Wu Enhui, Qiao Liang*
Umnyango Wezekhemikhali, iFudan University, Shanghai 200433, China
Ama-Microorganisms ahlobene eduze nezifo nempilo yabantu. Indlela yokuqonda ukubunjwa kwemiphakathi yamagciwane kanye nemisebenzi yawo kuyindaba enkulu edinga ukufundwa ngokuphuthumayo. Eminyakeni yamuva nje, i-metaproteomics isiphenduke indlela yobuchwepheshe ebalulekile yokufunda ukwakheka nokusebenza kwama-microorganisms. Kodwa-ke, ngenxa yobunkimbinkimbi kanye nokuhlukahluka okuphezulu kwamasampula omphakathi we-microbial, ukucubungula isampula, ukutholwa kwedatha ye-mass spectrometry kanye nokuhlaziywa kwedatha kube yizinselele ezintathu ezinkulu okwamanje ezibhekene ne-metaproteomics. Ekuhlaziyweni kwe-metaproteomics, kuvame ukudingekile ukuthuthukisa ukwelashwa kusengaphambili kwezinhlobo ezahlukene zamasampuli futhi kwamukele ukuhlukaniswa kwe-microbial ehlukene, ukunothisa, ukukhishwa kanye nezikimu ze-lysis. Ngokufana ne-proteome yohlobo olulodwa, izindlela zokutholwa kwedatha ye-spectrometry mass kuma-metaproteomics ahlanganisa imodi yokutholwa okuncike kudatha (i-DDA) kanye nemodi yokutholwa kwedatha ezimele (DIA). Imodi yokutholwa kwedatha ye-DIA ingaqoqa ngokuphelele ulwazi lwe-peptide lwesampula futhi inamandla amakhulu okuthuthuka. Kodwa-ke, ngenxa yobunkimbinkimbi bamasampula e-metaproteome, ukuhlaziya idatha yayo ye-DIA sekuyinkinga enkulu evimbela ukumbozwa okujulile kwe-metaproteomics. Mayelana nokuhlaziywa kwedatha, isinyathelo esibaluleke kakhulu ukwakhiwa kwesizindalwazi sokulandelana kwamaprotheni. Ubukhulu nokuphelela kwesizindalwazi akugcini nje ngokuba nomthelela omkhulu enanini lokuhlonza, kodwa futhi kunomthelela ekuhlaziyweni kwezinhlobo zezilwane namazinga okusebenza. Njengamanje, izinga legolide lokwakhiwa kwesizindalwazi se-metaproteome yisizindalwazi sokulandelana kwamaprotheni esisekelwe ku-metagenome. Ngesikhathi esifanayo, indlela yokuhlunga yesizindalwazi somphakathi esekwe ekusesheni okuphindaphindwayo nayo ifakazelwe ukuthi inenani elisebenzayo elinamandla. Ngokombono wamasu athile okuhlaziya idatha, izindlela zokuhlaziya idatha ye-DIA egxile ku-peptide zithathe okujwayelekile ngokuphelele. Ngokuthuthukiswa kokufunda okujulile kanye nobuhlakani bokwenziwa, kuzokhuthaza kakhulu ukunemba, ukuhlanganisa kanye nesivinini sokuhlaziya sokuhlaziywa kwedatha ye-macroproteomic. Mayelana nokuhlaziywa kwe-bioinformatics engezansi, uchungechunge lwamathuluzi esichasiselo lwenziwe eminyakeni yamuva nje, olungenza isichasiselo sezinhlobo zezilwane ezingeni lamaphrotheni, izinga le-peptide kanye neleveli yofuzo ukuze kutholwe ukubunjwa kwemiphakathi yamagciwane. Uma kuqhathaniswa nezinye izindlela ze-omics, ukuhlaziya okusebenzayo kwemiphakathi yamagciwane kuyisici esiyingqayizivele se-macroproteomics. I-Macroproteomics isiyingxenye ebalulekile yokuhlaziywa kwama-multi-omics emiphakathini yamagciwane, futhi isenamandla amakhulu okuthuthuka mayelana nokujula kokufakwa, ukuzwela kokutholwa, kanye nokuphelela kokuhlaziywa kwedatha.
