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Mekhoa e mecha ea immunological le mass spectrometric bakeng sa tlhahlobo e rarahaneng ea li-oligosaccharide tse sa feleng ka har'a corn stover e phekotsoeng pele ka AFEX. Lignocellulosic biomass ke mokhoa o tsitsitseng oa ho sebelisa mafura a mesaletsa ea lintho tsa khale 'me e sebelisoa haholo ho nts'etsapele theknoloji ea biotechnology bakeng sa tlhahiso ea lihlahisoa tse kang lijo, lijo, mafura le lik'hemik'hale. Senotlolo sa mahlale ana ke nts'etsopele ea lits'ebetso tse qothisanang lehlokoa le litšenyehelo bakeng sa ho fetola lik'habohaedreite tse rarahaneng tse teng maboteng a sele ea limela hore e be tsoekere e bonolo joalo ka glucose, xylose le arabinose. Hobane lignocellulosic biomass e manganga haholo, e tlameha ho etsoa kalafo ea thermochemical (mohlala, ammonia fiber exfoliation (AFEX), dilute acid (DA), ionic liquids (IL)) le kalafo ea baeloji (mohlala, enzymatic hydrolysis le microbial fermentation) hammoho ho fumana sehlahisoa se lakatsehang. Leha ho le jwalo, ha dienzyme tsa fungal tsa kgwebo di sebediswa tshebetsong ya hydrolysis, ke 75-85% feela ya tswekere e qhibilihang e bopilweng e leng di-monosaccharides, mme 15-25% e setseng ke di-oligosaccharides tse qhibilihang, tse ke keng tsa thijoa, tseo e seng kamehla di fumanehang ho dikokwana-hloko. Pele, re atlehile ho arola le ho hlwekisa di-oligosaccharides tse manganga tse qhibilihang ka ho sebedisa motswako wa karohano ya carbon le diatomaceous earth le chromatography ya ho tlosa boholo, mme hape ra batlisisa thepa ya tsona ya thibelo ya enzyme. Re fumane hore di-oligosaccharides tse nang le diphetoho tsa uronic acid tse nang le tekanyo e hodimo ya polymerization (DP) di thata ho di sebetsana le metswako ya dienzyme tsa kgwebo ho feta di-oligosaccharides tse tlase tsa DP le tse sa nke lehlakore. Mona re tlaleha tshebediso ya mekgwa e mmalwa e eketsehileng, ho kenyeletswa le ho etsa profiling ya glycan ho sebedisa di-antibodies tsa monoclonal (mAbs) tse ikgethang ho di-glycans tsa biomass tsa dimela ho hlalosa dibopeho tsa glycan maboteng a sele ya dimela le di-hydrolysates tsa enzymatic, ionization ya laser e thuswang ke matrix, nako ya ho fofa ya mass-spectrometry. MALDI-TOF-MS) e sebelisa litlhōrō tsa tlhahlobo tse fanang ka tlhahisoleseling tse fumanoeng ka spectroscopy kamora ho bola ha bobeli ha li-ion tse mpe, chromatography ea khase le spectrometry ea bongata (GC-MS) ho hlalosa litlamo tsa oligosaccharide tse nang le le tse se nang tsona. Ka lebaka la boholo bo bonyenyane ba li-oligosaccharide (DP 4-20), limolek'hule tsena li thata ho li sebelisa bakeng sa ho tlama le ho hlalosa mAb. Ho hlōla bothata bona, re sebelisitse mokhoa o mocha oa ho thibela ho sisinyeha ha oligosaccharide o thehiloeng ho biotin o ileng oa tšoaea ka katleho boholo ba li-oligosaccharide tse qhibilihang tse tlase tsa DP holim'a microplate, tse ileng tsa sebelisoa tsamaisong ea mAb e phahameng bakeng sa tlhahlobo e khethehileng ea ho ligation. Mokhoa ona o mocha o tla nolofatsa nts'etsopele ea liteko tsa glycome tse tsoetseng pele haholo nakong e tlang tse ka sebelisoang ho arola le ho hlalosa li-oligosaccharide tse teng ho li-biomarker bakeng sa merero ea tlhahlobo.
