Indwara ya Alzheimer (AD) ibura proteine biomarkers yerekana uburyo bwinshi bwa patrophysiologiya, bikabuza iterambere ryo gusuzuma no kuvura. Hano, dukoresha proteomics yuzuye kugirango tumenye ibinyabuzima byitwa cerebrospinal fluid (CSF) biomarkers byerekana intera nini ya AD pathophysiology. Multiplex mass spectrometrie yerekanaga proteine zigera ku 3.500 hamwe na poroteyine zigera ku 12.000 muri AD CSF n'ubwonko. Isesengura ry'urusobe rwa proteome yo mu bwonko ryakemuye 44 ibinyabuzima bitandukanye, 15 muri byo byuzuzanya na proteome cerebrospinal fluid proteome. Ibimenyetso bya CSF AD muribi byiciro byuzuzanya bigabanijwe mumatsinda atanu ya poroteyine, byerekana inzira zitandukanye za patrophysiologique. Synaps na metabolite mubwonko bwa AD biragabanuka, ariko CSF iriyongera, mugihe myelination ikungahaye kuri glial hamwe nitsinda ryumubiri mubwonko na CSF byiyongera. Guhuza hamwe nindwara yihariye yibihinduka byemejwe mubisubizo birenga 500 bya CSF. Aya matsinda yanagaragaje amatsinda mato yibinyabuzima muri AD idafite ibimenyetso. Muri rusange, ibisubizo nintambwe itanga icyizere kijyanye nurubuga rwibikoresho bya biomarker kubikoresho byubuvuzi muri AD.
Indwara ya Alzheimer (AD) niyo itera indwara yo guta umutwe ya neurodegenerative ku isi yose kandi ikarangwa n’imikorere myinshi ya sisitemu y’ibinyabuzima idakora neza, harimo kwanduza synaptic, ubudahangarwa bw’umubiri, hamwe na metabolism ya mitochondial (1-3). Nyamara, poroteyine ya biomarkers yashizweho iracyibanda ku kumenya poroteyine ya amyloide na tau, bityo ntishobora kwerekana iyi mitekerereze itandukanye. Izi poroteyine “yibanze” zapimwe cyane mu mazi ya cerebrospinal fluid (CSF) zirimo (i) amyloide beta peptide 1-42 (Aβ1-42), igaragaza ishyirwaho rya plaque cortical amyloide; (ii) tau yose, ikimenyetso cyo guta axon; (iii) fosifori-tau (p-tau), uhagarariye patologi tau hyperphosphorylation (4-7). Nubwo ibyo binyabuzima byitwa cerebrospinal fluid biomarkers byadufashije cyane kumenya indwara za poroteyine "zandikiwe" AD (4-7), zerekana gusa agace gato ka biologiya igoye inyuma yindwara.
Kubura itandukaniro rya pathophysiologique ya AD biomarkers ya AD byateje ibibazo byinshi, harimo (i) kutabasha kumenya no kugereranya ibinyabuzima bitandukanye by’abarwayi ba AD, (ii) gupima bidahagije uburemere bw’indwara n’iterambere, cyane cyane mu cyiciro kibanza, Kandi ( iii) guteza imbere imiti ivura yananiwe gukemura burundu ibintu byose byangiza imitsi. Kwishingikiriza kuri patologiya yibanze kugirango dusobanure AD indwara zifitanye isano gusa byongera ibyo bibazo. Ibimenyetso byinshi kandi byinshi byerekana ko abantu benshi bageze mu zabukuru bafite ikibazo cyo guta umutwe bafite ibimenyetso birenze kimwe biranga kugabanuka kwubwenge (8). Abagera kuri 90% cyangwa barenga kubantu bafite indwara ya AD nabo bafite uburwayi bwimitsi, TDP-43, cyangwa izindi ndwara zangirika (9). Umubare munini wibihuru byindwara byahungabanije gahunda yacu yo kwisuzumisha yo guta umutwe, kandi harakenewe ibisobanuro birambuye bya patrophysiologique byindwara.
Urebye ibikenewe byihutirwa bya biomarkers zitandukanye za AD, umurima uragenda ukoresha uburyo bwa "omics" bushingiye kuri sisitemu rusange yo kuvumbura biomarkers. Ihuriro ryihuse ry’imiti (AMP) -AD Ihuriro ryatangijwe mu 2014 kandi riri ku isonga muri gahunda. Izi mbaraga zinyuranye zakozwe n’ikigo cy’igihugu cy’ubuzima, amashuri, n’inganda zigamije gukoresha ingamba zishingiye kuri sisitemu kugira ngo hasobanurwe neza imiterere y’imiterere ya AD no guteza imbere isesengura ry’ibinyabuzima hamwe n’ingamba zo kuvura (10). Mugice cyuyu mushinga, proteomics y'urusobe yabaye igikoresho cyiza cyo guteza imbere sisitemu ishingiye kuri biomarkers muri AD. Ubu buryo butabogamye bushingiye ku makuru ategura amakuru ya poroteyine agizwe mu matsinda cyangwa “modules” za poroteyine zifatanije zifitanye isano n'ubwoko bw'utugingo ngengabuzima, ingirangingo, n'imikorere y'ibinyabuzima (11-13). Ubushakashatsi hafi ya 12 bukungahaye kuri proteomics ubushakashatsi bwakozwe mubwonko bwa AD (13-23). Muri rusange, iri sesengura ryerekana ko proteome ya AD ubwonko bwa proteome ikomeza ishyirahamwe ryigenga ryabitswe cyane mubice byigenga no mu turere twinshi twa cortique. Mubyongeyeho, amwe murayo masomo yerekana impinduka zororoka mubwinshi bwa AD bijyanye namakuru menshi, byerekana pathophysiologie yindwara nyinshi. Hamwe na hamwe, ubu bushakashatsi bwerekana ingingo itanga icyizere cyo kuvumbura proteome y'ubwonko bwa proteome nka sisitemu ishingiye kuri biomarker muri AD.
Kugirango duhindure proteome ya AD ubwonko bwa proteome muburyo bwa sisitemu yingirakamaro ya biomarkers, twahujije umuyoboro ukomoka mubwonko hamwe nisesengura rya proteomic ya AD CSF. Ubu buryo bukomatanyije bwatumye hamenyekana ibice bitanu bitanga ibimenyetso bya biomarkers ya CSF ifitanye isano nubwoko butandukanye bw’ubwonko bushingiye ku bwonko, harimo synaps, imiyoboro y'amaraso, myelination, inflammation, hamwe n'imikorere idahwitse y'imihindagurikire. Twatsindiye neza ibyo bikoresho bya biomarker dukoresheje isesengura ryinshi, harimo ingero zirenga 500 za CSF zatewe n'indwara zitandukanye zifata ubwonko. Isesengura ryemewe ririmo gusuzuma intego zitsinda muri CSF yabarwayi bafite AD idafite ibimenyetso (AsymAD) cyangwa kwerekana ibimenyetso byerekana kwirundanya kwa amyloide idasanzwe mubidukikije bisanzwe. Iri sesengura ryerekana itandukaniro rishingiye ku binyabuzima mu baturage ba AsymAD kandi rikerekana ibimenyetso byerekana ibimenyetso bishobora kwanduza abantu mu ntangiriro z’indwara. Muri rusange, ibisubizo byerekana intambwe yingenzi mugutezimbere ibikoresho bya protein biomarker bishingiye kuri sisitemu nyinshi zishobora gukemura neza ibibazo byinshi byubuvuzi byugarije AD.
Intego nyamukuru yubu bushakashatsi ni ukumenya ibimenyetso bishya bya cerebrospinal fluid biomarkers byerekana ubwonko butandukanye bushingiye ku bwonko buganisha kuri AD. Igicapo S1 kirerekana uburyo bwacu bwubushakashatsi, burimo (i) isesengura ryuzuye ryakozwe nubushakashatsi bwibanze bwa AD CSF hamwe na proteome yubwonko bwurusobe kugirango hamenyekane ibimenyetso byinshi byindwara ziterwa na CSF ziterwa na biomarkers, na (ii) kwigana gukurikira Izi biomarkers ziri mubwonko butandukanye bwigenga cohort. Ubushakashatsi bushingiye ku buvumbuzi bwatangiranye no gusesengura imvugo itandukanye ya CSF ku bantu 20 basanzwe bafite ubwenge ndetse n’abarwayi 20 ba AD ku kigo cy’ubushakashatsi ku ndwara ya Emory Goizueta Alzheimer (ADRC). Isuzuma rya AD risobanurwa nkubumuga bukomeye bwubwenge imbere ya Aβ1-42 kandi urwego rwo hejuru rwa tau na p-tau byuzuye mumazi ya cerebrospinal fluid [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA )]] (Imbonerahamwe S1A). Igenzura (bivuze MoCA, 26.7 ± 2.2) ryari rifite urwego rusanzwe rwa biomarkers ya CSF.
Umuntu CSF arangwa nurwego rwinshi rwa poroteyine nyinshi, aho albumine nizindi poroteyine nyinshi cyane zishobora kubuza kumenya poroteyine zinyungu (24). Kugirango twongere ubujyakuzimu bwa poroteyine, twakuyeho poroteyine 14 za mbere nyinshi cyane kuri buri cyitegererezo cya CSF mbere yo gusesengura ibintu rusange (MS) (24). Peptide 39,805 zamenyekanye na MS, zashyizwe kuri proteome 3691 muri 40. Kugereranya poroteyine bikorwa na tandem mass tag (TMT) yanditseho (18, 25). Kugirango dukemure amakuru yabuze, twashyizemo gusa poroteyine zapimwe byibuze 50% by'icyitegererezo mu isesengura ryakurikiyeho, bityo amaherezo tugereranya proteomes 2875. Bitewe nuko itandukaniro rinini ryurwego rwinshi rwa poroteyine, icyitegererezo cyo kugenzura cyafatwaga nkikirenga (13) kandi nticyashyizwe mubisesengura byakurikiyeho. Indangagaciro nyinshi za 39 zisigaye zahinduwe ukurikije imyaka, igitsina, hamwe na covariance (13-15, 17, 18, 20, 26).
