Wu Enhui, Qiao Liang*
Dipatimenti ya Chemistry, Fudan University, Shanghai 200433, China
Tizilombo tating'onoting'ono timagwirizana kwambiri ndi matenda amunthu komanso thanzi. Momwe mungamvetsetse kapangidwe ka magulu a tizilombo tating'onoting'ono ndi ntchito zawo ndi nkhani yayikulu yomwe iyenera kuphunziridwa mwachangu. M'zaka zaposachedwa, metaproteomics yakhala njira yofunikira yaukadaulo yophunzirira kapangidwe ndi magwiridwe antchito a tizilombo. Komabe, chifukwa cha zovuta komanso kusiyanasiyana kwa zitsanzo zamagulu a tizilombo tating'onoting'ono, kukonza zitsanzo, kupeza ma data a spectrometry ndi kusanthula deta zakhala zovuta zazikulu zitatu zomwe metaproteomics ikukumana nazo. Pakuwunika kwa metaproteomics, nthawi zambiri kumakhala kofunikira kukhathamiritsa kuyambika kwa mitundu yosiyanasiyana ya zitsanzo ndikutengera njira zosiyanasiyana zolekanitsa tizilombo tating'onoting'ono, kukulitsa, kuchotsa ndi kupanga ma lysis. Mofanana ndi ma proteome amtundu umodzi, njira zopezera deta za mass spectrometry mu metaproteomics zimaphatikizapo njira yopezera deta (DDA) ndi njira yopezera deta (DIA). Njira yopezera deta ya DIA imatha kusonkhanitsa zonse za peptide yachitsanzo ndipo ili ndi kuthekera kwakukulu kwachitukuko. Komabe, chifukwa cha zovuta za zitsanzo za metaproteome, kusanthula kwake deta ya DIA kwakhala vuto lalikulu lomwe limalepheretsa kubisala kwakuya kwa metaproteomics. Pankhani ya kusanthula deta, sitepe yofunika kwambiri ndi yomanga ndondomeko ya ndondomeko ya mapuloteni. Kukula ndi kukwanira kwa deta sikumangokhudza kwambiri chiwerengero cha zizindikiritso, komanso kumakhudza kusanthula pa zamoyo ndi machitidwe ogwira ntchito. Pakalipano, muyeso wa golide womanga nkhokwe ya metaproteome ndi ndondomeko yotsatizana ya mapuloteni yotengera metagenome. Pa nthawi yomweyi, njira yosefera pagulu la anthu potengera kufufuza kobwerezabwereza yatsimikiziridwanso kuti ili ndi phindu lamphamvu. Kuchokera pamalingaliro a njira zowunikira deta, njira zowunikira ma data za DIA zokhala ndi peptide zakhala zodziwika bwino kwambiri. Ndi chitukuko cha kuphunzira mwakuya ndi luntha lochita kupanga, zidzalimbikitsa kwambiri kulondola, kufalitsa ndi kusanthula liwiro la kusanthula kwa deta ya macroproteomic. Pankhani ya kusanthula kwa bioinformatics, zida zofotokozera zidapangidwa m'zaka zaposachedwa, zomwe zimatha kulongosola zamitundu pamlingo wa mapuloteni, mulingo wa peptide ndi mulingo wa majini kuti apeze magulu a tizilombo tating'onoting'ono. Poyerekeza ndi njira zina za omics, kusanthula kwa magwiridwe antchito a magulu a tizilombo tating'onoting'ono ndi gawo lapadera la macroproteomics. Macroproteomics yakhala gawo lofunikira pakuwunika kwamitundu yambiri yamagulu ang'onoang'ono, ndipo akadali ndi kuthekera kwakukulu kwachitukuko pokhudzana ndi kuzama kwa kufalitsa, kuzindikira kuzindikira, komanso kukwanira kusanthula deta.
01 Zitsanzo zothandizira
Pakalipano, teknoloji ya metaproteomics yakhala ikugwiritsidwa ntchito kwambiri pofufuza za microbiome yaumunthu, nthaka, chakudya, nyanja, sludge yogwira ntchito ndi zina. Poyerekeza ndi kusanthula kwa proteome kwa mtundu umodzi, chitsanzo cha pretreatment cha metaproteome cha zitsanzo zovuta chimakumana ndi zovuta zambiri. The tizilombo tating'onoting'ono mu zitsanzo zenizeni ndi zovuta, amphamvu osiyanasiyana kuchuluka ndi lalikulu, maselo khoma dongosolo la mitundu yosiyanasiyana ya tizilombo tating'onoting'ono ndi osiyana kwambiri, ndipo zitsanzo nthawi zambiri amakhala ndi kuchuluka kwa khamu mapuloteni ndi zonyansa zina. Chifukwa chake, pakuwunika kwa metaproteome, nthawi zambiri ndikofunikira kukhathamiritsa mitundu yosiyanasiyana ya zitsanzo ndikutengera njira zolekanitsa tizilombo tating'onoting'ono, kulemeretsa, kuchotsa ndi ma lysis.
