Wu Enhui, Qiao Liang*
Ngalaba Chemistry, Mahadum Fudan, Shanghai 200433, China
Microorganisms nwere njikọ chiri anya na ọrịa na ahụike mmadụ. Otu esi aghọta ihe mejupụtara obodo microbial na ọrụ ha bụ isi okwu nke kwesịrị ịmụ ngwa ngwa. N'ime afọ ndị na-adịbeghị anya, metaproteomics abụrụla ụzọ teknụzụ dị mkpa iji mụọ nhazi na ọrụ nke microorganisms. Otú ọ dị, n'ihi mgbagwoju anya na nnukwu ihe dị iche iche nke ihe nlele obodo microbial, nhazi sample, nnweta data spectrometry na nchịkọta data aghọwo isi ihe ịma aka atọ nke metaproteomics na-eche ugbu a. Na metaproteomics analysis, ọ na-adịkarị mkpa iji bulie pretreatment nke ụdị dị iche iche nke samples na ịmụta dị iche iche microbial nkewa, ịba ọgaranya, mmịpụta na lysis atụmatụ. Yiri proteome nke otu ụdị, ụdị ụdị enwetara data spectrometry dị na metaproteomics gụnyere ụdị nnweta dabere data (DDA) na ụdị nnweta data dabere (DIA). Ụdị nnweta data DIA nwere ike ịnakọta ozi peptide kpamkpam ma nwee nnukwu ikike mmepe. Agbanyeghị, n'ihi mgbagwoju anya nke ihe atụ metaproteome, nyocha data DIA ya aghọwo nnukwu nsogbu na-egbochi mkpuchi miri emi nke metaproteomics. N'ihe gbasara nyocha data, nzọụkwụ kachasị mkpa bụ iwu nke usoro nchekwa data usoro protein. Ogo na izu oke nke nchekwa data ọ bụghị naanị na-enwe mmetụta dị ukwuu na ọnụọgụ njirimara, kamakwa ọ na-emetụta nyocha na ụdị na ọkwa ọrụ. Ka ọ dị ugbu a, ọkọlọtọ ọla edo maka iwupụta nchekwa data metaproteome bụ usoro nchekwa data usoro protein dabere na metagenome. N'otu oge ahụ, usoro nzacha nchekwa data ọha na-adabere na nchọgharị ugboro ugboro egosikwara na ọ nwere uru bara uru siri ike. Site n'echiche nke atụmatụ nyocha data akọwapụtara, ụzọ nyocha data DIA dabere na peptide ejirila usoro zuru oke. Site na mmepe nke mmụta miri emi na ọgụgụ isi, ọ ga-akwalite nke ọma izi ezi, mkpuchi na ọsọ nyocha nke nyocha data macroproteomic. N'ihe gbasara nyocha bioinformatics nke ala, e mepụtala usoro nkọwapụta n'afọ ndị na-adịbeghị anya, nke nwere ike ịkọwa nkọwa ụdị na ọkwa protein, ọkwa peptide na ọkwa mkpụrụ ndụ ihe nketa iji nweta nhazi nke obodo microbial. E jiri ya tụnyere ụzọ omics ndị ọzọ, nyocha ọrụ nke obodo microbial bụ ihe pụrụ iche nke macroproteomics. Macroproteomics abụrụla akụkụ dị mkpa nke nyocha multi-omics nke obodo microbial, ma ka nwere ikike mmepe dị ukwuu n'ihe gbasara omimi mkpuchi, nchọpụta nchọpụta, na nyocha data zuru oke.
01 Nlereanya pretreatment
Ka ọ dị ugbu a, a na-eji teknụzụ metaproteomics eme ihe na nyocha nke microbiome mmadụ, ala, nri, oke osimiri, sludge na-arụ ọrụ na ubi ndị ọzọ. E jiri ya tụnyere nyocha proteome nke otu ụdị, ihe nlele pretreatment nke metaproteome nke ihe nlele dị mgbagwoju anya na-eche ihe ịma aka ndị ọzọ ihu. Na microbial mejupụtara n'ezie samples bụ mgbagwoju, ike nso nke ukwuu bụ nnukwu, cell mgbidi Ọdịdị nke dị iche iche nke microorganisms dị nnọọ iche, na sample mgbe nwere nnukwu ego nke usu-edozi ahụ na ndị ọzọ adịghị ọcha. Ya mere, na nyocha nke metaproteome, ọ na-adịkarị mkpa iji bulie ụdị ụdị dị iche iche ma nakwere nkewa microbial dị iche iche, ịba ụba, mmịpụta na atụmatụ lysis.
Mwepụta nke microbial metaproteomes sitere na ụdị dị iche iche nwere ụfọdụ myirịta yana ụfọdụ ọdịiche, mana ugbu a enweghị usoro nhazi tupu nhazi maka ụdị ụdị metaproteome dị iche iche.
