Wu Enhui, Qiao Liang*
Department of Chemistry, Fudan University, Shanghai 200433, Suav
Cov kab mob microorganisms zoo sib xws rau tib neeg cov kab mob thiab kev noj qab haus huv. Yuav ua li cas nkag siab txog qhov muaj pes tsawg leeg ntawm cov zej zog microbial thiab lawv txoj haujlwm yog qhov teeb meem loj uas yuav tsum tau kawm sai sai. Nyob rau hauv xyoo tas los no, metaproteomics tau dhau los ua ib qho tseem ceeb ntawm kev kawm txog kev muaj pes tsawg leeg thiab kev ua haujlwm ntawm cov kab mob. Txawm li cas los xij, vim qhov nyuaj thiab siab heterogeneity ntawm microbial cov qauv hauv zej zog, cov qauv ua qauv, cov ntaub ntawv loj spectrometry tau txais thiab kev txheeb xyuas cov ntaub ntawv tau dhau los ua peb qhov teeb meem loj tam sim no ntsib los ntawm metaproteomics. Hauv kev txheeb xyuas metaproteomics, feem ntau yuav tsum tau ua kom zoo dua qhov kev kho mob ntawm cov qauv sib txawv thiab txais kev sib cais microbial sib txawv, txhawb nqa, rho tawm thiab lysis schemes. Zoo ib yam li cov proteome ntawm ib hom, qhov loj spectrometry cov ntaub ntawv nrhiav tau hom hauv metaproteomics suav nrog cov ntaub ntawv-dependent acquisition (DDA) hom thiab cov ntaub ntawv-yooj yim nrhiav tau (DIA) hom. DIA cov ntaub ntawv tau txais hom tuaj yeem sau tag nrho cov ntaub ntawv peptide ntawm tus qauv thiab muaj peev xwm txhim kho tau zoo. Txawm li cas los xij, vim yog qhov nyuaj ntawm cov qauv metaproteome, nws cov ntaub ntawv DIA tsom xam tau dhau los ua qhov teeb meem loj uas cuam tshuam qhov sib sib zog nqus ntawm metaproteomics. Nyob rau hauv cov nqe lus ntawm kev txheeb xyuas cov ntaub ntawv, cov kauj ruam tseem ceeb tshaj plaws yog kev tsim kho cov ntaub ntawv protein ntau. Qhov loj thiab ua tiav ntawm cov ntaub ntawv tsis tsuas yog muaj kev cuam tshuam zoo rau cov naj npawb ntawm cov cim, tab sis kuj cuam tshuam rau kev txheeb xyuas ntawm hom thiab qib ua haujlwm. Tam sim no, tus qauv kub rau kev tsim kho cov ntaub ntawv metaproteome yog cov ntaub ntawv protein ntau raws li cov metagenome. Nyob rau tib lub sijhawm, pej xeem cov ntaub ntawv lim dej raws li kev tshawb nrhiav rov qab kuj tau raug pov thawj tias muaj txiaj ntsig zoo. Los ntawm qhov kev xav ntawm cov tswv yim kev tsom xam cov ntaub ntawv tshwj xeeb, peptide-centered DIA cov ntaub ntawv tsom xam txoj hauv kev tau nyob hauv qhov tseem ceeb. Nrog rau txoj kev loj hlob ntawm kev kawm sib sib zog nqus thiab kev txawj ntse, nws yuav txhawb nqa qhov tseeb, kev pab cuam thiab kev soj ntsuam ceev ntawm cov ntaub ntawv macroproteomic tsom xam. Nyob rau hauv cov nqe lus ntawm downstream bioinformatics tsom xam, ib tug series ntawm annotation cov cuab yeej tau tsim nyob rau hauv lub xyoo tsis ntev los no, uas muaj peev xwm ua tau hom annotation nyob rau hauv cov protein, qib peptide thiab qib noob kom tau cov muaj pes tsawg leeg ntawm microbial zej zog. Piv nrog rau lwm txoj hauv kev omics, kev tsom xam ua haujlwm ntawm cov zej zog microbial yog qhov tshwj xeeb ntawm macroproteomics. Macroproteomics tau dhau los ua ib feem tseem ceeb ntawm ntau qhov kev tshuaj ntsuam xyuas ntawm cov zej zog microbial, thiab tseem muaj peev xwm txhim kho tau zoo nyob rau hauv cov nqe lus ntawm kev pab cuam qhov tob, kev tshawb nrhiav rhiab heev, thiab kev tshawb xyuas cov ntaub ntawv tiav.
