Wu Enhui, Qiao Liang*
ISebe leKhemistri, iYunivesithi yaseFudan, eShanghai 200433, eChina
I-Microorganisms inxulumene ngokusondeleyo nezifo zabantu kunye nempilo. Indlela yokuqonda ukubunjwa koluntu lwe-microbial kunye nemisebenzi yabo ngumba omkhulu ekufuneka ufundwe ngokukhawuleza. Kwiminyaka yakutshanje, i-metaproteomics iye yaba yindlela ebalulekileyo yobugcisa yokufunda ukubunjwa kunye nomsebenzi we-microorganisms. Nangona kunjalo, ngenxa yobunzima kunye nokungafani okuphezulu kweesampulu zoluntu lwe-microbial, ukusetyenzwa kwesampulu, ukufunyanwa kwedatha ye-spectrometry kunye nohlalutyo lwedatha ibe yimiceli mngeni emithathu ngoku ejongene ne-metaproteomics. Kuhlalutyo lwe-metaproteomics, kudla ngokuba yimfuneko ukunyusa unyango lwangaphambili lweentlobo ezahlukeneyo zeesampulu kunye nokwamkela ukwahlukana kwe-microbial eyahlukeneyo, ukutyebisa, ukutsalwa kunye ne-lysis schemes. Ngokufana neproteome yohlobo olulodwa, iindlela zokufunyanwa kwedatha ye-mass spectrometry kwi-metaproteomics ziquka i-data-dependent acquisition (DDA) mode kunye ne-data-independent acquisition mode (DIA) mode. Imodi yokufumana idatha ye-DIA inokuqokelela ngokupheleleyo ulwazi lwepeptide yesampulu kwaye inamandla amakhulu ophuhliso. Nangona kunjalo, ngenxa yobunzima beesampulu ze-metaproteome, uhlalutyo lwayo lwedatha ye-DIA ibe yingxaki enkulu ethintela ukugubungela okunzulu kweemetaproteomics. Ngokumalunga nohlalutyo lwedatha, inyathelo elibaluleke kakhulu kukwakhiwa kwedatha yokulandelelana kweprotheyini. Ubungakanani kunye nokugqibeka kwedatha akukhona nje impembelelo enkulu kwinani lokuchongwa, kodwa kuchaphazela uhlalutyo kwiintlobo kunye namanqanaba okusebenza. Okwangoku, umgangatho wegolide wokwakhiwa kwedatha ye-metaproteome yi-database yokulandelelana kweprotheyini esekelwe kwi-metagenome. Kwangaxeshanye, indlela yokuhluza isiseko sesiseko sikawonke-wonke esekwe kukhangelo oluphinda-phindwayo nayo ibonakaliswe inexabiso elisebenzayo elinamandla. Ngokombono wezicwangciso ezithile zokuhlalutya idatha, iindlela zokuhlalutya idatha ye-DIA egxile kwi-peptide zithathe eyona nto iphambili. Ngokuphuhliswa kokufunda okunzulu kunye nobukrelekrele bokwenziwa, kuya kukhuthaza kakhulu ukuchaneka, ukugubungela kunye nesantya sohlalutyo lohlalutyo lwedatha ye-macroproteomic. Ngokumalunga nohlalutyo lwe-bioinformatics esezantsi, uchungechunge lwezixhobo zokuchaza luye lwaphuhliswa kwiminyaka yamuva nje, olunokwenza inkcazo yeentlobo kwinqanaba leprotheni, inqanaba le-peptide kunye nenqanaba lemfuza ukufumana ukubunjwa koluntu lwe-microbial. Xa kuthelekiswa nezinye iindlela ze-omics, uhlalutyo olusebenzayo lwee-microbial community luphawu olulodwa lwe-macroproteomics. I-Macroproteomics iye yaba yinxalenye ebalulekileyo yohlalutyo lwe-multi-omics yoluntu lwe-microbial, kwaye isenamandla amakhulu ophuhliso malunga nobunzulu bokugubungela, ukufumanisa uvakalelo, kunye nokuphelela kohlalutyo lwedatha.