02Nnweta data spectrometry Mass
Na nyocha proteome shotgun, ngwakọta peptide mgbe emechara ọgwụgwọ na-ebu ụzọ kewaa na kọlụm chromatographic, wee banye na spectrometer mass maka inweta data mgbe ionization gasịrị. N'otu aka ahụ nyocha proteome otu ụdị, ụdị nnabata data spectrometry dị na nyocha macroproteome gụnyere ụdị DDA na ọnọdụ DIA.
Site na nrụgharị na mmelite nke ngwa spectrometry na-aga n'ihu, a na-etinye akụrụngwa spectrometry nwere mmetụta dị elu na metaproteome, yana omimi mkpuchi nke nyocha metaproteome na-aga n'ihu. Ogologo oge gara aga, a na-eji usoro ihe nlere anya dị elu nke Orbitrap na-eduzi na metaproteome.
Tebụl 1 nke ederede izizi na-egosi ụfọdụ ọmụmụ ihe nnọchite anya na metaproteomics site na 2011 ruo ugbu a n'ihe gbasara ụdị nlele, atụmatụ nyocha, akụrụngwa spectrometry, usoro nnweta, ngwanrọ nyocha, na ọnụọgụ njirimara.
03 Nnyocha data Mass spectrometry
3.1 Atụmatụ nyocha data DDA
3.1.1 Ọchụchọ database
3.1.2de novousoro usoro
3.2 Atụmatụ nyocha data DIA
04 Nhazi ụdị na nkọwa ọrụ
Ngwakọta nke obodo microbial na ọkwa taxonomic dị iche iche bụ otu n'ime ebe nyocha dị na nyocha microbiome. N'ime afọ ndị na-adịbeghị anya, e mepụtala ọtụtụ ngwa nkọwa iji kọwaa ụdị dị na ọkwa protein, ọkwa peptide, na ọkwa mkpụrụ ndụ ihe nketa iji nweta nhazi nke obodo microbial.
Ihe dị mkpa nke nkọwa ọrụ bụ iji usoro protein ezubere iche tụnyere nchekwa data usoro protein na-arụ ọrụ. N'iji ọdụ data ọrụ mkpụrụ ndụ ihe nketa dị ka GO, COG, KEGG, eggNOG, wdg, enwere ike ịme nyocha nkọwa ọrụ dị iche iche na protein ndị macroproteomes chọpụtara. Ngwa nkọwa gụnyere Blast2GO, DAVID, KOBAS, wdg.
05 Nchịkọta na Outlook
Microorganisms na-arụ ọrụ dị mkpa na ahụike na ọrịa mmadụ. N'afọ ndị na-adịbeghị anya, metaproteomics abụrụla ụzọ ọrụ aka dị mkpa iji mụọ ọrụ nke obodo microbial. Usoro nyocha nke metaproteomics yiri nke otu ụdị proteomics, ma n'ihi mgbagwoju anya nke ihe nyocha nke metaproteomics, ọ dị mkpa ka a nabata usoro nyocha kpọmkwem na usoro nyocha ọ bụla, site na pretreatment sample, nnweta data na nyocha data. Ka ọ dị ugbu a, ekele maka mmelite nke ụzọ ọgwụgwọ mbụ, ihe ọhụrụ na-aga n'ihu nke teknụzụ uka spectrometry na mmepe ngwa ngwa nke bioinformatics, metaproteomics enweela ọganihu dị ukwuu na omimi njirimara na oke ngwa.
N'ime usoro ọgwụgwọ tupu ọgwụgwọ nke macroproteome samples, a ga-ebu ụzọ tụlee ọdịdị nke ihe nlele ahụ. Otu esi ekewa microorganisms na mkpụrụ ndụ gburugburu ebe obibi na protein bụ otu n'ime isi ihe ịma aka na-eche macroproteomes ihu, na nguzozi n'etiti arụmọrụ nkewa na mfu microbial bụ nsogbu dị ngwa a ga-edozi. Nke abuo, mmịpụta protein nke microorganisms ga-eburu n'uche ihe dị iche iche sitere n'ụdị ọdịdị nke nje bacteria dị iche iche kpatara. Ihe nlele macroproteome dị na nso nso a na-achọkwa ụzọ ọgwụgwọ tupu oge eruo.