Hifashishijwe isesengura ryibarurishamibare kugirango dusuzume imvugo itandukanye ku makuru yisubiraho, iri sesengura ryagaragaje poroteyine urwego rwinshi rwahinduwe ku buryo bugaragara (P <0.05) hagati y’ubugenzuzi n’imanza za AD (Imbonerahamwe S2A). Nkuko bigaragara ku gishushanyo 1A, ubwinshi bwa poroteyine 225 zose muri AD bwaragabanutse cyane, kandi ubwinshi bwa poroteyine 303 bwariyongereye cyane. Izi poroteyine zagaragajwe mu buryo butandukanye zirimo ibimenyetso byinshi byamenyekanye mu bwonko bwa AD, nka microtubule ifitanye isano na protein tau (MAPT; P = 3.52 × 10−8), neurofilament (NEFL; P = 6.56 × 10−3), Poroteyine 43 ikura. . P = 3.43 × 10−4) hamwe na VGF yo gukura kw'imitsi (VGF; P = 4.83 × 10−3) (4-6). Ariko, twabonye kandi izindi ntego zingenzi cyane, nka inhibitor ya GDP itandukanya 1 (GDI1; P = 1.54 × 10-10) hamwe na calcium ya modul ya calcium ihuza 1 (SMOC1; P = 6.93 × 10-9). Isesengura rya Gene Ontology (GO) ryagabanutse cyane kuri poroteyine 225 ryagaragaje cyane isano iri hagati yimikorere yumubiri wumubiri nka metabolism ya steroid, coagulation yamaraso, hamwe nibikorwa bya hormone (Igicapo 1B na Imbonerahamwe S2B). Ibinyuranye, poroteyine yiyongereye cyane ya 303 ifitanye isano rya bugufi n'imiterere y'ingirabuzimafatizo hamwe na metabolism.
. AD imanza za proteome ya CSF Muri poroteyine zose. Poroteyine zigabanutse cyane (P <0.05) muri AD zerekanwa mubururu, mugihe poroteyine ziyongereye cyane muburwayi zerekanwa mumutuku. Poroteyine yatoranijwe yanditseho. (B) Amagambo yo hejuru ya GO ajyanye na poroteyine yagabanutse cyane (ubururu) kandi yiyongera (umutuku) muri AD. Erekana amagambo atatu ya GO hamwe na z-amanota menshi murwego rwibinyabuzima, imikorere ya molekile, nibice bigize selile. (C) MS yapimye urwego rwa MAPT muri sample ya CSF (ibumoso) nisano ifitanye nicyitegererezo cya ELISA tau (iburyo). Coefficient ya Pearson hamwe nigiciro cya P irerekanwa. Bitewe no kubura amakuru ya ELISA kubibazo bimwe bya AD, iyi mibare ikubiyemo indangagaciro kuri 38 kuri 39 zasesenguwe. . Bisanzwe, usanzwe.
Urwego rwa proteomic rwa MAPT rufitanye isano rya bugufi nurwego rwa ELISA rwapimwe rwigenga (r = 0,78, P = 7.8 × 10-9; Ishusho 1C), rushyigikira agaciro k'ibipimo byacu bya MS. Nyuma yo gusya kwa trypsin kurwego rwa poroteyine ya amyloide prursor (APP), peptide yihariye ya isoform yashizwe kuri C-terminus ya Aβ1-40 na Aβ1-42 ntishobora kuba ioni neza (27, 28). Kubwibyo, peptide ya APP twabonye ntaho ihuriye nurwego rwa ELISA Aβ1-42. Kugirango dusuzume imvugo itandukanye ya buri rubanza, twakoresheje poroteyine zagaragajwe mu buryo butandukanye hamwe na P <0.0001 [igipimo cyo kuvumbura ibinyoma (FDR) yakosoye P <0.01] kugira ngo dukore isesengura rya cluster ikurikirana ingero (Imbonerahamwe S2A). Nkuko bigaragara ku gishushanyo cya 1D, izo poroteyine 65 zifite akamaro kanini zirashobora guhuza neza urugero ukurikije imiterere y’indwara, usibye ikibazo kimwe cya AD gifite imiterere-yo kugenzura. Muri izo poroteyine 65, 63 ziyongereye muri AD, mu gihe ebyiri gusa (CD74 na ISLR) zagabanutse. Muri rusange, iri sesengura ryubwonko bwerekanye proteine amagana muri AD zishobora kuba nk'ibinyabuzima byangiza indwara.
Noneho twakoze isesengura ryigenga rya proteome ya AD ubwonko. Ubwonko bwubuvumbuzi bwubuvumbuzi bwarimo cortex ibanziriza cortex (DLPFC) itagenzuwe (n = 10), indwara ya Parkinson (PD; n = 10), ivanze na AD / PD (n = 10) na AD (n = 10). ) Icyitegererezo. Emery Goizueta ADRC. Imibare y’izi manza 40 yasobanuwe mbere (25) kandi ikubiye mu mbonerahamwe ya S1B. Twifashishije TMT-MS mu gusesengura izi nyama 40 zo mu bwonko hamwe na cohort yo kwigana imanza 27. Muri rusange, aya makuru abiri yubwonko yabyaye 227.121 peptide idasanzwe, yashizwe kuri proteome 12.943 (25). Gusa izo poroteyine zapimwe byibuze 50% by'imanza zashyizwe mu iperereza ryakurikiyeho. Amakuru yanyuma yo kuvumbura arimo poroteyine 8817 zingana. Hindura urugero rwinshi rwa poroteyine ukurikije imyaka, igitsina, nintera ya nyuma yo gupfa (PMI). Isesengura ritandukanye ryisesengura ryamakuru yashyizweho nyuma yo gusubira inyuma ryerekanye ko> poroteyine 2000 zahinduwe ku buryo bugaragara [P <0.05, isesengura ry’ibitandukanye (ANOVA)] mu bice bibiri cyangwa byinshi by’indwara. Hanyuma, twakoze isesengura rya cluster yagenzuwe dushingiye kuri poroteyine zitandukanye, na P <0.0001 muri AD / kugenzura no / cyangwa AD / PD kugereranya (Ishusho S2, A na B, Imbonerahamwe S2C). Izi poroteyine 165 zahinduwe cyane zerekana neza ibibazo hamwe na AD patologi bivuye kugenzura hamwe na PD, byemeza impinduka zikomeye za AD muri proteome zose.
Twahise dukoresha algorithm yitwa Weighted Gene Co-expression Network Analysis (WGCNA) kugirango dukore isesengura ryurusobe kuri proteome yavumbuwe, itegura amakuru yashyizwe mubice bya poroteyine hamwe nuburyo busa (11-13). Isesengura ryagaragaje modules 44 (M) zifatanije na poroteyine, zitondekanya kandi zibarwa kuva kuri nini (M1, n = 1821 za poroteyine) kugeza kuri ntoya (M44, n = 34 za poroteyine) (Ishusho 2A na Imbonerahamwe S2D)). Nkuko byavuzwe haruguru (13) Kubara umwirondoro uhagarariye imvugo cyangwa poroteyine iranga buri module, hanyuma uyihuze na leta y’indwara na AD patologiya, ni ukuvuga, shiraho ihuriro ry’indwara z’indwara ya Alzheimer (CERAD) na Score ya Braak (Ishusho 2B). Muri rusange, module 17 yari ifitanye isano cyane na AD neuropathology (P <0.05). Inyinshi murizo modul zijyanye n'indwara nazo zikungahaye ku bwoko bwihariye bw'utugingo ngengabuzima (Ishusho 2B). Nkuko byavuzwe haruguru (13), gukungahaza ubwoko bwakagari bigenwa no gusesengura module ihuriweho hamwe nurutonde rwerekana ubwoko bwihariye bwa selile. Izi genes zikomoka kumakuru yatangajwe muri neuron yimbeba yihariye, endoteliyale na glial selile. Ikigereranyo cya RNA (RNA-seq) igerageza (29).
(A) Menya WGCNA ya proteome yubwonko. . Ubukomezi bwiza (umutuku) nibibi (ubururu) bifitanye isano n'ikarita y'ubushyuhe bw'amabara abiri, kandi inyenyeri zerekana akamaro k'ibarurishamibare (P <0.05). Koresha Hypergeometric Fisher Ikizamini Cyukuri (FET) (hepfo) kugirango usuzume ubwoko bwimikorere ya selile ya proteine module. Ubwinshi bwigicucu gitukura cyerekana urwego rwimitunganyirize yubwoko, naho inyenyeri yerekana akamaro k’imibare (P <0.05). Koresha uburyo bwa BH kugirango ukosore agaciro P gakomoka kuri FET. (C) Genda gusesengura poroteyine. Ibikorwa bya biologiya bifitanye isano cyane byerekanwe kuri buri module cyangwa itsinda rya module bifitanye isano. oligo, oligodendrocyte.