01 Sample pretreatment
Tam sim no, metaproteomics thev naus laus zis tau siv dav hauv kev tshawb fawb ntawm tib neeg microbiome, av, zaub mov, dej hiav txwv, sludge nquag thiab lwm yam. Piv nrog rau kev tsom xam proteome ntawm ib hom, cov qauv pretreatment ntawm metaproteome ntawm cov qauv nyuaj ntsib teeb meem ntau dua. Cov microbial muaj pes tsawg leeg nyob rau hauv cov qauv tiag tiag yog complex, dynamic ntau ntawm abundance yog loj, cell phab ntsa qauv ntawm ntau hom kab mob sib txawv heev, thiab cov qauv feem ntau muaj ib tug loj npaum li cas ntawm cov proteins thiab lwm yam impurities. Yog li ntawd, nyob rau hauv kev tsom xam ntawm metaproteome, nws yog ib qhov tsim nyog yuav tau optimize ntau hom qauv thiab txais yuav txawv microbial sib cais, enrichment, extraction thiab lysis schemes.
Kev rho tawm ntawm microbial metaproteomes los ntawm cov qauv sib txawv muaj qee qhov zoo sib xws nrog rau qee qhov sib txawv, tab sis tam sim no tsis muaj cov txheej txheem ua ntej ua ntej rau ntau hom metaproteome qauv.
02Mass spectrometry cov ntaub ntawv tau txais
Hauv kev tsom xam cov phom phom proteome, cov peptide sib tov tom qab pretreatment yog thawj zaug sib cais nyob rau hauv chromatographic kem, thiab ces nkag mus rau lub loj spectrometer rau cov ntaub ntawv tau tom qab ionization. Zoo ib yam li ib hom kev tshuaj ntsuam xyuas proteome, cov ntaub ntawv loj spectrometry tau txais hom hauv macroproteome tsom xam suav nrog DDA hom thiab DIA hom.
Nrog rau qhov txuas ntxiv thiab hloov tshiab ntawm cov cuab yeej spectrometry, huab hwm coj spectrometry cov cuab yeej nrog ntau dua rhiab heev thiab kev daws teeb meem yog siv rau metaproteome, thiab qhov kev pab cuam qhov tob ntawm metaproteome tsom xam kuj tseem txhim kho tas li. Rau lub sijhawm ntev, cov cuab yeej ua haujlwm siab loj spectrometry coj los ntawm Orbitrap tau siv dav hauv metaproteome.
Table 1 ntawm cov ntawv qub qhia txog qee cov kev tshawb fawb txog cov metaproteomics los ntawm 2011 txog rau tam sim no hais txog cov qauv qauv, kev tsom xam cov tswv yim, cov cuab yeej loj spectrometry, kev nrhiav tau, software tsom xam, thiab tus lej ntawm cov cim.
03Mass spectrometry cov ntaub ntawv tsom xam
3.1 DDA cov ntaub ntawv tsom xam cov tswv yim
3.1.1 Database Search
3.1.2de novsequence zoo
3.2 DIA cov ntaub ntawv tsom xam cov tswv yim
04 Hom kev faib tawm thiab kev ua haujlwm annotation
Kev sib xyaw ntawm cov zej zog microbial ntawm ntau theem taxonomic yog ib qho ntawm cov kev tshawb fawb tseem ceeb hauv kev tshawb fawb microbiome. Nyob rau hauv xyoo tas los no, ib tug series ntawm annotation cov cuab yeej tau tsim los piav txog hom ntawm cov protein, qib peptide, thiab qib noob kom tau txais cov kab ke ntawm cov zej zog microbial.
Lub ntsiab lus tseem ceeb ntawm kev ua haujlwm yog los sib piv cov hom phiaj protein ib ntus nrog cov ntaub ntawv ua haujlwm protein ntau. Siv cov gene muaj nuj nqi databases xws li GO, COG, KEGG, eggNOG, thiab lwm yam., sib txawv functional annotation soj ntsuam yuav ua tau rau cov proteins txheeb xyuas los ntawm macroproteomes. Cov cuab yeej piav qhia suav nrog Blast2GO, DAVID, KOBAS, thiab lwm yam.