01Isampuli yonyango lwangaphambili
Okwangoku, itekhnoloji ye-metaproteomics isetyenziswe ngokubanzi kuphando lwe-microbiome yabantu, umhlaba, ukutya, ulwandle, i-sludge esebenzayo kunye nezinye iindawo. Xa kuthelekiswa nohlalutyo lweproteome yohlobo olunye, iisampulu yonyango lwangaphambili lwemetaproteome yeesampulu ezintsonkothileyo ijongene nemingeni eyongezelelekileyo. Ukubunjwa kwe-microbial kwiisampuli zangempela kuyinkimbinkimbi, uluhlu oluguquguqukayo lobuninzi likhulu, isakhiwo sodonga lweeseli zeentlobo ezahlukeneyo ze-microorganisms zihluke kakhulu, kwaye iisampuli zihlala ziqulethe inani elikhulu leeprotheni zomkhosi kunye nezinye izinto ezingcolileyo. Ngoko ke, kuhlalutyo lwe-metaproteome, kudla ngokufuneka ukuba kwandiswe iintlobo ezahlukeneyo zeesampulu kunye nokwamkela ukuhlukana kwe-microbial eyahlukeneyo, ukutyebisa, ukutsalwa kunye ne-lysis schemes.
Ukutsalwa kwe-microbial metaproteomes kwiisampulu ezahlukeneyo kunento efanayo kunye nomahluko othile, kodwa okwangoku kukho ukunqongophala kwenkqubo edibeneyo yokucubungula kwangaphambili kwiintlobo ezahlukeneyo zeesampulu zemetaproteome.
02Ukufumana idatha yespectrometry yobunzima
Kuhlalutyo lwe-shotgun proteome, umxube we-peptide emva konyango lwangaphambili uye wahlulwa kuqala kwikholamu yechromatographic, kwaye emva koko ungene kwi-mass spectrometer yokufumana idatha emva kwe-ionization. Ngokufana nohlalutyo lweproteome yohlobo olulodwa, iindlela zokufumana idatha ye-mass spectrometry kuhlalutyo lwe-macroproteome ziquka imo ye-DDA kunye ne-DIA mode.
Ngokuphindaphinda ngokuqhubekayo kunye nohlaziyo lwezixhobo ze-mass spectrometry, izixhobo ze-mass spectrometry ezinovakalelo oluphezulu kunye nesisombululo zisetyenziswa kwi-metaproteome, kwaye ubunzulu bokugubungela uhlalutyo lwe-metaproteome bubuye buphuculwe ngokuqhubekayo. Ixesha elide, uthotho lwezixhobo ze-spectrometry zobunzima obuphezulu ezikhokelwa yi-Orbitrap zisetyenziswe ngokubanzi kwi-metaproteome.
Itheyibhile 1 yombhalo wokuqala ibonisa ezinye izifundo ezimele kwi-metaproteomics ukusuka kwi-2011 ukuza kuthi ga ngoku malunga nohlobo lwesampula, isicwangciso sokuhlalutya, isixhobo se-mass spectrometry, indlela yokufumana, isofthiwe yokuhlalutya, kunye nenani lokuchongwa.
03Uhlalutyo lwedatha ye-spectrometry yobunzima
3.1 Isicwangciso sokuhlalutya idatha ye-DDA
3.1.1 Ukukhangela kwiDatabase
3.1.2de novoiqhinga lokulandelelana
3.2 Isicwangciso sokuhlalutya idatha ye-DIA
04Ukuhlelwa kweentlobo kunye nenkcazo yokusebenza
Ukubunjwa koluntu lwe-microbial kumanqanaba ahlukeneyo e-taxonomic yenye yeendawo eziphambili zophando kuphando lwe-microbiome. Kwiminyaka yakutshanje, uthotho lwezixhobo zokuchaza luye lwaphuhliswa ukucacisa iintlobo kwinqanaba leprotheyini, inqanaba le-peptide, kunye nenqanaba lemfuza ukufumana ukubunjwa koluntu lwe-microbial.