Urutonde rwibintu bitanu bifitanye isano rya hafi na astrocyte na microglia ikungahaye cyane (M30, M29, M18, M24, na M5) byerekanaga isano ikomeye na AD neuropathology AD (Ishusho 2B). Isesengura rya Ontologiya rihuza iyi moderi yisi niterambere ryimikorere, ikwirakwizwa, nubudahangarwa (Ishusho 2C na Imbonerahamwe S2E). Module ebyiri ziyongera, M8 na M22, nazo zigengwa cyane nindwara. M8 ifitanye isano cyane n'inzira ya Toll imeze nk'iyakira, inzira yerekana kasike igira uruhare runini mugukingira indwara (30). Mugihe kimwe, M22 ifitanye isano rya bugufi no guhindura nyuma yubusobanuro. M2, ikungahaye kuri oligodendrocytes, yerekana isano ikomeye hamwe na AD patologiya hamwe na ontologiya hamwe na synthesis ya nucleoside hamwe no kwigana ADN, byerekana ubwiyongere bukabije bw'uturemangingo mu ndwara. Muri rusange, ibi bivumbuwe bishyigikira kuzamuka kwa glial modules twabonye mbere muri proteome ya AD (13, 17). Kugeza ubu usanga ko modul nyinshi zijyanye na AD zijyanye na glial murusobe zerekana imvugo yo hasi mukugenzura no kwandura PD, bikagaragaza umwihariko windwara zabo zazamutse muri AD (Igicapo S2C).
Module enye gusa murusobe rwacu proteome (M1, M3, M10, na M32) zifitanye isano ribi na AD patologiya (P <0.05) (Ishusho 2, B na C). M1 na M3 zombi zikungahaye ku bimenyetso bya neuronal. M1 ifitanye isano cyane nibimenyetso bya synaptique, mugihe M3 ifitanye isano rya hafi nimikorere ya mito-iyambere. Nta kimenyetso cyerekana ubukire bwubwoko bwa M10 na M32. M32 yerekana isano iri hagati ya M3 na metabolism selile, mugihe M10 ifitanye isano cyane no gukura kwingirabuzimafatizo n'imikorere ya microtubule. Ugereranije na AD, modul zose uko ari enye zongerewe imbaraga mugucunga na PD, zibaha indwara yihariye ya AD (Ishusho S2C). Muri rusange, ibisubizo bishyigikira kugabanuka kwinshi kwa moderi ikungahaye kuri neuron twabonye mbere muri AD (13, 17). Muncamake, isesengura ryurusobe rwa proteome yubwonko twavumbuye rwakoze AD yihariye yahinduwe muburyo buhuye nibyo twabonye mbere.
AD irangwa nicyiciro cya mbere kidafite ibimenyetso (AsymAD), aho abantu bagaragaza kwirundanya kwa amyloide nta kugabanuka kwubwenge (5, 31). Icyiciro kidafite ibimenyetso cyerekana idirishya rikomeye ryo gutahura hakiri kare. Twerekanye mbere uburyo bukomeye bwo kubungabunga uburyo bwa AsymAD na AD ubwonko bwubwonko bwa proteome murwego rwigenga (13, 17). Kugirango tumenye neza ko urusobe rw'ubwonko twavumbuye muri iki gihe ruhuye n'ibyavuye mu bushakashatsi bwabanje, twasesenguye uburyo bwo kubika module 44 mu makuru yakuwe mu miryango 27 DLPFC. Aya mashyirahamwe arimo kugenzura (n = 10), AsymAD (n = 8) Na AD (n = 9). Kugenzura hamwe na AD byashyizwe mubisesengura ryubwonko twavumbuye (Imbonerahamwe S1B), mugihe ibibazo bya AsymAD byari byihariye gusa muri kopi yo kwigana. Izi manza za AsymAD nazo zaturutse muri banki yubwonko ya Emory Goizueta ADRC. Nubwo kumenya byari bisanzwe mugihe cyurupfu, urugero rwa amyloide rwari hejuru cyane (bivuze CERAD, 2.8 ± 0.5) (Imbonerahamwe S1B).
TMT-MS isesengura ryiyi ngingo 27 yubwonko yatumye habaho proteomes 11,244. Ibiharuro byanyuma birimo izo poroteyine zingana byibuze 50% byintangarugero. Iyi mibare ikoporora ikubiyemo 8638 (98.0%) ya poroteyine 8817 zagaragaye mu isesengura ry’ubwonko twavumbuye, kandi ifite hafi 3000 zahinduye poroteyine hagati y’ubugenzuzi hamwe na AD (P <0.05, nyuma y’ikizamini cya Tukey cyakozwe kugirango isesengure itandukaniro) ( Imbonerahamwe S2F). Muri izo poroteyine zagaragajwe mu buryo butandukanye, 910 yanagaragaje impinduka zikomeye hagati ya AD n'indwara zo kugenzura ubwonko bwa proteome (P <0.05, nyuma ya ANOVA Tukey yahuje t-test). Birakwiye ko tumenya ko ibyo bimenyetso 910 bihuye cyane mubyerekezo byimpinduka hagati ya proteomes (r = 0,94, P <1.0 × 10-200) (Igicapo S3A). Muri poroteyine ziyongereye, poroteyine zifite impinduka zihamye hagati yamakuru ni cyane cyane abanyamuryango ba moderi ikungahaye kuri M5 na M18 (MDK, COL25A1, MAPT, NTN1, SMOC1, na GFAP). Muri poroteyine zagabanutse, abafite impinduka zihamye ni abanyamuryango bonyine ba module ya M1 (NPTX2, VGF, na RPH3A) ifitanye isano na synaps. Twongeye kugenzura impinduka zijyanye na AD ya midkine (MDK), CD44, isohora poroteyine 1 (SFRP1) hamwe na VGF ikoresheje iburengerazuba (Ishusho S3B). Isesengura ryo kubungabunga module ryerekanye ko hafi 80% ya poroteyine (34/44) muri proteome yo mu bwonko yabitswe cyane mu makuru yo kwigana (z-amanota> 1.96, FDR yakosoye P <0.05) (Ishusho S3C). Cumi na bane muri izi module zabitswe byumwihariko hagati ya proteome zombi (z-amanota> 10, FDR yakosoye P <1.0 × 10−23). Muri rusange, kuvumbura no kwigana urwego rwo hejuru rwo guhuzagurika mu mvugo itandukanye no guhuza modular hagati ya proteome yo mu bwonko byerekana imyororokere yimpinduka muri poroteyine ya AD imbere. Byongeye kandi, yemeje kandi ko AsymAD nindwara zateye imbere zifite imiterere isa nubwonko bwubwonko.
Isesengura rirambuye ku mvugo itandukanye mu mibare yo kwigana ubwonko yerekana urwego rugaragara rw’imihindagurikire ya poroteyine ya AsymAD, harimo 151 zahinduye poroteyine hagati ya AsymAD no kugenzura (P <0.05) (Ishusho S3D). Bikwiranye numutwaro wa amyloide, APP mubwonko bwa AsymAD na AD yiyongereye cyane. MAPT ihinduka gusa muri AD, ibyo bikaba bihujwe no kwiyongera kurwego rwa tangles hamwe no kumenyekana bizwi no kugabanuka kwubwenge (5, 7). Module ikungahaye cyane ku isi (M5 na M18) igaragara cyane muri poroteyine ziyongereye muri AsymAD, mu gihe module ya M1 ifitanye isano na neuron niyo ihagarariye poroteyine zagabanutse muri AsymAD. Byinshi muribi bimenyetso bya AsymAD byerekana impinduka nini mu ndwara zerekana ibimenyetso. Muri ibyo bimenyetso harimo SMOC1, proteine glial ya M18, ifitanye isano n'ibibyimba byo mu bwonko ndetse no gukura kw'amaso n'amaguru (32). MDK ni ikintu gikura cya heparin ihuza gukura no gukura kwa selile na angiogenezi (33), undi munyamuryango wa M18. Ugereranije nitsinda rishinzwe kugenzura, AsymAD yiyongereye cyane, ikurikirwa no kwiyongera kwa AD. Ibinyuranye, synaptic protein neuropentraxin 2 (NPTX2) yagabanutse cyane mubwonko bwa AsymAD. NPTX2 mbere yari ifitanye isano na neurodegeneration kandi ifite uruhare ruzwi muguhuza synapses ishimishije (34). Muri rusange, ibisubizo birerekana impinduka zitandukanye za poroteyine zitandukanye muri AD zisa naho zitera imbere hamwe nuburemere bwindwara.
Urebye ko tumaze kugera ku bujyakuzimu bwa poroteyine mu kuvumbura proteome yo mu bwonko, turagerageza kumva neza ihuriro ryayo hamwe n'urusobe rwa AD transcriptome. Kubwibyo, twagereranije proteome yubwonko twavumbuye hamwe na module twabanje gukora duhereye kuri microarray yapimye genes 18,204 muri AD (n = 308) no kugenzura (n = 157) tissue ya DLPFC (13). kurengerwa. Muri rusange, twabonye modules 20 zitandukanye za RNA, inyinshi muri zo zerekanaga ubutunzi bwubwoko bwihariye, harimo neuron, oligodendrocytes, astrocytes, na microglia (Ishusho 3A). Impinduka nyinshi ziyi module muri AD zerekanwa mubishusho 3B. Dukurikije isesengura ryambere rya poroteyine-RNA dukoresheje proteome yimbitse ya MS proteome (hafi 3000) kuvumbura kwacu no kwigana moduline 34 za poroteyine zigumana cyane muri proteome yo mu bwonko, 14 (~ 40%) gusa ni bo batsinze ikizamini nyacyo cya Fisher (FET) byagaragaye ko gifite imibare ifatika hamwe na transcriptome (Ishusho 3A). Bihujwe no gusana ibyangiritse kuri ADN (P-M25 na P-M19), ibisobanuro bya poroteyine (P-M7 na P-M20), RNA guhuza / gutera (P-M16 na P-M21) hamwe na poroteyine (P-M13 na P- M23) ntabwo ihuzagurika na modul muri transcriptome. Kubwibyo, nubwo amakuru yimbitse ya proteome yashyizweho akoreshwa muburyo bwo gusesengura ibintu (13), ibyinshi muri AD proteome ya proteome ntabwo byashizwe kumurongo wanditse.