05 Cov ntsiab lus thiab Outlook
Microorganisms ua lub luag haujlwm tseem ceeb hauv tib neeg kev noj qab haus huv thiab kab mob. Nyob rau hauv xyoo tas los no, metaproteomics tau dhau los ua ib qho tseem ceeb hauv kev kawm txog kev ua haujlwm ntawm cov zej zog microbial. Cov txheej txheem tshuaj ntsuam xyuas ntawm metaproteomics zoo ib yam li ib hom kab mob proteomics, tab sis vim yog qhov nyuaj ntawm kev tshawb fawb cov khoom ntawm metaproteomics, cov tswv yim kev tshawb fawb tshwj xeeb yuav tsum tau muab coj los siv hauv txhua kauj ruam kev tshuaj ntsuam, los ntawm cov qauv ua ntej, cov ntaub ntawv tau txais mus rau cov ntaub ntawv tsom xam. Tam sim no, ua tsaug rau kev txhim kho cov txheej txheem pretreatment, kev nruam ntej ntawm huab hwm coj spectrometry thev naus laus zis thiab kev loj hlob sai ntawm bioinformatics, metaproteomics tau ua tiav zoo hauv kev txheeb xyuas qhov tob thiab daim ntawv thov.
Nyob rau hauv tus txheej txheem ntawm kev kho ua ntej ntawm cov qauv macroproteome, qhov xwm ntawm tus qauv yuav tsum tau txiav txim siab ua ntej. Yuav ua li cas cais cov kab mob los ntawm ib puag ncig cov hlwb thiab cov proteins yog ib qho ntawm cov teeb meem tseem ceeb uas ntsib cov macroproteomes, thiab qhov sib npaug ntawm kev sib cais kev ua tau zoo thiab microbial poob yog qhov teeb meem ceev uas yuav tsum tau daws. Qhov thib ob, cov protein rho tawm ntawm cov kab mob yuav tsum tau coj mus rau hauv tus account qhov sib txawv los ntawm cov txheej txheem heterogeneity ntawm cov kab mob sib txawv. Cov qauv macroproteome hauv cov kab mob kuj tseem xav tau cov kev kho mob ua ntej.
Nyob rau hauv cov nqe lus ntawm huab hwm coj spectrometry instruments, mainstream mass spectrometry instruments tau hloov pauv los ntawm huab hwm coj spectrometers raws li Orbitrap mass analyzers xws li LTQ-Orbitrap thiab Q Exactive rau huab hwm coj spectrometers raws li ion mobility ua ke nrog lub sij hawm ntawm-flight huab cua analyzers xws li timsTOF Pro. . TimsTOF series ntawm cov twj paj nruag nrog cov ntaub ntawv ion txav mus los muaj cov ntaub ntawv muaj txiaj ntsig zoo, tsis tshua pom kev txwv, thiab rov ua tau zoo. Lawv tau maj mam dhau los ua cov cuab yeej tseem ceeb hauv ntau yam kev tshawb fawb uas yuav tsum tau kuaj pom ntau yam, xws li proteome, metaproteome, thiab metabolome ntawm ib hom tsiaj. Nws yog ib nqi sau cia hais tias nyob rau hauv lub sij hawm ntev, lub dynamic ntau yam ntawm huab hwm coj spectrometry ntsuas tau txwv cov protein kev pab cuam qhov tob ntawm metaproteome kev tshawb fawb. Nyob rau hauv lub neej yav tom ntej, huab hwm coj spectrometry cov cuab yeej nrog ib tug loj dynamic ntau yam yuav ua tau kom lub rhiab heev thiab raug ntawm cov protein qhia nyob rau hauv metaproteomes.
