Txhim kho Sab saum toj-Down Proteomics Ntawm Lub Hlwb Npog Nrog FAIMS Part 4
Aug 28, 2024
Peb kuj tau txheeb xyuas ntau qhov tawg tau muab los ntawm cov tsis-canonical splice isoforms ntawm tauprotein paub tias tau hais nyob rau hauv tib neeg lub hlwb.83 luv luv, tau isoforms yog txhais los ntawm tus naj npawb ntawm N-terminal inserts vim lwm txoj kev splicing ntawm exon 2 thiab/los yog 3 (xa mus. rau li 0N, 1N, thiab 2N) thiab tus naj npawb ntawm microtubule-binding rov ua dua vim lwm txoj kev splicing ntawm exon 10 (hu ua 3R lossis 4R).84
Tau protein ntau yog cov protein tseem ceeb hauv cov hlwb neuronal, uas ua lub luag haujlwm tseem ceeb hauv kev tswj cov qauv thiab kev ua haujlwm ntawm cov hlwb neuronal. Cov kev tshawb fawb tau pom tias tau cov protein muaj feem cuam tshuam nrog kev nco, thiab nws qhov kev qhia txawv txav tuaj yeem ua rau kev paub tsis meej thiab txawm tias kev paub tsis meej.
Cov kws tshawb fawb tau ua ntau txoj kev tshawb fawb txog kev sib raug zoo ntawm tau protein thiab kev nco. Raws li kev tshawb fawb, tau protein tuaj yeem pab tswj kev ruaj ntseg ntawm cov paj hlwb, yog li txhawb cov ntaub ntawv xa mus thiab nco tsim ntawm cov hlwb neuronal. Txawm li cas los xij, ib zaug tau cov protein ntau txawv txav, xws li cov protein ntau ntxiv, nws yuav ua rau cov hlab ntsha tawg thiab tuag ntawm ntau cov hlwb neuronal, uas yuav cuam tshuam rau kev tsim thiab kev saib xyuas ntawm kev nco thiab tseem ua rau kev nco tsis zoo. Yog li ntawd, kev tswj hwm qhov kev qhia ib txwm muaj thiab kev ua haujlwm ntawm tau protein yog qhov tseem ceeb heev rau kev tswj hwm kev noj qab haus huv ntawm kev nco.
Cov kev tshawb fawb tsis ntev los no kuj tau pom tias cov khoom noj muaj nyob hauv qee cov zaub mov thiab dej haus kuj tseem muaj txiaj ntsig zoo rau kev tswj hwm qhov ua haujlwm ntawm tau protein ntau. Piv txwv li, antioxidants, Omega -3 fatty acids, thiab caffeine-nplua nuj zaub thiab txiv hmab txiv ntoo tuaj yeem pab tswj kev qhia thiab kev ua haujlwm ntawm tau cov protein, uas pab txhim kho kev nco thiab kev txawj ntse.
Yog li ntawd, peb yuav tsum tau ua tib zoo saib xyuas kev noj qab haus huv zoo, tswj kev noj zaub mov, txo kev ntxhov siab, thiab lwm yam, kom tswj tau qhov kev coj ua thiab kev ua haujlwm ntawm tau protein. Nyob rau tib lub sijhawm, kev tswj hwm tus cwj pwm zoo thiab ua ib qho kev tawm dag zog tuaj yeem pab txhawb kev xa xov xwm thiab kev nco tsim ntawm cov hlwb neuronal. Kuv ntseeg tias tsuav yog peb ua tib zoo saib xyuas kev noj qab haus huv, peb muaj peev xwm nco ntsoov noj qab nyob zoo thiab muaj kev zoo siab rau lub neej. Qhov no qhia tau hais tias peb yuav tsum txhim kho peb lub cim xeeb. Cistanche tuaj yeem txhim kho kev nco zoo vim Cistanche yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm Cistanche los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv hauv ntau txoj hauv kev.