. (hepfo). Ubukomezi bwigicucu gitukura byerekana urwego rwo gukungahaza ubwoko bwakagari mumwanya wo hejuru hamwe nuburemere bwuzuzanya bwa module muburyo bwo hasi. Inyenyeri zerekana akamaro k'imibare (P <0.05). (B) Urwego rwo guhuza hagati ya genes ziranga buri module transcriptome na AD imiterere. Module ibumoso ihujwe cyane na AD (ubururu), naho iburyo ni byiza cyane bifitanye isano na AD (umutuku). Log-yahinduwe BH yakosowe P agaciro yerekana urwego rwibisobanuro byimibare ya buri sano. . . Coefficient ya Pearson hamwe nigiciro cya P irerekanwa. Micro, microglia; imibiri yo mu kirere, astrocytes. CT, kugenzura.
Poroteyine nyinshi zuzuzanya hamwe na RNA modules zisangiye ubwoko bwubwoko butandukanye bwo gutunganyiriza hamwe nicyerekezo gihinduka cya AD (Ishusho 3, B na C). Mu yandi magambo, module ya synaps ijyanye na M1 ya proteome yo mu bwonko (PM 1) yashizwe hamwe na moderi eshatu zikungahaye kuri neurologiya ikungahaye kuri RNA (R-M1, R-M9 na R-M16), ziri muri AD Byombi byerekanwe urwego rugabanijwe. Mu buryo nk'ubwo, M5 na M18 bikungahaye kuri poroteyine byuzuye hamwe na RNA ikungahaye kuri astrocytes hamwe na mikorobe (R-M3, R-M7, na R-M10) kandi bigira uruhare runini mu ndwara Kwiyongera. Ibi bisangiwe muburyo bwa modular hagati yamakuru yombi yunganira kurushaho gushyigikira ubwoko bwingirabuzimafatizo hamwe nimpinduka ziterwa nindwara twabonye muri proteome yubwonko. Ariko, twabonye itandukaniro ryinshi ryingenzi hagati ya RNA nurwego rwa poroteyine zerekana ibimenyetso byihariye muribi bisangiwe. Isesengura rifitanye isano n’imvugo itandukanye ya proteomics na transcriptomics ya molekile iri muri ubwo buryo bwuzuye (Ishusho ya 3D) yerekana uku kudahuza. Kurugero, APP hamwe nizindi poroteyine nyinshi za glial module (NTN1, MDK, COL25A1, ICAM1, na SFRP1) byagaragaje ubwiyongere bukomeye muri proteome ya AD, ariko nta mpinduka zigeze zihinduka mumyandikire ya AD. Izi mpinduka zihariye za poroteyine zishobora kuba zifitanye isano rya hafi na plaque amyloide (23, 35), bikerekana proteome nkisoko yimpinduka zindwara, kandi izi mpinduka ntizishobora kugaragara mumyandikire.
Nyuma yo gusesengura ubwigenge ubwonko na proteome za CSF twavumbuye, twakoze isesengura ryuzuye ryamakuru abiri kugirango tumenye biomarkers ya AD CSF ijyanye na pathophysiologiya y'urusobe rw'ubwonko. Tugomba kubanza gusobanura guhuzagurika kwa proteome ebyiri. Nubwo byemewe cyane ko CSF igaragaza impinduka zifata ubwonko mu bwonko bwa AD (4), urugero nyarwo rwo guhuzagurika hagati yubwonko bwa AD na proteome ya CSF ntirusobanutse. Mugereranije umubare wibicuruzwa bya gene bisangiwe byagaragaye muri proteomes zacu ebyiri, twasanze hafi 70% (n = 1936) za poroteyine zagaragaye mumazi yubwonko nazo zapimwe mubwonko (Ishusho 4A). Inyinshi murizo poroteyine zuzuzanya (n = 1721) zashizwe kuri imwe muri 44 zifatanije zerekana kuva mubwonko bwavumbuwe (Ishusho 4B). Nkuko byari byitezwe, ibice bitandatu binini byubwonko (M1 kugeza M6) byagaragaje umubare munini wa CSF. Ariko, hariho modules ntoya yubwonko (urugero, M15 na M29) igera kurwego rwo hejuru rutunguranye rwo guhuzagurika, runini kuruta ubwonko bwikubye kabiri ubunini. Ibi bidutera imbaraga zo gukoresha uburyo burambuye, bushingiye ku mibare yo kubara guhuza ubwonko n'amazi yo mu bwonko.
(A na B) Poroteyine zagaragaye mu bwonko bwavumbuwe hamwe namakuru ya CSF aruzuzanya. Inyinshi muri izo poroteyine zuzuzanya zifitanye isano na imwe muri 44 yo guhuza imvugo yo mu bwonko ihuza imvugo. (C) Menya isano iri hagati ya cerebrospinal fluid proteome na proteome y'ubwonko. Buri murongo wikarita yubushyuhe yerekana isesengura ryihariye rya hypergeometric FET. Umurongo wo hejuru werekana guhuzagurika (imvi / umukara igicucu) hagati yubwonko na proteome yose ya CSF. Umurongo wa kabiri werekana ko guhuzagurika hagati yubwonko na proteine ya CSF (igicucu gitukura) bigengwa cyane muri AD (P <0.05). Umurongo wa gatatu werekana ko guhuzagurika hagati yubwonko bwubwonko na proteine ya CSF (igicucu cyubururu) bigabanutse cyane muri AD (P <0.05). Koresha uburyo bwa BH kugirango ukosore agaciro P gakomoka kuri FET. . Izi panne zirimo poroteyine 271 zose zijyanye n'ubwonko, zifite imvugo itandukanye muri proteome ya CSF.
Twifashishije FET imwe umurizo, twasuzumye akamaro ko guhuzagurika kwa poroteyine hagati ya proteome ya CSF na modul yubwonko. Isesengura ryerekanye ko modul 14 yubwonko muri data CSF yashyizweho ifite imibare ihuriweho cyane (FDR yahinduye P <0.05), hamwe na module yinyongera (M18) ihuzagurika ryayo rifite akamaro (FDR yahinduye P = 0.06) (Igicapo 4C , umurongo wo hejuru). Dushishikajwe kandi na module ihuza cyane na poroteyine za CSF zitandukanye. Kubwibyo, twakoresheje isesengura ryinyongera rya FET kugirango tumenye imwe muri (i) proteine ya CSF yiyongereye cyane muri AD kandi (ii) poroteyine ya CSF yagabanutse cyane muri AD (P <0.05, byombi t test AD / igenzura) Module yubwonko hamwe no guhuzagurika bifite akamaro. hagati yabo. Nkuko bigaragara kumurongo wo hagati no hepfo yishusho ya 4C, iri sesengura ryinyongera ryerekana ko 8 muri 44 module yubwonko ihuza cyane na proteyine yongewe muri AD CSF (M12, M1, M2, M18, M5, M44, M33, na M38) . ), mugihe module ebyiri gusa (M6 na M15) zerekanye guhuzagurika hamwe na proteine yagabanutse muri AD CSF. Nkuko byari byitezwe, modules 10 zose ziri muri modul 15 hamwe na hejuru cyane hamwe na proteome ya CSF. Kubwibyo, twibwira ko izi module 15 nisoko itanga umusaruro mwinshi wa AD ikomoka mubwonko bwa CSF biomarkers.
Twakubye modul 15 zuzuzanya mubice bitanu binini bya poroteyine dushingiye ku kuba hafi y’igishushanyo cy’ibiti bya WGCNA no guhuza ubwoko bw’utugingo na gene ontologiya (Ishusho 4D). Umwanya wambere urimo modules zikungahaye kuri neuron na proteine zijyanye na synaps (M1 na M12). Umwanya wa synaptique urimo poroteyine zose hamwe 94, kandi urwego muri proteome ya CSF rwahindutse ku buryo bugaragara, bituma ruba isoko nini yerekana ibimenyetso bifitanye isano n'ubwonko bwa CSF mubice bitanu. Itsinda rya kabiri (M6 na M15) ryerekanye isano ya hafi n'ibimenyetso by'uturemangingo twa endoteliyale hamwe n'umubiri w'amaraso, nka “gukira ibikomere” (M6) no “kugenzura uburyo bwo gukingira indwara” (M15). M15 nayo ifitanye isano cyane na metabolism ya lipoprotein, ifitanye isano rya hafi na endotelium (36). Umuyoboro w'amaraso urimo ibimenyetso 34 bya CSF bijyanye n'ubwonko. Itsinda rya gatatu ririmo modules (M2 na M4) zifitanye isano cyane nibimenyetso bya oligodendrocyte no gukwirakwiza selile. Kurugero, amagambo yo murwego rwohejuru ya ontologiya ya M2 arimo "amabwiriza meza yo kwigana ADN" na "purine biosynthesis inzira". Hagati aho, izo M4 zirimo "gutandukanya selile glial" na "gutandukanya chromosome". Umwanya wa myelination urimo ibimenyetso 49 bya CSF bijyanye n'ubwonko.
Itsinda rya kane ririmo module nyinshi (M30, M29, M18, M24, na M5), kandi hafi ya modules zose zikungahaye cyane kuri microglia na marike ya astrocyte. Bisa na myelination panel, panel ya kane nayo irimo modules (M30, M29, na M18) zifitanye isano rya bugufi no gukwirakwiza selile. Izindi module ziri muri iri tsinda zifitanye isano cyane n’amagambo y’ubudahangarwa, nka “inzira y’ubudahangarwa bw'umubiri” (M5) na “amabwiriza yo gukingira indwara” (M24). Itsinda ririnda umubiri ririmo ibimenyetso 42 bya CSF bijyanye n'ubwonko. Hanyuma, akanama kanyuma karimo ibimenyetso 52 bifitanye isano n'ubwonko kuri module enye (M44, M3, M33, na M38), zose ziri kumubiri zijyanye no kubika ingufu na metabolism. Ikinini muri izi module (M3) gifitanye isano rya hafi na mitochondria kandi gikungahaye ku bimenyetso byihariye bya neuron. M38 numwe mubagize module ntoya muriyi metabolome kandi ikanagaragaza umwihariko wa neuron.