Kuchotsa ma metaproteomes ang'onoang'ono kuchokera ku zitsanzo zosiyanasiyana kumakhala ndi zofanana zina komanso zosiyana, koma pakali pano pali kusowa kwa njira yogwirizana yokonzekera mitundu yosiyanasiyana ya zitsanzo za metaproteome.
02Kupeza kwa data ya Mass spectrometry
Pakuwunika kwamfuti ya proteome, kusakaniza kwa peptide pambuyo poti kuyambika kumasiyanitsidwa koyamba mugawo la chromatographic, kenako ndikulowa mu mass spectrometer kuti apeze deta pambuyo pa ionization. Mofanana ndi kusanthula kwamtundu umodzi wa proteome, njira zopezera deta ya mass spectrometry mu kusanthula kwa macroproteome kumaphatikizapo DDA mode ndi DIA mode.
Ndi kubwereza kosalekeza ndi kusinthidwa kwa zida za mass spectrometry, zida za mass spectrometry zokhala ndi chidwi chachikulu komanso kusamvana zimagwiritsidwa ntchito ku metaproteome, ndipo kuya kwa kuwunika kwa metaproteome kumakulitsidwanso mosalekeza. Kwa nthawi yayitali, zida zingapo zowoneka bwino kwambiri zotsogozedwa ndi Orbitrap zakhala zikugwiritsidwa ntchito kwambiri mu metaproteome.
Gulu 1 lazolemba zoyambirira likuwonetsa maphunziro ena oyimira metaproteomics kuyambira 2011 mpaka pano malinga ndi mtundu wa zitsanzo, njira yowunikira, chida cha misa spectrometry, njira yopezera, mapulogalamu owunikira, ndi kuchuluka kwa zizindikiritso.
03Mass spectrometry data kusanthula
3.1 Njira yowunikira deta ya DDA
3.1.1 Kusaka kwa Database
3.1.2ndi novokutsatira njira
3.2 Njira yowunikira deta ya DIA
04 Magulu amitundu ndi kafotokozedwe kantchito
Kapangidwe ka madera ang'onoang'ono pamagawo osiyanasiyana a taxonomic ndi amodzi mwamagawo ofunikira pakufufuza kwa microbiome. M'zaka zaposachedwa, zida zingapo zofotokozera zapangidwa kuti zifotokozere zamoyo pamlingo wa mapuloteni, mulingo wa peptide, ndi mulingo wa majini kuti apeze magulu a tizilombo tating'onoting'ono.
Chofunikira cha kafotokozedwe kantchito ndikufanizira motsatana ndi mapuloteni omwe amatsatana ndi nkhokwe yama protein. Pogwiritsa ntchito nkhokwe za jini monga GO, COG, KEGG, eggNOG, ndi zina zotero, kusanthula kosiyanasiyana kwa magwiridwe antchito kumatha kuchitidwa pamapuloteni odziwika ndi ma macroproteomes. Zida zofotokozera zikuphatikiza Blast2GO, DAVID, KOBAS, ndi zina.
05Chidule ndi Outlook
Tizilombo tating'onoting'ono timagwira ntchito yofunika kwambiri paumoyo wamunthu komanso matenda. M'zaka zaposachedwa, metaproteomics yakhala njira yofunikira yaukadaulo yophunzirira ntchito zamagulu ang'onoang'ono. Njira yowunikira ya metaproteomics ndi yofanana ndi yamtundu umodzi wa proteomics, koma chifukwa cha zovuta za chinthu chofufuzira cha metaproteomics, njira zenizeni zofufuzira ziyenera kutengedwa pa sitepe iliyonse yowunikira, kuchokera ku pretreatment pretreatment, kupeza deta kusanthula deta. Pakalipano, chifukwa cha kusintha kwa njira zopangira mankhwala, kusinthika kosalekeza kwa teknoloji ya spectrometry ya mass spectrometry ndi chitukuko chofulumira cha bioinformatics, metaproteomics yapita patsogolo kwambiri pakuzama kwa chizindikiritso ndi kukula kwa ntchito.
Pokonzekera chithandizo cha zitsanzo za macroproteome, chikhalidwe cha chitsanzo chiyenera kuganiziridwa poyamba. Momwe mungalekanitsire tizilombo toyambitsa matenda ku maselo a chilengedwe ndi mapuloteni ndi imodzi mwazovuta zazikulu zomwe macroproteomes akukumana nazo, ndipo kulinganiza pakati pa kulekanitsa bwino ndi kuwonongeka kwa tizilombo toyambitsa matenda ndi vuto lofulumira lomwe liyenera kuthetsedwa. Kachiwiri, mapuloteni m'zigawo za tizilombo tiyenera kuganizira kusiyana structural heterogeneity osiyana mabakiteriya. Zitsanzo za macroproteome pamndandanda wazotsatira zimafunikiranso njira zapadera zochizira.
