiri sa SpeedVac Plus (Eppendorf) ho fihlela mokelikeli o setseng o nyamela ka ho feletseng.Li-peptide tse hlakotsoeng li ile tsa qhaloa ka 100 μl ea 0.08% trifluoroacetic acid ho 2.5% AcN mme likhakanyo li ile tsa lekanngoa ho NanoDrop 2000 (Thermo Scientific).Hoo e ka bang 1 μg ea peptide e kopantsoeng ka sampuli ka 'ngoe e ile ea kenngoa tsamaisong ea LC-MS/MS.
Li-peptide tse amanang le sefapano li ile tsa aroloa ka UltiMate 3000 RSLCnano LC system (Thermo Scientific) e hokahantsoeng le Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Li-peptide tse hokahaneng li ile tsa bokelloa ho 300 µm ID, 5 mm bolelele µ-pele ho kholumo ea C18 e nang le C18 PepMap100 sorbent le 5 µm PepMap sorbent (Thermo Scientific).Laola phallo ea pompo e behiloeng ho 5 µl/min 0.08% trifluoroacetic acid e qhibilihisitsoeng ho 2.5% AcN.Li-peptide tse amanang le sefapano li ile tsa aroloa holim'a kholomo ea silica e kopantsoeng e nang le bophara bo ka hare ba 75 μm le bolelele ba 150 mm, e tletseng 2 μm PepMap sorbent (Thermo Scientific).Mehato ea mobile A le B e ne e e-na le 0.1% FA metsing le 0.1% FA ho acetonitrile, ka ho latellana.The gradiente e qala ho 2.5% B 'me e eketseha ka tatellano ho 40% B ho feta metsotso e 90, ebe ho ea ho 90% B metsotsong e 2 e latelang.Sebopeho sa karolo ea selefouno se ile sa bolokoa ho 90% B bakeng sa metsotso ea 10 ebe se fokotseha ka ho toba ho 2.5% B ka metsotso e 2.Kholomo e ile ea lekanngoa ho 2.5% B bakeng sa metsotso e 8 pele ho potoloho e latelang.Li-peptide tse hokahaneng tse hokahaneng ho tsoa kholomong ea tlhahlobo li ile tsa kenngoa mohloling oa nanoelectrospray ionization (NSI) 'me tsa kenngoa ho Exploris 480 mass spectrometer (Thermo Scientific).
Orbitrap Exploris 480 mass spectrometer e sebetsa ka mokhoa o nepahetseng oa ho hokahanya ha data.Tlhahlobo e feletseng e ile ea etsoa ka mokhoa oa karolo ka qeto ea 120,000 e nang le mefuta e mengata ea ho tloha m / z 350 Th ho ea ho m / z 2000 Th.Sepheo se tloaelehileng sa AGC se behiloe ho 300% ka nako e ngata ea ho kenya letsoho ea 50ms.Ho fumaneha ha tlhoro ea Monoisotopic ho thehiloe bakeng sa li-peptide.Paramethara ea ho phomola e behiloe ho 'nete haeba ho fumanoa li-precursor tse fokolang haholo.Bonyane matla a ionic a selelekela a ne a behiloe ho 5.0e3 mme tefiso ea pele e bolela ho fihla ho +8 e kenyelelitsoe litekong.
Nako ea potoloho pakeng tsa litlhahlobo tse kholo ka mokhoa oa ho hokahanya ha data e ne e behiloe ho metsotsoana e 2.5.Ho kenyelletsoa ha boima bo matla ho ile ha behoa metsotsoana e 20 ka mor'a karohano ea pele ea ion ea pele.Fesetere ea ho itšehla thajana e ne e behiloe ho 2 Th.Mofuta oa matla a tloaelehileng a ho thulana ka mokhoa o tsitsitseng oa matla a ho thulana o khethiloe ho data e itšetlehileng ka MS/MS scan.Matla a ho thulana a behiloe ho 30%.Qeto ea Orbitrap e behiloe ho 15,000 le sepheo sa AGC ho 100%.Nako e tloaelehileng ea ente e behiloe ho 60 milliseconds.