Muri rusange, utwo tubaho dutanu twerekana ubwoko butandukanye bwimikorere nimikorere muri cortex ya AD, kandi hamwe hamwe harimo ibimenyetso 271 bifitanye isano n'ubwonko bwa CSF (Imbonerahamwe S2G). Kugirango dusuzume agaciro kibi bisubizo bya MS, twakoresheje uburyo bwo kwagura hafi (PEA), tekinoroji ya antibody ishingiye kuri orthogonal ifite ubushobozi bwo kugwiza ibintu byinshi, ibyiyumvo bihanitse kandi byihariye, kandi twongeye gusesengura ibyitegererezo byamazi yo mu bwonko twasanze agace kamwe ka 271 biomarkers (n = 36). Izi ntego 36 zerekana impinduka muri AD nyinshi za PEA, zifitanye isano rya bugufi nubushakashatsi bwakozwe na MS (r = 0.87, P = 5.6 × 10-12), bwagenzuye neza ibyavuye mu isesengura ryuzuye rya MS (Ishusho S4 ).
Insanganyamatsiko yibinyabuzima yashimangiwe nitsinda ryacu ritanu, kuva ibimenyetso bya synaptique kugeza imbaraga za metabolisme, byose bifitanye isano nindwara ya AD (1-3). Kubwibyo, modul zose uko ari 15 zirimo izo panne zifitanye isano na AD patologiya muri proteome yubwonko twavumbuye (Ishusho 2B). Ikigaragara cyane ni ihuriro ryiza ryiza rya pathologiya hagati ya glial modules yacu hamwe ningaruka mbi ya patologi ihuza hagati ya moderi nini nini ya neuronal (M1 na M3). Isesengura ritandukanye ryisesengura ryubwonko bwakorewe ubwonko (Igicapo S3D) nacyo cyerekana proteine za glial M5 na M18. Muri AsymAD hamwe nibimenyetso bya AD, poroteyine ziyongera cyane hamwe na synaps M1 bifitanye isano na poroteyine igabanuka cyane. Ubu bushakashatsi bwerekana ko ibimenyetso 271 byubwonko bwa cerebrospinal twabonye mumatsinda atanu bifitanye isano nibikorwa byindwara muri cortex ya AD, harimo nibibaho mugihe cyambere kidafite ibimenyetso.
Kugirango dusesengure neza icyerekezo cyo guhindura intungamubiri za poroteyine mu bwonko no mu mazi y’umugongo, twashushanyije ibi bikurikira kuri buri cyiciro 15 cyuzuzanya: (i) twasanze urwego rwinshi rwa module mu bwonko bwashyizweho na (ii) module poroteyine Itandukaniro rigaragarira mu bwonko bwa cerebrospinal (Igicapo S5). Nkuko byavuzwe haruguru, WGCNA ikoreshwa mu kumenya ubwinshi bwa module cyangwa agaciro ka poroteyine iranga ubwonko (13). Ikarita y'ibirunga ikoreshwa mu gusobanura imvugo itandukanye ya poroteyine ya modular mu mazi yo mu bwonko (AD / kugenzura). Iyi mibare yerekana ko bitatu muri bitanu byerekana uburyo butandukanye bwo kwerekana ubwonko n'amazi y'uruti rw'umugongo. Modules ebyiri zumwanya wa synaps (M1 na M12) zerekana igabanuka ryurwego rwinshi mubwonko bwa AD, ariko bihuza cyane na proteyine yiyongereye muri AD CSF (Ishusho S5A). Modul ifitanye isano na neuron irimo metabolome (M3 na M38) yerekanaga ubwonko nubwonko bwubwonko bwerekana imiterere idahuye (Igicapo S5E). Umwanya wamaraso werekanye kandi uburyo butandukanye bwo kwerekana imvugo, nubwo modules zayo (M6 na M15) ziyongereye muburyo bwubwonko bwa AD kandi zigabanuka muri CSF irwaye (Igicapo S5B). Ibice bibiri bisigaye birimo imiyoboro minini ya glial proteyine zihora zigenzurwa mubice byombi (Igicapo S5, C na D).
Nyamuneka menya ko iyi nzira idasanzwe kubimenyetso byose biri muriyi mbaho. Kurugero, akanama ka synaptique karimo poroteyine nyinshi zigabanuka cyane mubwonko bwa AD na CSF (Igicapo S5A). Muri ibyo bimenyetso byerekana ubwonko bwa cerebrospinal fluid harimo NPTX2 na VGF ya M1, na chromogranine B ya M12. Nubwo bimeze bityo ariko, nubwo ibyo bidasanzwe, ibyinshi mubimenyetso bya synaptique byashyizwe hejuru mumazi ya AD. Muri rusange, iri sesengura ryashoboye gutandukanya imibare igaragara mubyerekezo byubwonko nubwonko bwubwonko muri buri kibaho. Izi mpinduka zigaragaza isano igoye kandi akenshi itandukanye hagati yubwonko na CSF ya proteine muri AD.
Hanyuma, twifashishije isesengura ryinshi rya MS kwigana (kwigana CSF 1) kugirango tugabanye 271 ya biomarkers kumurongo ugamije ibyiringiro kandi byororoka (Ishusho 5A). Kopi ya CSF 1 ikubiyemo ingero 96 zose za Emory Goizueta ADRC, harimo kugenzura, AsymAD, na AD cohort (Imbonerahamwe S1A). Izi ndwara za AD zirangwa no kugabanuka kwubwenge buke (bivuze MoCA, 20.0 ± 3.8), hamwe nimpinduka muri biomarkers ya AD yemejwe mumazi ya cerebrospinal fluid (Imbonerahamwe S1A). Bitandukanye nisesengura rya CSF twasanze, uku kwigana bikorwa hakoreshejwe uburyo bunoze kandi bwinjiza cyane "uburyo bumwe-bumwe" bwa MS (butagabanijwe kumurongo), harimo protocole yoroshye yo gutegura protocole ikuraho ibikenerwa byo gukingira indwara zintangarugero. . Ahubwo, "umuyoboro wongera imbaraga" umwe ukoreshwa mu kongera ibimenyetso bya poroteyine nkeya (37). Nubwo igabanya proteome yuzuye, ubu buryo bwo kurasa bumwe bugabanya cyane igihe cyimashini kandi byongera umubare wicyitegererezo cya TMT gishobora gusesengurwa neza (17, 38). Muri rusange, isesengura ryagaragaje peptide 6.487, zishushanya proteome 1,183 mu manza 96. Kimwe n’isesengura rya CSF twasanze, izo poroteyine zonyine zingana byibuze 50% by'icyitegererezo zashyizwe mu mibare yakurikiyeho, kandi amakuru yasubijwe inyuma kubera ingaruka z'imyaka n'uburinganire. Ibi byatumye umubare wanyuma wa proteome 792, 95% muri zo nazo zagaragaye mu makuru ya CSF yabonetse.
. . T-ibizamini byombi cyangwa ANOVA hamwe na Tukey nyuma yo gukosorwa byakoreshejwe mugusuzuma akamaro k'imibare y'impinduka mubwinshi muri buri isesengura ryigana. CT, kugenzura.
Kubera ko dushishikajwe cyane no kugenzura intego 271 zijyanye na CSF zijyanye n'ubwonko dukoresheje isesengura ryuzuye, tuzagabanya gukomeza gusuzuma iyi proteome yigana kuri ibi bimenyetso. Muri izo poroteyine 271, 100 zagaragaye mu kwigana CSF 1. Igicapo S6A cerekana imvugo itandukanye yibi bimenyetso 100 byuzuzanya hagati yubugenzuzi hamwe nicyitegererezo cya AD. Amateka ya Synaptic na metabolite yiyongera cyane muri AD, mugihe poroteyine zo mu maraso zigabanya cyane indwara. Byinshi mubimenyetso 100 byuzuzanya (n = 70) byakomeje icyerekezo kimwe cyimpinduka mubice bibiri byamakuru (Igicapo S6B). Ibi bimenyetso 70 byemewe byubwonko bifitanye isano na CSF (Imbonerahamwe S2H) byerekana cyane cyane ibyerekanwe mbere byerekanwe, ni ukuvuga kugabanuka kwa poroteyine zo mu mitsi no kugenzura-izindi ngingo zose. 10 gusa muri 70 za poroteyine zemewe zerekanye impinduka mubwinshi bwa AD zivuguruza iyi nzira. Kugirango tubyare akanama kagaragaza neza icyerekezo rusange cyubwonko hamwe nubwonko bwubwonko, twakuyemo poroteyine 10 murwego rwinyungu twarangije kugenzura (Ishusho 5A). Kubwibyo, itsinda ryacu ririmo poroteyine 60 zose zagenzuwe mubice bibiri byigenga bya CSF AD dukoresheje uburyo butandukanye bwo gutegura no gusesengura urubuga rwa MS. Imigambi ya z-amanota yibi bibanza byanyuma muri CSF ikopi ya 1 igenzura hamwe na AD byemeje icyerekezo cyagaragaye muri cohort ya CSF twabonye (Ishusho 5B).