Nyem paub 10 txoj hauv kev los txhim kho kev nco
Nws kuj tseem tsim nyog sau cia tias cov fragments endogenous ntawm tau yog ib qho kev tshawb pom nyob rau hauv tib neeg lub hlwb cov ntaub so ntswg thiab cerebrospinal kua, thiab sib txawv fragmentation ntawm tau tuaj yeem ua lub luag haujlwm hauv AD progression.85,86
Peb tau pom ntau qhov sib txawv uas tuaj yeem paub qhov txawv ntawm {{0}}N thiab 1N tau (Figures 10A, S7, thiab S8). Qhov tseeb tias peb saib cov spectral siab suav rau 0N (86 PrSMs) thiab 1N (91 PrSMs), tab sis tsis txhob saib ib qho PrSMs rau 2N, yog ua raws li qhov ntau ntawm kev tiv thaiv kab mob yav dhau los uas pom0N thiab 1N ua cov ntawv tseem ceeb thiab ua ∼ 91% ntawm tag nrho tau thaum 2N suav nrog ∼ 9% 87
Hais txog qhov kev ua haujlwm tsis meej ntawm microtubule-binding rov ua dua, peb tau pom cov spectra uas tuaj yeem muab rau ob qho tib si 3R (Figures 10A thiab S9) thiab 4R tau (Figures 10A thiab S10) ib yam nkaus.
Parsimonious inference tau tso cai rau peb los txiav txim siab tias braintau feem ntau sawv cev los ntawm kev sib xyaw ntawm 1N3R (Tau-B) ua raws los ntawm 0N4R (Tau-D) splicevariants, uas yog pom zoo nrog kev soj ntsuam yav dhau los.87,88
Thaum lwm cov splice isoformsal kuj muaj cov khoom tawg yooj yim, qhov tsawg ntawm cov spectral ntais ntawv (<4) did not allow us to confidently conclude about their existence.
Kuj ceeb tias, N-termini ntawm cov tseem ceeb R-domain uas muaj cov khoom tawg yog txuas ntxiv los ntawm C-termini ntawm qhov tseem ceeb N-domain uas muaj cov seem, los yog lwm lo lus, lawv zoo li cov khoom sib txuas uas tsim los ntawm kev tshwm sim proteolyticcleavage (hu ua. site #1 hauv daim duab 10A). Tsis tas li ntawd, C-termini ntawm 3Rand 4R domain-muaj proteoforms qhia tib qhov chaw cleavage (xa mus rau qhov chaw # 2 hauv daim duab 10A), txawm hais tias cov sequences lawv tus kheej txawv vim splice variation ntawm exon 10.
Txawm li cas los xij, qhov xav tsis thoob, tag nrho cov chaw cleavage no nyob hauv peb qhov kev piav qhia zoo dhexapeptide motifs.89–95 Daim duab 10B qhia tag nrho cov cim uas peb tau txheeb xyuas los ntawm peb cov ntaub ntawv teev tseg uas tuaj yeem kos rau 1N3R tau isoform, nrog rau qhov chaw ntawm qhov cleavage. qhov chaw.
Cleavage site #1 lies nyob rau hauv lub thib ob proline-nplua nuj cheeb tsam ntawm tau, uas faib cov KVAVVR hexapeptide ib theem zuj zus thiab muab ib tug ntawm cov muaj zog binding qhov chaw rau microtubules (Daim duab 10B).89–91 Cleavage site #2 lies nyob rau hauv lub 3R thiab 4R hexapeptide motifs VQIVYK thiab VQIINK, uas tau ua kom pom kev sib sau ua ke ntawm tau (Figure 10B).92–95
Interestingly, ntau ntawm cov fragments, raws li pom nyob rau hauv daim duab 10B, pib los yog xaus nyob rau hauv qhov sib thooj ntawm ob qhov chaw cleavage. Txhua yam ntawm cov hexapeptide motifs yog paub los tsim ib tug -structure, feem ntau nyob rau hauv daim ntawv ntawm -hairpins uas underlie lub aggregation koom nyob rau hauv ntau yam neurodegenerative kab mob.89,93,96 Intriguingly, cov kev soj ntsuam no qhia tau hais tias muaj peev xwm ntawm ib tug sib koom proteolytic degradation pathwayamong tau isoforms uas yog. muaj peev xwm cuam tshuam lawv cov aggregation-seedingregions.25,85,86
Thaum kawg, peb pom ntau qhov sib tw spectral rau A uas tuaj yeem pom tsuas yog siv FAIMS.