Pele re latela marang-rang a protheine-protheine ka lisampole tse amanang le sefapano, re ile ra sebetsana le lifaele tse tala re sebelisa sephutheloana sa MaxQuant (mofuta oa 1.6.12.0) 26,27 ho khetholla li-peptide / liprotheine tse ka fumanoang lisampoleng.Ho phaella moo, litlhahlobo tse tšoanang tsa proteomic li ile tsa etsoa ka lisampole tse sa amaneng tsa Flo-1 tse tšoaroang le tse sa tšoaroeng ka IFNα.Lintlha tsa MS/MS li ile tsa batlisisoa polokelong ea batho ea UniProt (www.uniprot.org) (e kentsoeng ka la 12 Phato, 2020, e na le likenyelletso tse 75,093) ho sebelisoa enjine ea ho batla ea Andromeda27.Patlisiso e ile ea etsoa ntle le ho bonts'a ho nepahala ha enzyme le liphetoho tse fapaneng tsa deamidation (N, Q) le oxidation (M).Mamello ea boima ba precursor e behiloe ho 20 ppm le li-ion tsa lihlahisoa ho 0.02 Da.Phapang ea pele le e phahameng ea boima e ne e behiloe ho 10 ppm.Boima bo boholo ba peptide bo behiloe ho 4600 Da mme ho tšoana ho latellana ho behiloe pakeng tsa 7 le 25 amino acid (aa).Tlhahlobo e eketsehileng ea lipalo-palo e ile ea etsoa ho sebelisoa lenaneo la Perseus (version 1.6.10.45).Likahare tsa protheine li ne li baloa ka ho tloaeleha matla a spectral a protheine (LFQ intensity; unlabeled quantification)27 mme boleng ba matla bo fetotsoe ho Log2.Khokahano ea maemo a holimo ea liprotheine tse khetholloang ke matla a tsona a peptide e hahiloe ho sebelisoa sephutheloana sa pheatmap (v1.0.12) ho R (v 4.1.2).Tlhahlobo ea ntlafatso ea litsela e ile ea etsoa ho sebelisoa database ea tsela ea Reactome bakeng sa liprotheine tse tšoaroang ke IFNα tse neng li sebelisoa ka makhetlo a fetang a mane ha li bapisoa le lisampole tse sa phekoloang.
Ho tsebahatsa li-lysine (K) kapa li-serine (S) tse khethehileng tsa lik'hemik'hale tsa lik'hemik'hale tsa lik'hemik'hale tsa protheine tse hlahlojoang ke LC-MS/MS li ile tsa etsoa ho sebelisoa mochine oa ho tsebahatsa oa spectroscopic (SIM-XL) bakeng sa li-peptide tse amanang le sefapano (SIM-XL)29.Ntlha ea pele, litšebelisano tse ka khonehang pakeng tsa liphatsa tsa lefutso tsa interferon-associated (IFN) tsa DNA tse hanyetsanang le tšenyo ea DNA (IRDS) li ile tsa batlisisoa ho sebelisoa dataset ea protheine ea IRDS e hlalositsoeng Padariya et al.28.Ho hlahloba maemo ohle le ho pheta-pheta ha UniProt eohle ea motho ho matla haholo, kahoo polokelo ea batho ea UniProt (www.uniprot.org) (e jarollotsoeng la 12 Phato 2020, e na le likenyo tsa 75,093) khahlano le pheta-pheto e phekoletsoeng ke IFNα.E 'ngoe ea li-filters tsa litšebelisano tse kholo.Litšebelisano tsena tsa bohlokoa bo phahameng tse fumanoeng li ile tsa atolosoa le ho lekoa ka ho pheta-pheta le maemo ohle.
Ho SIM-XL, DSS e ne e sebelisoa bakeng sa crosslinker (XL) 'me phetoho ea boima ba XL le phetoho ea boima ba phetoho e behiloe ho 138.06 le 156.07, ka ho latellana.Ho nahanoa ka libaka tse latelang tse kopanyang maikutlo: KK, KS le KN-TERM, ntle le li-ion tsa moqolotsi.Ka bobeli precursor le fragment ppm li behiloe ho 20 mme moeli oa Xrea o behiloe ho 0.15.Trypsin e ne e nkoa e le e tobileng ka ho feletseng, 'me mokhoa o matla oa ho arola C-trap (HCD) o ile oa kenngoa ts'ebetsong.Maemo a phokotso ea XCorr DB le palo e tlase ea li-peptide bakeng sa phokotso ea DB e matla e behiloe ho 2.5 le 2, ka ho latellana.Mekhahlelo e meng ke: monyetla oa monoisotope le peak coincidence cutoff, bonyane masala a 4 AA ka khoele e le 'ngoe le tefiso e phahameng ea likhoele, le boholo ba 3 ba likarohano tse fetiloeng.Limmapa tse rokiloeng tsa 2D li ile tsa hlahlobjoa ho (SIM-XL) 'me setšoantšo sa xQuest28 sa sebelisoa ho aha limmapa tsa 2D.Li-crosslink tsa protheine holim'a mehaho ea liprotheine li fanoa ka PyMol (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC).
Mehaho ea mohlala oa protheine e entsoe ka ho sebelisa seva sa Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 ho sebelisa melao-motheo ea mokhoa oa homology le ts'ebetsong ea "Mokhoa o patiloeng oa Markov".Phyre2 e hlahisa meaho ea mohlala e ipapisitseng le tatellano ea tatellano le meaho e tsebahalang ea protheine.Bakeng sa liprotheine tsa H2BFS, HLA-A, HMGA1, LRCH4, le MDN1, li-template structures 1kx552, 1kj349, 2eze55, 6hlu62, le 6i2665 li ile tsa sebelisoa.Ho phaella moo, sebopeho sa AlphaFold71 MX1, UBP18 le ROBO1 le sona se ile sa nkoa.Sebopeho sa protheine se ne se bonoa ho sebelisoa sephutheloana sa BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, USA) le sephutheloana sa Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).