Rau cov ntaub ntawv loj spectrometry tau, txawm hais tias DIA cov ntaub ntawv tau txais hom tau txais kev pom zoo nyob rau hauv proteome ntawm ib hom, feem ntau tam sim no macroproteome tsom xam tseem siv DDA cov ntaub ntawv nrhiav tau hom. DIA cov ntaub ntawv tau txais hom tuaj yeem tau txais cov ntaub ntawv tawg ion ntawm cov qauv, thiab piv nrog DDA cov ntaub ntawv tau txais hom, nws muaj peev xwm kom tau txais cov ntaub ntawv peptide ntawm cov qauv macroproteome. Txawm li cas los xij, vim qhov nyuaj ntawm DIA cov ntaub ntawv, kev tshuaj xyuas ntawm DIA macroproteome cov ntaub ntawv tseem tab tom ntsib teeb meem loj. Kev txhim kho ntawm kev txawj ntse txawj ntse thiab kev kawm tob yuav tsum txhim kho qhov raug thiab ua tiav ntawm DIA cov ntaub ntawv txheeb xyuas.
Hauv kev txheeb xyuas cov ntaub ntawv ntawm metaproteomics, ib qho ntawm cov kauj ruam tseem ceeb yog kev tsim cov protein ntau cov ntaub ntawv. Rau cov chaw tshawb fawb nrov xws li cov hnyuv, cov hnyuv microbial databases xws li IGC thiab HMP tuaj yeem siv, thiab cov txiaj ntsig zoo tau ua tiav. Rau feem ntau lwm yam kev tshuaj ntsuam metaproteomics, qhov zoo tshaj plaws database tsim lub tswv yim tseem yog los tsim kom tau ib qho piv txwv tshwj xeeb protein sequence database raws li metagenomic sequencing cov ntaub ntawv. Rau cov qauv hauv zej zog microbial nrog kev nyuaj siab thiab loj dynamic ntau yam, nws yog ib qho tsim nyog yuav tsum tau ua kom qhov sib npaug ntawm qhov tob kom nce qhov kev txheeb xyuas ntawm cov tsiaj tsis tshua muaj, yog li kev txhim kho cov kev pab them nqi ntawm cov protein ib ntus database. Thaum tsis muaj cov ntaub ntawv sib txuas, ib txoj kev tshawb nrhiav rov qab tuaj yeem siv los txhim kho cov ntaub ntawv pej xeem. Txawm li cas los xij, kev tshawb nrhiav rov qab tuaj yeem cuam tshuam rau FDR kev tswj hwm zoo, yog li cov txiaj ntsig tshawb fawb yuav tsum tau ua tib zoo xyuas. Tsis tas li ntawd, kev siv tau ntawm cov qauv FDR zoo tswj cov qauv hauv metaproteomics tsom xam tseem tsim nyog tshawb nrhiav. Nyob rau hauv cov nqe lus ntawm kev tshawb fawb lub tswv yim, hybrid spectral lub tsev qiv ntawv zoo tuaj yeem txhim kho qhov tob ntawm DIA metaproteomics. Nyob rau hauv xyoo tas los no, lub tsev qiv ntawv spectral kwv yees uas tsim los ntawm kev kawm tob tau qhia txog kev ua tau zoo hauv DIA proteomics. Txawm li cas los xij, cov ntaub ntawv metaproteome feem ntau muaj ntau lab cov protein nkag, uas ua rau muaj qhov loj ntawm cov tsev qiv ntawv spectral uas tau kwv yees, siv ntau cov ntaub ntawv suav, thiab ua rau muaj qhov chaw tshawb nrhiav loj. Tsis tas li ntawd, qhov zoo sib xws ntawm cov protein nyob rau hauv metaproteomes txawv heev, ua rau nws nyuaj los xyuas kom meej qhov tseeb ntawm lub tsev qiv ntawv spectral kwv yees qauv, yog li kwv yees spectral tsev qiv ntawv tsis tau dav siv nyob rau hauv metaproteomics. Tsis tas li ntawd, cov txheej txheem tshiab ntawm cov protein thiab kev faib tawm cov ntsiab lus yuav tsum tau tsim los siv rau kev txheeb xyuas metaproteomics ntawm cov protein zoo sib xws.
Hauv cov ntsiab lus, raws li kev tshawb fawb microbiome technology, metaproteomics thev naus laus zis tau ua tiav cov txiaj ntsig kev tshawb fawb tseem ceeb thiab tseem muaj peev xwm loj hlob.
Post lub sij hawm: Aug-30-2024