N'ihe gbasara ngwá ọrụ spectrometry mass, ihe ndị na-ahụ maka ihe ndị na-eme ihe na-eme ka a gbanwee site na spectrometers mass spectrometers dabeere na Orbitrap mass analyzers dị ka LTQ-Orbitrap na Q Exactive to mass spectrometers dabeere na ion agagharị tinyere oge-of-flight mass analyzers dị ka timsTOF Pro. . Usoro nke timsTOF nwere ozi ion agagharị agagharị nwere ezigbo nchọpụta dị elu, oke nchọpụta dị ala, yana ezigbo nkwughachi. Ha ejirila nke nta nke nta bụrụ ngwa dị mkpa n'ụdị nyocha dị iche iche nke chọrọ nchọpụta spectrometry dị ka proteome, metaproteome, na metabolome nke otu ụdị. Ọ dị mma ịmara na ruo ogologo oge, ụdị ike dị iche iche nke akụrụngwa spectrometry egbochila mkpuchi mkpuchi protein nke nyocha metaproteome. N'ọdịnihu, akụrụngwa spectrometry nke nwere oke ike nwere ike imeziwanye nghọta na izi ezi nke njirimara protein na metaproteomes.
Maka inweta data spectrometry mass spectrometry, ọ bụ ezie na anabatala ọnọdụ nnweta data DIA na proteome nke otu ụdị, ọtụtụ nyocha macroproteome dị ugbu a ka na-eji ụdị nnweta data DDA. Ụdị nnweta data DIA nwere ike nweta ozi ion nke ọma nke ihe nlele ahụ, ma e jiri ya tụnyere ụdị nnweta data DDA, ọ nwere ike nweta ozi peptide zuru oke nke nlele macroproteome. Otú ọ dị, n'ihi nnukwu mgbagwoju anya nke data DIA, nyocha nke data macroproteome DIA ka na-eche nnukwu nsogbu ihu. A na-atụ anya na mmepe nke ọgụgụ isi na mmụta miri emi ga-emeziwanye izi ezi na izu oke nke nyocha data DIA.
Na nyocha data nke metaproteomics, otu n'ime usoro dị mkpa bụ iwu nchekwa data usoro protein. Maka mpaghara nyocha a ma ama dị ka ahịhịa intestinal, enwere ike iji ọdụ data microbial intestinal dị ka IGC na HMP, na enwetala nsonaazụ njirimara dị mma. Maka ọtụtụ nyocha metaproteomics ndị ọzọ, atụmatụ nrụpụta nchekwa data kacha dị irè ka bụ iji guzobe nchekwa data usoro protein akọwapụtara nke dabere na data usoro metagenomic. Maka ihe atụ nke obodo microbial nwere nnukwu mgbagwoju anya na nnukwu ike ike, ọ dị mkpa ịbawanye omimi nke usoro iji mee ka njirimara nke ụdị dị ala dị ala, si otú a na-eme ka mkpuchi nke usoro nchekwa data protein dịkwuo elu. Mgbe enweghị usoro data enweghị usoro, enwere ike iji usoro nchọgharị ugboro ugboro iji kwalite nchekwa data ọha. Agbanyeghị, nchọgharị ugboro ugboro nwere ike imetụta njikwa mma FDR, yabụ ọ dị mkpa ka ejiri nlezianya lelee nsonaazụ ọchụchọ. Na mgbakwunye, ojiji nke ụdị njikwa mma FDR ọdịnala na nyocha metaproteomics ka bara uru inyocha. N'ihe gbasara atụmatụ ọchụchọ, atụmatụ ọbá akwụkwọ ngwakọ nwere ike melite omimi mkpuchi nke metaproteomics DIA. N'afọ ndị na-adịbeghị anya, ọbá akwụkwọ ahụ e buru n'amụma emepụtara dabere na mmụta miri emi egosila arụmọrụ dị elu na proteomics DIA. Otú ọ dị, ọdụ data metaproteome na-enwekarị ndenye protein, nke na-ebute nnukwu ọba akwụkwọ ndị a na-ebu amụma, na-eri ọtụtụ ihe mgbakọ na mwepụ, na-ebutekwa nnukwu ohere ọchụchọ. Na mgbakwunye, myirịta dị n'etiti usoro protein na metaproteomes dịgasị iche iche nke ukwuu, na-eme ka o sie ike ịhụ izi ezi nke ụdị amụma ọbá akwụkwọ spectral, ya mere, ejibeghị akwụkwọ ọba akwụkwọ ndị e buru amụma n'ọtụtụ ebe na metaproteomics. Na mgbakwunye, ọ dị mkpa ka ewepụta nrịbama protein ọhụrụ na atụmatụ nkọwa nhazi ọkwa iji tinye na nyocha metaproteomics nke protein ndị yiri ya nke ukwuu.
Na nchịkọta, dị ka teknụzụ nyocha microbiome na-apụta, teknụzụ metaproteomics enwetala nsonaazụ nyocha dị ukwuu ma nweekwa nnukwu ikike mmepe.
Oge nzipu: Ọgọst-30-2024