Cov A proteoforms no, uas feem ntau yuav tsum tau tshwj xeeb fractionation orhandling cov tswv yim los txhim kho kev rov qab los vim lawv cov hydrophobic thiab aggregation-prone properties, 9,16,97,98 tau pom zoo siv FAIMS nyob rau hauv −40 txog −50 CVrange.
Qhov no suav nrog cov canonical A 1–42 thiab A 1–40 (Daim duab 11A, B, feem), nrog rau ntau daim ntawv N-terminally truncated (tshwj xeeb A 2–42 thiab A 4–42, Daim duab 11C, D).
Nws yog ib nqi sau cia hais tias A proteoforms nrog ntau yam N- thiab C-terminal cleavages yog tsis yooj yim sua kom paub nyob rau hauv lub hauv qab-up tsom xam vim huab cua hydrophobicity ntawm tryptic cov khoom, uas qhia txog qhov zoo ntawm cov protein tsis zoo.
Peb ntseeg tias FAIMS-TDP txheej txheem muaj txoj hauv kev zoo rau kev ua tiav qhov kev txheeb xyuas tsis zoo ntawm cov A proteoforms, tso cai rau kev txiav txim siab sib txawv ntawm cov khoom sib txawv ntawm N thiab C-terminal cleavage khoom uas muaj nyob.
Cov lus xaus
Peb tau piav qhia txog yuav ua li cas nano-LC thim rov qab-theem sib cais ntawm cov qauv sib xyaw ua ke uas muaj cov protein ntau, dhau los ntawm ntau qhov loj, tuaj yeem tau txais txiaj ntsig los ntawm kev siv cov gasphase fractionation los ntawm FAIMS nyob rau hauv cov ntsiab lus ntawm sab saum toj-down mass spectrometry.
FAIMSwas tau pom tias muaj kev cuam tshuam rau kev sib kis ntawm cov proteoforms los ntawm qhov loj me thiab / lossis nqi, txo MS1 qhov nyuaj thiab tso cai rau qhov tob ntawm kev pab cuam ntawm proteome.
FAIMS ntawm asingle CV (−50) tau qhib qhov kev txheeb xyuas ntawm 1833 ± 17 cim proteoforms ntawm qhov nruab nrab, ntau dua ob npaug piv rau tsis muaj FAIMS (754 ± 35). Qhov sib ntxiv ntawm FAIMS tsis tau ua rau muaj qhov tsis zoo ntawm kev tsim cov khoom lag luam thiab tseem nyob ze 20% RSD, uas yog piv nrog cov ntawv sau tsis pub dhau hauv qab.
Kev txo qis CV ntawm FAIMS tau sau tseg kom nce qhov molecular loj ntawm hom ion raug xa mus rau qhov ntsuas (qhov nruab nrab MWof ∼5 kDa ntawm −50 CV vs ∼15 kDa ntawm −20 CV), thiab kev hloov pauv ntawm CV kuj tau pom los cuam tshuam kev sib kis ntawm ib lub hnab ntawv proteoform tus nqi them lub xeev txawv.
Peb kuj tau txiav txim siab qhov sib xyaw ua ke ntawm CVs uas tuaj yeem tsim qhov loj tshaj plaws theoretical siab tshaj plaws ntawm proteoforms / noob lossis proteome sequence coverage. Piv rau peb qhov "Tsis Muaj FAIMS" cov ntaub ntawv teev tseg, sab nraud CV nce qib ntawm −50, −40, thiab −30 V tuaj yeem muaj ntau tshaj ob npaug ntawm cov cim proteoforms, cov noob tshwj xeeb, thiab cov kev pab cuam sib luag.