Pankhani ya zida za misa spectrometry, zida zamtundu wa spectrometry zasintha kuchokera ku ma spectrometer ambiri kutengera zowunikira misa ya Orbitrap monga LTQ-Orbitrap ndi Q Exactive kupita ku ma spectrometer ambiri kutengera kusuntha kwa ion kuphatikiza kuwunika kwa misa ya nthawi yowuluka monga timsTOF Pro. . Zida za timsTOF zokhala ndi chidziwitso cha ion mobility dimension ili ndi kulondola kwakukulu, malire ozindikira, komanso kubwereza kwabwino. Pang'onopang'ono akhala zida zofunika kwambiri m'mafukufuku osiyanasiyana omwe amafunikira kufufuza kwa spectrometry, monga proteome, metaproteome, ndi metabolome ya mtundu umodzi. Ndizofunikira kudziwa kuti kwa nthawi yayitali, kusinthasintha kwa zida za spectrometry kwachepetsa kuya kwa mapuloteni a kafukufuku wa metaproteome. M'tsogolomu, zida za ma spectrometry zokhala ndi mawonekedwe okulirapo zitha kukulitsa chidwi komanso kulondola kwa chidziwitso cha mapuloteni mu metaproteomes.
Pakupeza deta ya mass spectrometry, ngakhale njira yopezera deta ya DIA yavomerezedwa kwambiri mu proteome ya mtundu umodzi, kusanthula kwamakono kwa macroproteome kumagwiritsabe ntchito njira yopezera deta ya DDA. Njira yopezera deta ya DIA imatha kupeza chidziwitso cha fragment ion chachitsanzo, ndipo poyerekeza ndi njira yopezera deta ya DDA, ili ndi mwayi wopeza chidziwitso cha peptide cha chitsanzo cha macroproteome. Komabe, chifukwa chazovuta kwambiri za data ya DIA, kusanthula kwa DIA macroproteome data ikukumanabe ndi zovuta zazikulu. Kukula kwa luntha lochita kupanga komanso kuphunzira mozama kukuyembekezeka kuwongolera kulondola komanso kukwanira kwa kusanthula kwa data ya DIA.
Pakuwunika kwa data ya metaproteomics, imodzi mwamagawo ofunikira ndikumanga nkhokwe zama protein. Kwa malo ofufuzira otchuka monga zomera za m'mimba, matumbo a tizilombo toyambitsa matenda monga IGC ndi HMP angagwiritsidwe ntchito, ndipo zotsatira zodziwika bwino zapezeka. Pakuwunika kwina kwa ma metaproteomics, njira yabwino kwambiri yopangira ma database ikadali kukhazikitsa nkhokwe yachitsanzo cha protein yotsatizana potengera data ya metagenomic. Kwa zitsanzo zamagulu ang'onoang'ono okhala ndi zovuta zambiri komanso kusinthasintha kwakukulu, ndikofunikira kuwonjezera kuzama kotsatizana kuti muwonjezere kuzindikirika kwa mitundu yocheperako, potero kuwongolera kufalikira kwa nkhokwe zamapuloteni. Pamene kusanja deta kulibe, njira yofufuzira yobwerezabwereza ingagwiritsidwe ntchito kukhathamiritsa malo osungira anthu. Komabe, kusaka mobwerezabwereza kungakhudze kuwongolera kwamtundu wa FDR, kotero zotsatira zake ziyenera kuyang'aniridwa mosamala. Kuphatikiza apo, kugwiritsa ntchito mitundu yachikhalidwe ya FDR yowongolera khalidwe pakuwunika kwa metaproteomics ndikofunikirabe kufufuzidwa. Pankhani ya njira yosaka, njira ya hybrid spectral library imatha kupititsa patsogolo kuya kwa DIA metaproteomics. M'zaka zaposachedwa, laibulale yowoneratu yomwe idanenedweratu yopangidwa potengera kuphunzira mozama yawonetsa magwiridwe antchito apamwamba mu DIA proteomics. Komabe, nkhokwe za metaproteome nthawi zambiri zimakhala ndi mamiliyoni ambiri a mapuloteni, zomwe zimapangitsa kuti zikhale zonenedweratu zowerengera mabuku, zimagwiritsa ntchito makompyuta ambiri, ndipo zimabweretsa malo ambiri osaka. Kuonjezera apo, kufanana pakati pa machitidwe a mapuloteni mu metaproteomes kumasiyana kwambiri, zomwe zimapangitsa kuti zikhale zovuta kutsimikizira kulondola kwa chitsanzo chowonetseratu laibulale ya spectral, kotero kuti malaibulale owonetseratu owonetseratu sanagwiritsidwe ntchito kwambiri mu metaproteomics. Kuphatikiza apo, malingaliro atsopano a mapuloteni ndi njira zofotokozera zamagulu ziyenera kupangidwa kuti zigwiritsidwe ntchito pakuwunika kwa metaproteomics zamapuloteni otsatizana kwambiri.
Mwachidule, monga ukadaulo wofufuza wa microbiome, ukadaulo wa metaproteomics wapeza zotsatira zazikulu zofufuza komanso uli ndi kuthekera kwakukulu kwachitukuko.
Nthawi yotumiza: Aug-30-2024