01 Isampula yokwelashwa
Njengamanje, ubuchwepheshe be-metaproteomics busetshenziswe kabanzi ocwaningweni lwe-microbiome yomuntu, inhlabathi, ukudla, ulwandle, udaka olusebenzayo neminye imikhakha. Uma kuqhathaniswa nokuhlaziywa kwe-proteome yohlobo olulodwa, isampula yokwelashwa kwangaphambili kwe-metaproteome yamasampuli ayinkimbinkimbi ibhekana nezinselele ezengeziwe. Ukubunjwa kwe-microbial kumasampuli wangempela kuyinkimbinkimbi, ububanzi obuguquguqukayo benala bukhulu, ukwakheka kodonga lwamaseli ezinhlobo ezahlukene zama-microorganisms kuhluke kakhulu, futhi amasampula ngokuvamile aqukethe inani elikhulu lamaprotheni abamba kanye nokunye ukungcola. Ngakho-ke, ekuhlaziyweni kwe-metaproteome, ngokuvamile kudingekile ukwandisa izinhlobo ezahlukene zamasampuli futhi kwamukele ukuhlukaniswa kwe-microbial ehlukene, ukucebisa, ukukhishwa kanye nezikimu ze-lysis.
Ukukhishwa kwama-microbial metaproteomes kumasampuli ahlukene kunokufana okuthile kanye nomehluko othile, kodwa okwamanje kukhona ukuntuleka kwenqubo ehlanganisiwe yokucubungula kusengaphambili yezinhlobo ezahlukene zamasampuli e-metaproteome.
02Ukutholwa kwedatha ye-spectrometry enkulu
Ekuhlaziyweni kwe-shotgun proteome, ingxube ye-peptide ngemva kokwelashwa kusengaphambili ihlukaniswa kuqala kukholamu ye-chromatographic, bese ingena ku-spectrometer enkulu ukuze kutholwe idatha ngemva kwe-ionization. Ngokufana nokuhlaziywa kwe-proteome yohlobo olulodwa, izindlela zokuthola idatha ye-spectrometry enkulu ekuhlaziyeni kwe-macroproteome zihlanganisa imodi ye-DDA kanye nemodi ye-DIA.
Ngokuphindaphinda okuqhubekayo kanye nokubuyekezwa kwamathuluzi e-mass spectrometry, amathuluzi e-mass spectrometry anokuzwela okuphezulu nokulungiswa asetshenziswa ku-metaproteome, futhi ukujula kokufakwa kokuhlaziywa kwe-metaproteome nakho kuthuthukiswa ngokuqhubekayo. Isikhathi eside, uchungechunge lwamathuluzi e-high-resolution mass spectrometry aphethwe yi-Orbitrap asetshenziswe kabanzi ku-metaproteome.
Ithebula 1 lombhalo wangempela libonisa ezinye izifundo ezimele i-metaproteomics kusukela ngo-2011 kuze kube manje mayelana nohlobo lwesampula, isu lokuhlaziya, ithuluzi le-mass spectrometry, indlela yokutholwa, isofthiwe yokuhlaziya, nenombolo yokuhlonza.
03Ukuhlaziywa kwedatha ye-spectrometry yobuningi
3.1 Isu lokuhlaziya idatha ye-DDA
3.1.1 Usesho Lwesizindalwazi
3.1.2de novoisu lokulandelanisa
3.2 Isu lokuhlaziya idatha ye-DIA
04Ukuhlukaniswa kwezinhlobo zezilwane nesichasiselo esisebenzayo
Ukubunjwa kwemiphakathi ye-microbial emazingeni ahlukene e-taxonomic ingenye yezindawo zocwaningo ezibalulekile ocwaningweni lwe-microbiome. Eminyakeni yakamuva, uchungechunge lwamathuluzi esichasiselo luye lwathuthukiswa ukuze luchaze izinhlobo zezilwane ezingeni lamaphrotheni, izinga le-peptide, kanye nezinga lofuzo ukuze kutholwe ukubunjwa kwemiphakathi yamagciwane.