Muri izo poroteyine 60, harimo molekile zizwiho kuba zifitanye isano na AD, nka osteopontin (SPP1), ikaba ari cytokine itera umuriro wahujwe na AD mu bushakashatsi bwinshi (39-41), na GAP43, poroteyine ya synaptic. ibyo bifitanye isano neza na neurodegeneration (42). Poroteyine zagenzuwe neza ni ibimenyetso bifitanye isano n'izindi ndwara zifata ubwonko, nka amyotrophique lateral sclerose (ALS) ifitanye isano na superoxide dismutase 1 (SOD1) n'indwara ya Parkinson ifitanye isano na desaccharase (PARK7). Twagenzuye kandi ko ibindi bimenyetso byinshi, nka SMOC1 hamwe nubwonko bukungahaye mu bwonko bwerekana protein 1 (BASP1), bifite aho bihurira na neurodegeneration. Birakwiye ko tumenya ko kubera ubwinshi bwabo muri rusange muri proteome ya CSF, biragoye kuri twe gukoresha ubu buryo bwinjiza cyane-uburyo bumwe bwo gutahura uburyo bwo kumenya neza MAPT hamwe nizindi poroteyine zimwe na zimwe zijyanye na AD (urugero, NEFL na NRGN ) (43, 44).
Twahise dusuzuma ibi bimenyetso 60 byibanze muburyo butatu bwo kwigana. Muri CSF Copy 2, twakoresheje TMT-MS imwe kugirango dusesengure itsinda ryigenga rya 297 igenzura hamwe nicyitegererezo cya AD cyatanzwe na Emory Goizueta ADRC (17). Kwigana CSF 3 harimo gusubiramo amakuru ya TMT-MS aboneka kubigenzura 120 hamwe nabarwayi ba AD baturutse i Lausanne, mubusuwisi (45). Twabonye ibirenze bibiri bya gatatu bya 60 byibanze muri buri dataset. Nubwo ubushakashatsi bwu Busuwisi bwakoresheje uburyo butandukanye bwa MS hamwe nuburyo bwo kubara TMT (45, 46), twerekanye cyane imigendekere yacu muburyo bubiri bwasesenguwe (Ishusho 5, C na D, na Imbonerahamwe S2, I, na J). Kugirango dusuzume indwara yihariye yitsinda ryacu, twakoresheje TMT-MS kugirango dusesengure amakuru ya kane yo kwigana (CSF replication 4), atarimo kugenzura gusa (n = 18) na AD (n = 17), ariko na PD ( n = 14)), ALS (n = 18) hamwe no guta umutwe imbere (FTD) ingero (n = 11) (Imbonerahamwe S1A). Twatsinze neza hafi bibiri bya gatatu bya poroteyine zo muri iyi kipe (38 kuri 60). Ibisubizo byerekana impinduka zihariye za AD muburyo butanu bwa biomarker (Igicapo 5E na Imbonerahamwe S2K). Ubwiyongere bwitsinda rya metabolite bwerekanaga AD ikomeye cyane, hanyuma ikurikirwa na myelination na glial group. Ku rugero ruto, FTD irerekana kandi kwiyongera hagati yibi bice, bishobora kwerekana impinduka zishobora guhinduka (17). Ibinyuranye, ALS na PD berekanye hafi ya myelination, glial, na metabolome imyirondoro nkitsinda rishinzwe kugenzura. Muri rusange, nubwo hari itandukaniro mugutegura icyitegererezo, urubuga rwa MS, hamwe nuburyo bwo kugereranya TMT, iri sesengura ryasubiwemo ryerekana ko ibimenyetso byibanze byibanze bifite impinduka zihamye za AD yihariye mubisobanuro birenga 500 bya CSF.
AD neurodegeneration yamenyekanye cyane mumyaka itari mike mbere yuko ibimenyetso byubwenge bitangira, bityo hakenewe byihutirwa abakora biomarkers ya AsymAD (5, 31). Nyamara, ibimenyetso byinshi kandi byinshi byerekana ko ibinyabuzima bya AsymAD biri kure y’abantu bahuje ibitsina, kandi imikoranire igoye y’ingaruka no kwihangana itera itandukaniro rinini ryabantu ku giti cyabo mu iterambere ry’indwara (47). Nubwo ikoreshwa mugutahura ibibazo bya AsymAD, urwego rwibanze rwa biomarkers ya CSF (Aβ1-42, tau yose hamwe na p-tau) ntabwo byagaragaye ko rushobora guhanura byimazeyo uzatera imbere guta umutwe (4, 7), byerekana byinshi Birashoboka nkenerwa gushyiramo ibikoresho bya biomarker byuzuye bishingiye kubintu byinshi bya physiologiya yubwonko kugirango tumenye neza ibyago byabaturage. Kubwibyo, twakurikiranye gusesengura itsinda ryacu ryemewe na biomarker AD mubaturage ba AsymAD ya kopi ya CSF 1. Izi manza 31 za AsymAD zerekanye urwego rwibanze rwa biomarker (Aβ1–42 / igereranyo cya tau ELISA, <5.5) no kumenya byuzuye (bivuze MoCA, 27.1 ± 2.2) (Imbonerahamwe S1A). Byongeye kandi, abantu bose bafite AsymAD bafite amanota yo guta umutwe ya 0, byerekana ko nta kimenyetso cyerekana ko igabanuka ryimikorere ya buri munsi cyangwa imikorere.
Twabanje gusesengura urwego rwibikoresho byemewe muri 96 CSF yigana 1, harimo na AsymAD cohort. Twabonye ko panele nyinshi mumatsinda ya AsymAD zagize impinduka zikomeye zisa na AD zimeze nkinshi, imitsi yimitsi yerekanaga inzira yo kumanuka muri AsymAD, mugihe izindi panne zose zerekanaga inzira yo kuzamuka (Ishusho 6A). Kubwibyo, ibice byose byerekanaga isano ikomeye na ELISA Aβ1-42 hamwe nurwego rwa tau rwose (Ishusho 6B). Ibinyuranye, isano iri hagati yitsinda n amanota ya MoCA irakennye. Kimwe mubitangaje byagaragaye muri iri sesengura ni intera nini ya paneli nyinshi muri cohort ya AsymAD. Nkuko bigaragara ku gishushanyo cya 6A, urwego rwumurwi witsinda rya AsymAD rusanzwe rwambukiranya urwego rwitsinda rishinzwe kugenzura hamwe nitsinda rya AD, ryerekana guhinduka cyane. Kugirango turusheho gucukumbura ubudahangarwa bwa AsymAD, twakoresheje isesengura rya Multidimensional Scaling (MDS) kuri 96 CSF yo kwigana 1. Isesengura rya MDS ryemerera kwiyumvisha isano iri hagati yimanza zishingiye kubihinduka bimwe mumibare yashyizweho. Kuri iri sesengura rya cluster, dukoresha gusa ibyo bimenyetso byemewe bifite impinduka zifatika zifatika (P <0.05, AD / kugenzura) mubuvumbuzi bwa CSF no kwigana proteome 1 (n = 29) (Imbonerahamwe S2L). Isesengura ryatanze ibisobanuro bigaragara hagati yubugenzuzi bwacu na AD (Ishusho 6C). Ibinyuranye, imanza zimwe za AsymAD zegeranye neza mumatsinda yo kugenzura, mugihe izindi ziri mubibazo bya AD. Kugirango turusheho gucukumbura ubudahangarwa bwa AsymAD, twakoresheje ikarita ya MDS kugirango dusobanure amatsinda abiri yizo manza za AsymAD. Itsinda rya mbere ryarimo imanza za AsymAD zegeranye zegeranye no kugenzura (n = 19), mugihe itsinda rya kabiri ryaranzwe nimanza za AsymAD zifite ikimenyetso cyerekana hafi ya AD (n = 12).
. Isesengura ryibitandukanye na Tukey nyuma yo gukosorwa byakoreshejwe kugirango harebwe akamaro k'imibare y'impinduka nyinshi. . Coefficient ya Pearson hamwe nigiciro cya P irerekanwa. . Isesengura ryakoreshejwe mu kugabanya itsinda rya AsymAD kugenzura (n = 19) na AD (n = 12) matsinda mato. . Ikibaho biomarkers gifite amabara. . Isesengura rya Tukey nyuma yo guhindura itandukaniro ryakoreshejwe mugusuzuma akamaro k'imibare.
Twasuzumye imvugo itandukanye ya poroteyine hagati yubu bugenzuzi na AD-imeze nka AsymAD (Ishusho 6D na Imbonerahamwe S2L). Ikarita y'ibirunga yavuyemo yerekana ko ibimenyetso 14 byahindutse cyane hagati yaya matsinda yombi. Byinshi muribi bimenyetso ni synaps na metabolome. Nyamara, SOD1 na myristoylated alanine ikungahaye kuri protein kinase C substrate (MARCKS), bagize itsinda rya myelin na glial immunite, nabo bari muri iri tsinda (Ishusho 6, D na E). Itsinda ryimitsi ryatanze kandi ibimenyetso bibiri byagabanutse cyane mumatsinda ya AD isa na AsymAD, harimo na AE ihuza protein 1 (AEBP1) kandi ikuzuza umuryango C9. Nta tandukaniro rikomeye ryari hagati yubugenzuzi na AD-nka matsinda mato ya AsymAD muri ELISA AB1-42 (P = 0.38) na p-tau (P = 0.28), ariko mubyukuri hari itandukaniro rikomeye murwego rwose rwa tau (P = 0.0031 ) (Ishusho S7). Hano hari ibimenyetso byinshi byerekana ko impinduka hagati yitsinda rya AsymAD zombi zifite akamaro kanini kurenza urwego rwa tau (urugero, YWHAZ, SOD1, na MDH1) (Ishusho 6E). Muri rusange, ibisubizo byerekana ko akanama kacu kemewe gashobora kuba karimo biomarkers zishobora kwandikwa hamwe n’ingaruka zishobora guterwa n’abarwayi bafite indwara zidasanzwe.