Ingundoqo yenkcazo esebenzayo kukuthelekisa ulandelelwano lweprotheyini ekujoliswe kuyo kunye nesiseko sedatha yeprotheyini esebenzayo. Ukusebenzisa i-database ye-gene function ezifana ne-GO, i-COG, i-KEGG, i-eggNOG, njl., uhlalutyo oluhlukeneyo lwe-annotation lunokwenziwa kwiiprotheni ezichongiweyo ngama-macroproteomes. Izixhobo zochazo ziquka iBlast2GO, DAVID, KOBAS, njl.
05Isishwankathelo kunye ne-Outlook
Ii-Microorganisms zidlala indima ebalulekileyo kwimpilo yabantu nakwizifo. Kwiminyaka yakutshanje, i-metaproteomics ibe yindlela ebalulekileyo yobugcisa yokufunda umsebenzi we-microbial community. Inkqubo yokuhlalutya ye-metaproteomics ifana neyodwa yeproteomics, kodwa ngenxa yobunzima bento yophando ye-metaproteomics, izicwangciso ezithile zophando kufuneka zamkelwe kwinqanaba ngalinye lokuhlalutya, ukusuka kwisampuli yonyango, ukufumana idatha ukuya kuhlalutyo lwedatha. Okwangoku, ngenxa yokuphuculwa kweendlela zonyango, ukusungulwa okuqhubekayo kobuchwepheshe be-mass spectrometry kunye nophuhliso olukhawulezayo lwe-bioinformatics, i-metaproteomics yenze inkqubela phambili enkulu kubunzulu bokuchonga kunye nomda wokusetyenziswa.
Kwinkqubo yonyango lwangaphambili lweesampuli ze-macroproteome, uhlobo lwesampuli kufuneka luqwalaselwe kuqala. Indlela yokwahlula i-microorganisms kwiiseli zokusingqongileyo kunye neeprotheni enye yeengxaki eziphambili ezijongene ne-macroproteomes, kwaye ukulinganisela phakathi kokusebenza kakuhle kokuhlukana kunye nokulahlekelwa kwe-microbial yingxaki engxamisekileyo yokusombulula. Okwesibini, i-protein extraction of microorganisms kufuneka ithathele ingqalelo iyantlukwano ebangelwa yi-heterogeneity yesakhiwo seebhaktheriya ezahlukeneyo. Iisampulu zeMacroproteome kuluhlu lomkhondo nazo zifuna iindlela ezithile zonyango lwangaphambili.
Ngokumalunga nezixhobo ze-mass spectrometry, izixhobo ze-mass spectrometry ezingundoqo ziye zatshintsha ukusuka kwi-spectrometers yobunzima ngokusekwe kuhlalutyi lobunzima lwe-Orbitrap njenge-LTQ-Orbitrap kunye ne-Q Exactive ukuya kwi-spectrometers enkulu esekwe kuhambo lwe-ion oludityaniswe nohlalutyi lobuninzi bexesha lokubhabha njenge-timsTOF Pro. . Uluhlu lwe-timsTOF lwezixhobo ezinolwazi lwe-ion mobility dimension lunokuchaneka okuphezulu kokuchongwa, umda wokubona ophantsi, kunye nokuphindaphinda okulungileyo. Ziye ngokuthe ngcembe zibe zizixhobo ezibalulekileyo kwiinkalo ezahlukeneyo zophando ezifuna ukubonwa kwe-mass spectrometry, njengeproteome, i-metaproteome, kunye ne-metabolome yohlobo olulodwa. Kuyaphawuleka ukuba ixesha elide, uluhlu oluguquguqukayo lwezixhobo ze-mass spectrometry luthintele ubunzulu bokhuseleko lweprotheyini yophando lwemetaproteome. Kwixesha elizayo, izixhobo ze-mass spectrometry ezinoluhlu olukhulu oluguquguqukayo lunokuphucula uvakalelo kunye nokuchaneka kokuchongwa kweprotheyini kwiimetaproteomes.