Ingqikithi yesichasiselo esisebenzayo iwukuqhathanisa ukulandelana kwephrotheni okuqondiwe nesizindalwazi sokulandelana kwamaprotheni. Kusetshenziswa imininingo egciniwe yokusebenza kofuzo efana ne-GO, COG, KEGG, eggNOG, njll., ukuhlaziya okuhlukile kokusebenza kwezichasiselo kungenziwa kumaphrotheni akhonjwe ama-macroproteome. Amathuluzi ezichasiselo afaka iBlast2GO, DAVID, KOBAS, njll.
05Isifinyezo kanye ne-Outlook
Ama-microorganisms adlala indima ebalulekile empilweni yabantu nasezifweni. Eminyakeni yamuva nje, i-metaproteomics isiphenduke indlela yezobuchwepheshe ebalulekile yokufunda umsebenzi wemiphakathi yamagciwane. Inqubo yokuhlaziya ye-metaproteomics ifana neye-proteomics yohlobo olulodwa, kodwa ngenxa yobunzima bento yocwaningo ye-metaproteomics, amasu athile ocwaningo adinga ukwamukelwa esinyathelweni ngasinye sokuhlaziya, kusukela ekusetshenzisweni kwangaphambili kwesampula, ukutholwa kwedatha kuya ekuhlaziyweni kwedatha. Njengamanje, ngenxa yokuthuthukiswa kwezindlela zokwelapha, ukusungulwa okusha okuqhubekayo kobuchwepheshe be-mass spectrometry kanye nokuthuthukiswa okusheshayo kwe-bioinformatics, i-metaproteomics yenze inqubekelaphambili enkulu ekujuleni kokuhlonza kanye nobubanzi bokusebenza.
Enqubweni yokwelashwa kwangaphambili kwamasampula e-macroproteome, uhlobo lwesampula kufanele lucatshangelwe kuqala. Indlela yokuhlukanisa ama-microorganisms kumangqamuzana emvelo namaprotheni kungenye yezinselelo ezibalulekile ezibhekene nama-macroproteomes, futhi ibhalansi phakathi kokusebenza kahle kokuhlukana nokulahlekelwa kwamagciwane kuyinkinga ephuthumayo okufanele ixazululwe. Okwesibili, ukukhishwa kwamaprotheni kwama-microorganisms kufanele kucabangele umehluko obangelwa ukuhlukahluka kwesakhiwo samabhaktheriya ahlukene. Amasampula e-Macroproteome kububanzi bokulandelela nawo adinga izindlela ezithile zokwelapha zangaphambi kokwelashwa.
Ngokuya ngamathuluzi e-mass spectrometry, amathuluzi e-mass spectrometry ajwayelekile abe nenguquko esuka kuma-spectrometer amaningi asekelwe kubahlaziyi besisindo se-Orbitrap njenge-LTQ-Orbitrap kanye ne-Q Exactive ukuya kuma-spectrometer amaningi asekelwe ekuhambeni kwe-ion okuhambisana nesikhathi sendiza esihlaziya isisindo njenge-timsTOF Pro. . Uchungechunge lwe-timsTOF lwamathuluzi anolwazi lobukhulu bokunyakaza kwe-ion banokunemba kokutholwa okuphezulu, umkhawulo wokutholwa ophansi, nokuphindaphinda okuhle. Kancane kancane ziye zaba amathuluzi abalulekile emikhakheni ehlukahlukene yocwaningo edinga ukutholwa kwe-spectrometry enkulu, njenge-proteome, i-metaproteome, ne-metabolome yohlobo olulodwa. Kuyaphawuleka ukuthi isikhathi eside, uhla oluguqukayo lwamathuluzi e-mass spectrometry lukhawulele ukujula kokufakwa kwamaprotheni ocwaningweni lwe-metaproteome. Ngokuzayo, amathuluzi e-mass spectrometry anobubanzi obukhulu obuguqukayo angathuthukisa ukuzwela nokunemba kokuhlonza amaprotheni kuma-metaproteome.