Hano harakenewe byihutirwa ibikoresho bishingiye kuri biomarker sisitemu yo gupima neza no kwibasira patrophysiologiya itandukanye inyuma ya AD. Ibi bikoresho biteganijwe ko bidahindura gusa uburyo bwo gusuzuma AD, ahubwo binateza imbere ingamba zifatika, zihariye zo kuvura abarwayi (1, 2). Kugira ngo ibyo bishoboke, twakoresheje uburyo butabogamye bwa proteomics kubwonko bwa AD na CSF kugirango tumenye urubuga rwa biomarkers rwa CSF rugaragaza imiterere myinshi ya patrophysiologiya ishingiye ku bwonko. Isesengura ryacu ryakoze ibice bitanu bya CSF biomarker, (i) byerekana synaps, imiyoboro y'amaraso, myelin, immunite na metabolic dysfunction; (ii) kwerekana imyororokere ikomeye ku mbuga zitandukanye za MS; (iii) Erekana impinduka zindwara zigenda zitera intambwe yambere na nyuma ya AD. Muri rusange, ibyagaragaye byerekana intambwe itanga icyizere cyo guteza imbere ibikoresho bitandukanye, byizewe, bishingiye ku mbuga za biomarker ibikoresho byubushakashatsi bwa AD hamwe nubuvuzi bukoreshwa.
Ibisubizo byacu byerekana ishyirahamwe ryabungabunzwe cyane rya AD ubwonko bwa proteome kandi dushyigikira ikoreshwa ryayo nka ankeri yo guteza imbere biomarker. Isesengura ryacu ryerekana ko imibare ibiri yigenga ya TMT-MS irimo ubwonko bwa AD na AsymAD ifite modularite ikomeye. Ibyavuye mu bushakashatsi byongereye akazi kambere, byerekana kubungabunga imbaraga zikomeye zingingo zirenga 2000 zubwonko zivuye mubice byinshi byigenga imbere, imbere, parietal, na cortex yigihe gito (17). Uru rusobe rwumvikanyweho rugaragaza impinduka zinyuranye ziterwa nindwara zigaragara mubushakashatsi bugezweho, harimo kwiyongera kwa moderi ikungahaye ku isi no kugabanuka kwa modul ikungahaye kuri neuron. Kimwe nubushakashatsi bugezweho, uru rusobe runini narwo rugaragaza impinduka zikomeye muri AsymAD, yerekana ibintu bitandukanye bitandukanye bya patrophysiologiya (17).
Nyamara, muri ubu buryo bushingiye ku buryo bukomeye bushingiye ku buryo bushingiye ku bidukikije, hariho byinshi by’ibinyabuzima bitandukanye cyane cyane mu bantu mu ntangiriro za AD. Akanama kacu ka biomarker karashoboye kwerekana amatsinda abiri muri AsymAD, yerekana imvugo itandukanye itandukanye yibimenyetso byinshi bya CSF. Itsinda ryacu ryashoboye kwerekana itandukaniro ryibinyabuzima hagati yaya matsinda yombi, bitagaragaye kurwego rwibanze rwa biomarkers. Ugereranije nitsinda rishinzwe kugenzura, Aβ1-42 / igereranyo cya tau igipimo cyaba bantu ba AsymAD bari hasi bidasanzwe. Nyamara, urwego rwose rwa tau rwatandukanye cyane hagati yitsinda ryombi rya AsymAD, mugihe urwego Aβ1-42 na p-tau rwagumye kugereranywa. Kubera ko CSF tau ndende isa nkaho ihanura neza ibimenyetso byubwenge kuruta urwego Aβ1-42 (7), turakeka ko amakipe yombi ya AsymAD ashobora kuba afite ibyago bitandukanye byo kwandura indwara. Urebye ingano ntoya ya AsymAD yacu no kubura amakuru maremare, ubushakashatsi buracyakenewe kugirango twizere neza iyi myanzuro. Nyamara, ibisubizo byerekana ko itsinda rya CSF rishingiye kuri sisitemu rishobora kongera ubushobozi bwacu bwo gutandukanya abantu neza mugihe cyindwara idasanzwe.
Muri rusange, ibyo twabonye bishyigikira uruhare rwibikorwa byinshi byibinyabuzima mu gutera AD. Nyamara, imbaraga zidasanzwe za metabolisme zahindutse insanganyamatsiko yibintu bitanu byemewe byemewe. Poroteyine za metabolike, nka hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) na lactate dehydrogenase A (LDHA), ni biomarkers ya synaptic yemewe cyane, byerekana ko kwiyongera kwa AD CSF ari igitsina cyororoka cyane. Imiyoboro y'amaraso hamwe na panele glial nayo irimo ibimenyetso byinshi bigira uruhare muri metabolism yibintu bya okiside. Ibyavuye mu bushakashatsi bihuye n’uruhare runini inzira ya metabolike igira mu bwonko bwose, ntabwo ari uguhuza ingufu nyinshi za neurone, ahubwo inuzuza ingufu nyinshi za astrocytes hamwe nizindi selile (17, 48). Ibisubizo byacu bishyigikira ibimenyetso bigenda byiyongera byerekana ko impinduka ziterwa na redox hamwe no guhagarika inzira zingufu zishobora kuba ihuriro ryibanze hagati yinzira zingenzi zagize uruhare mu gutera indwara ya AD, harimo indwara ziterwa na mitochondial, indwara ziterwa na glial-mediya, hamwe no kwangirika kwamaraso (49). Byongeye kandi, metabolike cerebrospinal fluid biomarkers irimo umubare munini wa poroteyine zikungahaye zitandukanye hagati yubugenzuzi bwacu na AD-nka AsymAD matsinda mato, byerekana ko guhagarika izo mbaraga n'inzira za redox bishobora kuba ingenzi mugihe cyambere cyindwara.
Ubwonko butandukanye nubwonko bwa cerebrospinal fluid panel twabonye nabyo bifite ingaruka zishimishije kubinyabuzima. Synaps na metabolomes ikungahaye kuri neurone yerekana kugabanuka k'ubwonko bwa AD kandi byiyongera cyane mumazi ya cerebrospinal. Urebye ko neuron ikungahaye kuri mitochondriya itanga ingufu kuri synaps kugirango itange ingufu kubimenyetso byabo byihariye (50), biteganijwe ko isano ihuza imvugo yaya matsinda yombi ya neuron. Gutakaza neuron no gukuramo ingirangingo zangiritse birashobora gusobanura ubu bwonko hamwe na CSF bigenda byindwara nyuma, ariko ntibishobora gusobanura impinduka zambere twabonye (13). Igisobanuro kimwe gishoboka kuri ubu bushakashatsi mu ndwara ya asimptomatic kare ni ugukata synaptic idasanzwe. Ibimenyetso bishya byerekana imiterere yimbeba byerekana ko microglia-yunganirwa na synaptic phagocytose ishobora gukoreshwa muburyo budasanzwe muri AD kandi bigatera gutakaza synaps hakiri kare mubwonko (51). Ibikoresho bya synaptique byajugunywe birashobora kwegeranya muri CSF, niyo mpamvu tubona ubwiyongere bwa CSF muburyo bwa neuron. Gukata synaptique ikingira indwara birashobora kandi gusobanura igice kwiyongera kwa poroteyine zo mu kirere tubona mu bwonko no mu bwonko bwa cerebrospinal mugihe cyose cyindwara. Usibye gukata synaptic, muri rusange ibintu bidasanzwe munzira ya exocytic irashobora no gutuma ubwonko butandukanye hamwe na CSF byerekana ibimenyetso bya neuronal. Ubushakashatsi butari buke bwerekanye ko ibikubiye muri exosomes mu gutera ubwonko bwa AD byahindutse (52). Inzira idasanzwe nayo igira uruhare mu gukwirakwiza Aβ (53, 54). Birakwiye ko tumenya ko guhagarika ururenda rwa exosomal bishobora kugabanya AD-isa na AD muri moderi yimbeba ya AD transgenic (55).
Muri icyo gihe, poroteyine iri mu mitsi y'amaraso yerekanaga ubwiyongere bukabije mu bwonko bwa AD, ariko byagabanutse cyane muri CSF. Inzitizi y'amaraso n'ubwonko (BBB) idakora neza irashobora gusobanura igice. Ubushakashatsi bwinshi bwigenga bwa postmortem bwerekanye ko BBB isenyuka muri AD (56, 57). Ubu bushakashatsi bwemeje ibikorwa bitandukanye bidasanzwe bikikije uru rugingo rufunze cyane rwa selile endothelia, harimo ubwonko bwa capillary leakage ndetse no kwirundanya kwa poroteyine ziva mu maraso (57). Ibi birashobora gutanga ibisobanuro byoroshye kuri poroteyine zo mu maraso zashyizwe hejuru mu bwonko, ariko ntishobora gusobanura byimazeyo igabanuka rya poroteyine zimwe mu mazi yo mu bwonko. Ikintu kimwe gishoboka nuko sisitemu yo hagati yo hagati itandukanya byimazeyo molekile kugirango ikemure ikibazo cyo kongera umuriro hamwe na stress ya okiside. Kugabanuka kwa zimwe muri poroteyine zikomeye za CSF muri iyi nteko, cyane cyane izagira uruhare mu kugena lipoproteine, bifitanye isano no kubuza urwego rwangiza rw’umuriro ndetse n’inzira ya neuroprotective y’ubwoko bwa ogisijeni ikora. Ibi ni ukuri kuri Paroxonase 1 (PON1), lipoprotein ihuza enzyme ishinzwe kugabanya urugero rwa stress ya okiside mu kuzenguruka (58, 59). Alpha-1-microglobulin / bikunin precursor (AMBP) nikindi kimenyetso cyamanutse kigengwa cyane nitsinda ryimitsi. Nibibanjirije gutwara lipid itwara bikunin, nayo igira uruhare mukurinda umuriro no kurinda imitsi (60, 61).