Nws tseem tsim nyog taw qhia tias peb txoj haujlwm tsuas yog tshawb nrhiav qhov CV me me uas peb xav tias zoo tshaj plaws peb txoj hauv kev TDP siv cov qauv nyuaj. Txij li thaum FAIMS tuaj yeem hloov kho tau yooj yim rau lwm txoj hauv kev, qhov kev daws teeb meem MS1 tsis tshua muaj kev soj ntsuam nrog Orbitrap analyzers tuaj yeem txhim kho ntawm kev txheeb xyuas cov proteoforms loj dua nyob rau hauv CV ntau yam uas peb tau sim, nrog rau dhau-20 V qhov twg loj dua proteoforms yuav pom.
Nws kuj tseem tuaj yeem pom tias yav tom ntej pawg ntsuas ntsuas ntsuas uas txhim kho ntawm qhov tau txais cov proteoforms loj dua thiab sib haum nrog FAIMS tuaj yeem tau txais txiaj ntsig los ntawm kev tshawb nrhiav CVs hauv qab −20, txuas mus rau qhov zoo voltage ntau.
Peb txoj haujlwm TDP tau tso cai rau peb txheeb xyuas thiab ua tus yam ntxwv tshwj xeeb proteoforms muab los ntawm cov noob nrog lub luag haujlwm paub hauv cov kab mob neurodegenerative.
Qhov no suav nrog kev txiav txim siab ntawm qhov tsis paub txog kev hloov pauv loj tam sim no nyob ze ntawm cov hlau khi ntawm -synuclein, uas tau siv los txheeb xyuas cov chaw muaj peev xwm ntawm cov hlau txuas hauv - thiab synuclein ib yam nkaus. PARK7 kuj tau pom tias tau hloov kho nrog succinyl groupon nws qhov chaw ua haujlwm Cys residue.
Peb muaj peev xwm paub qhov txawv ntawm tau fragments sib xws rau 0N, 1N, 3R, thiab 4R splice variant isoforms ib tus zuj zus thiab piav qhia txog qhov chaw tshiab proteolytic cleavage nyob rau hauv lossis ze rau ntau qhov sib sau ua ke-seeding hexapeptiderepeats.
Thaum kawg, FAIMS tau qhib qhov kev txheeb xyuas ntawm ntau qhov tsis zoo A proteoforms, suav nrog kev sib sau ua ke-nruab nrab A 1-42, tsis tas yuav tsum muaj kev faib ua feem ntau lossis kev ua kom huv.
Hauv cov ntsiab lus, peb ntseeg tias qhov sib ntxiv ntawm FAIMS los tshawb pom TDP yuav muab txoj hauv kev muaj zog thiab rov tsim dua tshiab rau kev nce cov proteome muaj rau kev tshawb nrhiav thiab kev ua tus cwj pwm.
Khoom siv ntxiv
Xa mus rau Web version ntawm PubMed Central rau cov khoom siv ntxiv.
TXOJ CAI
Cov kws sau ntawv xav ua tsaug rau Tao Liu thiab Richard Smith rau lawv cov lus qhia thiab kev sib tham muaj txiaj ntsig.
Qhov haujlwm no tau txais kev txhawb nqa los ntawm U01 AG061356 (PLDJ) thiab R01 AG015819 (DAB). Ib feem ntawm qhov kev tshawb fawb tau ua tiav siv EMSL (grid.436923.9), DOE Lub Chaw Haujlwm Saib Xyuas Kev Tshawb Fawb Kev Tshawb Fawb tau txhawb nqa los ntawm Biologicaland Environmental Research program. Graphical abstract thiab duab cov cuab tam nyob rau hauv daim duab 1 tau tsim sivBiorender.com.
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