Ukuze uthole idatha ye-spectrometry enkulu, nakuba imodi yokutholwa kwedatha ye-DIA iye yamukelwa kabanzi ku-proteome yohlobo olulodwa, ukuhlaziya okuningi kwamanje kwe-macroproteome kusasebenzisa imodi yokutholwa kwedatha ye-DDA. Imodi yokutholwa kwedatha ye-DIA ingathola ngokugcwele ulwazi lwe-ion yesiqephu sesampula, futhi uma iqhathaniswa nemodi yokutholwa kwedatha ye-DDA, inamandla okuthola ngokugcwele ulwazi lwe-peptide lwesampula ye-macroproteome. Nokho, ngenxa yobunkimbinkimbi obukhulu bedatha ye-DIA, ukuhlaziya idatha ye-DIA macroproteome kusabhekene nobunzima obukhulu. Ukuthuthukiswa kobuhlakani bokwenziwa nokufunda okujulile kulindeleke ukuthi kuthuthukise ukunemba nokuphelela kokuhlaziywa kwedatha ye-DIA.
Ekuhlaziyeni idatha ye-metaproteomics, esinye sezinyathelo ezibalulekile ukwakhiwa kwesizindalwazi sokulandelana kwamaprotheni. Ezindaweni zocwaningo ezidumile ezifana nezitshalo zamathumbu, isizindalwazi se-microbial yamathumbu njenge-IGC ne-HMP singasetshenziswa, futhi imiphumela emihle yokuhlonza ifinyelelwe. Kokunye ukuhlaziya okuningi kwe-metaproteomics, isu elisebenza kahle kakhulu lokwakhiwa kwesizindalwazi kusewukusungula isampula yolwazi lokulandelana kwamaprotheni olususelwe kudatha yokulandelana kwe-metagenomic. Kumasampuli omphakathi wezinambuzane ezincane ezinobunzima obuphezulu kanye nobubanzi obukhulu obuguquguqukayo, kuyadingeka ukwandisa ukujula kokulandelana ukuze kwandiswe ukuhlonzwa kwezinhlobo ezinensada ephansi, ngaleyo ndlela kuthuthukiswe ukumbozwa kwesizindalwazi sokulandelana kwamaprotheni. Uma ukulandelana kwedatha kuntuleka, indlela yokusesha ephindaphindwayo ingasetshenziswa ukuthuthukisa isizindalwazi somphakathi. Kodwa-ke, ukusesha okuphindaphindiwe kungase kuthinte ukulawulwa kwekhwalithi ye-FDR, ngakho imiphumela yosesho idinga ukuhlolwa ngokucophelela. Ngaphezu kwalokho, ukusebenza kwamamodeli okulawula ikhwalithi ye-FDR okuvamile ekuhlaziyweni kwe-metaproteomics kusafanele ukuhlola. Ngokwesu lokusesha, isu le-hybrid spectral library lingathuthukisa ukujula kokufakwa kwe-DIA metaproteomics. Eminyakeni yamuva nje, umtapo wolwazi obikezelwe we-spectral owenziwe ngokusekelwe ekufundeni okujulile ubonise ukusebenza okuphakeme ku-DIA proteomics. Kodwa-ke, imininingwane egciniwe ye-metaproteome ivamise ukuqukatha izigidi zamaphrotheni okufakiwe, okuholela esikalini esikhulu semitapo yolwazi ye-spectral ebikezelwe, kudla izinsiza eziningi zekhompuyutha, futhi kuphumele esikhaleni esikhulu sokusesha. Ngaphezu kwalokho, ukufana phakathi kokulandelana kwamaprotheni kuma-metaproteomes kuyahlukahluka kakhulu, okwenza kube nzima ukuqinisekisa ukunemba kwemodeli yokubikezela umtapo we-spectral, ngakho-ke imitapo yolwazi ye-spectral ebikezelwe ayizange isetshenziswe kabanzi kuma-metaproteomics. Ukwengeza, i-protein inference entsha kanye namasu esichasiselo sezigaba kudingeka athuthukiswe ukuze asetshenziswe ekuhlaziyweni kwe-metaproteomics yamaprotheni alandelanayo kakhulu.
Kafushane, njengobuchwepheshe bocwaningo lwe-microbiome olusafufusa, ubuchwepheshe be-metaproteomics buzuze imiphumela ebalulekile yocwaningo futhi bunamandla amakhulu okuthuthuka.
Isikhathi sokuthumela: Aug-30-2024