Ukufumana idatha ye-mass spectrometry, nangona imowudi yokufumana idatha ye-DIA yamkelwe ngokubanzi kwiproteome yohlobo olulodwa, uhlalutyo lwangoku lwe-macroproteome lusasebenzisa indlela yokufumana idatha ye-DDA. Imodi yokufumana idatha ye-DIA inokufumana ngokupheleleyo ulwazi lwe-ion yesampulu, kwaye xa kuthelekiswa nemodi yokufumana idatha ye-DDA, inokukwazi ukufumana ngokupheleleyo ulwazi lwe-peptide yesampuli ye-macroproteome. Nangona kunjalo, ngenxa yobunzima obuphezulu bedatha ye-DIA, uhlalutyo lwedatha ye-DIA macroproteome lusajongene nobunzima obukhulu. Ukuphuhliswa kwengqondo yokwenziwa kunye nokufunda okunzulu kulindeleke ukuba kuphuculwe ukuchaneka nokugqibelela kohlalutyo lwedatha ye-DIA.
Kuhlalutyo lwedatha ye-metaproteomics, elinye lamanyathelo aphambili kukwakhiwa kwedatha yokulandelelana kweprotheyini. Kwiindawo zophando ezidumileyo ezifana neentyantyambo zamathumbu, i-intestinal microbial databases ezifana ne-IGC kunye ne-HMP zingasetyenziswa, kwaye iziphumo ezilungileyo zokuchonga zifunyenwe. Kuninzi lolunye uhlalutyo lwe-metaproteomics, esona sicwangciso sisebenzayo sokwakhiwa kwesiseko sedatha kusekuseka isiseko sedatha yokulandelelana kweprotheyini ethile esekwe kwidatha yokulandelelana kwemetagenomic. Kwiisampulu zoluntu lwe-microbial ezinobunzima obuphezulu kunye noluhlu oluguquguqukayo olukhulu, kuyafuneka ukuba kwandiswe ubunzulu bolandelelwano ukunyusa ukuchongwa kweentlobo eziphantsi kobuninzi, ngaloo ndlela kuphuculwe ukugqunywa kwesiseko sedatha yeprotein. Xa ulandelelwano lwedatha lunqongophele, indlela yokukhangela ephindaphindwayo ingasetyenziselwa ukwandisa isiseko sedatha yoluntu. Nangona kunjalo, ukukhangela okuphindaphindiweyo kunokuchaphazela ulawulo lwekhwalithi ye-FDR, ngoko ke iziphumo zokukhangela kufuneka zihlolwe ngononophelo. Ukongeza, ukusetyenziswa kweemodeli zemveli zokulawula umgangatho we-FDR kuhlalutyo lwe-metaproteomics kusafanele ukuphononongwa. Ngokubhekiselele kwisicwangciso sokukhangela, isicwangciso selayibrari ye-hybrid spectral inokuphucula ubunzulu bogubungela i-DIA metaproteomics. Kwiminyaka yakutshanje, ithala leencwadi eliqikelelweyo elenziwe ngokusekwe kufundo olunzulu libonise ukusebenza okuphezulu kwi-DIA proteomics. Nangona kunjalo, i-database ye-metaproteome ihlala iqulethe izigidi zeeprotheyini ezingeniswayo, ezikhokelela kumlinganiselo omkhulu weelayibrari ze-spectral eziqikelelweyo, zisebenzisa izixhobo ezininzi zekhompyutha, kwaye ziphumela kwindawo enkulu yokukhangela. Ukongeza, ukufana phakathi kokulandelelana kweprotheyini kwi-metaproteomes kuyahluka kakhulu, okwenza kube nzima ukuqinisekisa ukuchaneka kwemodeli yethala leencwadi le-spectral, ngoko ke iilayibrari ze-spectral eziqikelelweyo azizange zisetyenziswe ngokubanzi kwi-metaproteomics. Ukongeza, iiprotheyini ezintsha zokungeniswa kunye nezicwangciso zenkcazo yokuhlelwa kufuneka ziphuhliswe ukuze zisetyenziswe kuhlalutyo lwe-metaproteomics yeeprotheyini ezilandelelana kakhulu.
Isishwankathelo, njengetekhnoloji yophando lwe-microbiome esakhulayo, itekhnoloji ye-metaproteomics izuze iziphumo ezibalulekileyo zophando kwaye inamandla amakhulu ophuhliso.
Ixesha lokuposa: Aug-30-2024