Nubwo hari ibitekerezo bitandukanye bishimishije, kutabasha kumenya neza uburyo bwindwara yibinyabuzima ni inzitizi izwi cyane yo kugabanya ubushakashatsi bwakozwe na proteomics. Kubwibyo, ubushakashatsi buracyakenewe kugirango dusobanure neza uburyo bwihishe inyuma yibi bikoresho bya biomarker. Kugirango tugere ku majyambere y’isesengura ry’amavuriro ashingiye kuri MS, icyerekezo kizaza nacyo gisaba gukoresha uburyo bugereranijwe bwo kugereranya ibinyabuzima binini bigenzurwa, nko gukurikirana ibyatoranijwe cyangwa bigereranywa (62). Muminsi ishize twakoresheje parallel reaction yo kugenzura (63) kugirango twemeze impinduka nyinshi za poroteyine za CSF zasobanuwe hano. Intego nyinshi zingenzi zibanze zagereranijwe hamwe nukuri, harimo YWHAZ, ALDOA, na SMOC1, ikarita yerekana synaps, metabolism, hamwe na panneux yumuriro (63). Kwigenga Kwigenga (DIA) hamwe nizindi ngamba zishingiye kuri MS nazo zishobora kuba ingirakamaro mugusuzuma intego. Bud n'abandi. . Ubu bushakashatsi buherutse bushyigikira ubushobozi bwibikoresho byacu kugirango bihinduke muburyo bwizewe bushingiye kuri MS. Antibody gakondo hamwe na aptamer-gutahura nabyo ni ngombwa mugutezimbere kurushaho iterambere ryibinyabuzima bya AD. Bitewe n'ubwinshi bwa CSF, biragoye kumenya izi biomarkers ukoresheje uburyo bwa MS bwinjiza cyane. NEFL na NRGN ni ingero ebyiri nkizo za biomarkers ya CSF nkeya, yashizwe kumurongo mugice cyisesengura ryacu ryuzuye, ariko ntishobora kumenyekana neza dukoresheje ingamba zacu imwe ya MS. Kwibanda ku ngamba zishingiye kuri antibodi nyinshi, nka PEA, zishobora guteza imbere ivuriro ryibimenyetso.
Muri rusange, ubu bushakashatsi butanga uburyo bwihariye bwa proteomics bwo kumenya no kugenzura ibinyabuzima bya CSF AD bishingiye kuri sisitemu zitandukanye. Kunonosora ibyo bimenyetso hejuru yinyongera ya AD hamwe na MS platform birashobora kwerekana ko biteza imbere AD ibyiciro no kuvura. Ubushakashatsi bwerekana urwego rurerure rwibi bikoresho mugihe cyingirakamaro nabyo kugirango hamenyekane guhuza ibimenyetso byerekana neza ibyago byindwara hakiri kare nimpinduka zuburemere bwindwara.
Usibye ingero 3 zandukuwe na CSF, ingero zose za CSF zikoreshwa muri ubu bushakashatsi zegeranijwe ziyobowe na Emory ADRC cyangwa ibigo by'ubushakashatsi bifitanye isano rya bugufi. Ibice bine byintangarugero za Emory CSF byakoreshejwe murubwo bushakashatsi bwa proteomics. Itsinda rya CSF ryasanze ririmo ingero zivuye mu buzima 20 n’abarwayi 20 AD. Kopi ya CSF 1 ikubiyemo ingero zivuye 32 zifite ubuzima bwiza, 31 AsymAD, nabantu 33 AD. Kopi ya CSF 2 ikubiyemo 147 igenzura hamwe na AD AD 150. Indwara nyinshi CSF yigana 4 cohort yarimo 18 igenzura, 17 AD, 19 ALS, 13 PD, na 11 FTD. Dukurikije amasezerano yemejwe n’inama ishinzwe isuzuma rya kaminuza ya Emory, abitabiriye kwiga Emory bose babonye uruhushya rubimenyeshejwe. Nk’uko Ikigo cy'igihugu gishinzwe gusaza mu mwaka wa 2014 kibyerekana neza uburyo bwiza bwo kwimenyereza ibigo bya Alzheimer (https://alz.washington.edu/BiospecimenTaskForce.html), amazi yo mu bwonko yakusanyirijwe hamwe abikwa no gucumita. Igenzura na AsymAD na AD abarwayi bahawe isuzuma ryubwenge risanzwe kuri Emory Cognitive Neurology Clinic cyangwa Goizueta ADRC. Ingero zabo zo mu bwonko zapimwe na INNO-BIA AlzBio3 Luminex kuri ELISA Aβ1-42, isesengura rya tau na p-tau (65). Indangagaciro za ELISA zikoreshwa mugushigikira ibyiciro byo gusuzuma ibyiciro hashingiwe kubipimo bya AD biomarker byaciwe (66, 67). Amakuru yibanze ya demokarasi no kwisuzumisha kubindi bisuzumwa bya CSF (FTD, ALS, na PD) nayo aboneka muri Emory ADRC cyangwa ibigo byubushakashatsi bishamikiyeho. Incamake y'urubanza metadata kuriyi manza za Emory CSF murayisanga mumeza S1A. Ibiranga kwigana CSF yo mu Busuwisi 3 cohort byashyizwe ahagaragara mbere (45).
CSF yabonye icyitegererezo. Kugirango twongere ubujyakuzimu bw'ivumburwa ryamakuru ya CSF, gukoresha ubudahangarwa bwa poroteyine nyinshi byakozwe mbere yo kugerageza. Muri make, 130 μl ya CSF kuva kuri 40 ya CSF hamwe nubunini bungana (130 μl) ya High Select Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) yashyizwe mu nkingi izunguruka (Thermo Fisher Scientific, A89868) mucyumba ubushyuhe Incubate). Nyuma yo kuzunguruka muminota 15, centrifuge icyitegererezo kuri 1000g muminota 2. Igikoresho cya 3K ultracentrifugal filter (Millipore, UFC500396) yakoreshejwe muguhuriza hamwe icyitegererezo cy'amazi hifashishijwe centrifuging kuri 14,000g muminota 30. Koresha urugero rwicyitegererezo kuri 75 μl hamwe na saline ya fosifate. Intungamubiri za poroteyine zasuzumwe n’uburyo bwa acide ya bicinchoninike (BCA) hakurikijwe protocole yuwabikoze (Thermo Fisher Scientific). CSF ikingira umubiri (60 μl) uhereye kuri 40 zose zashizwemo na lysyl endopeptidase (LysC) na trypsin. Muri make, icyitegererezo cyaragabanutse kandi gishyirwa hamwe na 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosifine na 3 μl 0.8 M chloroacetamide kuri 90 ° C muminota 10, hanyuma ikabyara ubwogero bwamazi muminota 15. Icyitegererezo cyavanze na 193 μl 8 M urea buffer [8 M urea na 100 mM NaHPO4 (pH 8.5)] kugeza kuri 6 M urea. LysC (4.5 μg; Wako) ikoreshwa mugusya nijoro mubushyuhe bwicyumba. Icyitegererezo cyahise kivangwa muri 1 M urea hamwe na 50 mM amonium bicarbonate (ABC) (68). Ongeramo amafaranga angana (4.5 μg) ya trypsin (Promega), hanyuma ushireho icyitegererezo kumasaha 12. Kwemeza igisubizo cya peptide igogora kugeza kuri 1% ya acide formique (FA) na 0.1% trifluoroacetic aside (TFA) (66), hanyuma ukayungurura hamwe na mg 50 mg Sep-Pak C18 (Amazi) nkuko byasobanuwe haruguru (25) . Peptide yahise ikurwa muri ml 1 ya 50% acetonitrile (ACN). Kugirango ibipimo bya poroteyine bigabanuke mu byiciro (25), 100 μl aliquots zo mu ngero 40 zose za CSF zahujwe kugira ngo zitange icyitegererezo kivanze, hanyuma kigabanywa mu bice bitanu by’imbere mu gihugu (GIS) (48). Ingero zose kugiti cye hamwe nibipimo byahujwe byumishwa na vacuum yihuta (Labconco).
CSF ikoporora icyitegererezo. Dayon na bagenzi be babanje gusobanura ubudahangarwa bw'umubiri no gusya kwa CSF kopi 3 (45, 46). Ingero zisigaye zo kwigana ntabwo zigeze zikingirwa. Kora ingero zidakuweho muri trypsin nkuko byasobanuwe mbere (17). Kuri buri sesengura ryasubiwemo, 120 μl aliquots za peptide zivuye kuri buri cyitegererezo zahurijwe hamwe hanyuma zigabanywamo ibice bingana na alikoti kugirango bikoreshwe nka TMT yanditseho isi yose imbere (48). Ingero zose kugiti cye hamwe nibipimo byahujwe byumishwa na vacuum yihuta (Labconco). Mu rwego rwo kuzamura ibimenyetso bya poroteyine nkeya ya CSF, mu guhuza 125 μl kuri buri cyitegererezo, hateguwe icyitegererezo “cyongerewe” kuri buri isesengura ryigana [ni ukuvuga icyitegererezo cy’ibinyabuzima cyigana icyitegererezo cy’ubushakashatsi, ariko amafaranga aboneka arahari binini cyane (37, 69)] byahujwe na sample ya CSF ivanze (17). Icyitegererezo kivanze noneho cyakingiwe hakoreshejwe ml 12 ya High Select Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), igogorwa nkuko byasobanuwe haruguru, ikanashyirwa mubimenyetso byinshi byakurikiyeho TMT.
Igihe cyo kohereza: Kanama-27-2021