Lignocellulosic biomass, e entsoeng ka thepa ea temo, meru, joang le patsi, ke sesebelisoa se ka sebelisoang bakeng sa tlhahiso ea lihlahisoa tse thehiloeng linthong tse phelang, ho kenyeletsoa lijo, lijo, mafura le lik'hemik'hale tse tlang pele ho hlahisa lihlahisoa tsa boleng bo holimo1. Lik'habohaedreite (tse kang cellulose le hemicellulose) tse teng maboteng a sele ea limela lia fokotsoa hore e be li-monosaccharides ka ts'ebetso ea lik'hemik'hale le phetoho ea biotransformation (tse kang enzymatic hydrolysis le microbial fermentation). Mekhoa e tloaelehileng ea pele ho kalafo e kenyelletsa katoloso ea ammonia fiber (AFEX), dilute acid (DA), ionic liquid (IL), le mouoane oa ho phatloha (SE), tse sebelisang motsoako oa lik'hemik'hale le mocheso ho fokotsa tlhahiso ea lignocellulose ka ho bula mabota a sele ea limela3,4. manganga a lintho tse bonahalang, 5. Enzymatic hydrolysis e etsoa ka moroalo o phahameng oa lintho tse tiileng ho sebelisoa li-enzyme tse sebetsang tsa khoebo tse nang le lik'habohaedreite (CAZymes) le microbial fermentation ho sebelisoa tomoso ea transgenic kapa libaktheria ho hlahisa mafura le lik'hemik'hale tse thehiloeng linthong tse phelang 6.
Li-CAZyme ka har'a li-enzyme tsa khoebo li entsoe ka motsoako o rarahaneng oa li-enzyme tse arolang litlamo tse rarahaneng tsa lik'habohaedreite le tsoekere ho theha li-monosaccharides2,7. Joalokaha re tlalehile pejana, marang-rang a rarahaneng a li-polymer tse nkhang hamonate tsa lignin le lik'habohaedreite a li etsa hore li se ke tsa rarolloa haholo, e leng se lebisang phetohong e sa fellang ea tsoekere, e bokellang 15-25% ea li-oligosaccharides tsa bong tse sa hlahisoeng nakong ea hydrolysis ea enzymatic ea biomass e phekotsoeng esale pele. Ena ke bothata bo tloaelehileng ka mekhoa e fapaneng ea pele ho phekolo ea biomass. Mabaka a mang a bothata bona a kenyelletsa thibelo ea li-enzyme nakong ea hydrolysis, kapa ho ba sieo kapa maemo a tlase a li-enzyme tsa bohlokoa tse hlokahalang ho roba litlamo tsa tsoekere ka har'a biomass ea limela. Ho utloisisa sebopeho le litšobotsi tsa sebopeho sa tsoekere, joalo ka litlamo tsa tsoekere ka har'a li-oligosaccharides, ho tla re thusa ho ntlafatsa phetoho ea tsoekere nakong ea hydrolysis, ho etsa hore lits'ebetso tsa biotechnology li qothisane lehlokoa le lihlahisoa tse tsoang ho peterole.
Ho fumana sebopeho sa lik'habohaedreite ho thata 'me ho hloka motsoako oa mekhoa e kang chromatography ea metsi (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 le mass spectrometry (MS)17. ,18. Mekhoa ea MS e kang time-of-flight mass spectrometry ka laser desorption le ionization ho sebelisoa matrix (MALDI-TOF-MS) ke mokhoa o feto-fetohang oa ho khetholla meaho ea lik'habohaedreite. Haufinyane tjena, Collision-Induced Dissociation (CID) tandem MS ea sodium ion adducts e sebelisitsoe haholo ho khetholla menoana ea letsoho e tsamaellanang le maemo a ho hokela a oligosaccharide, litlhophiso tsa anomeric, tatellano, le maemo a makala 20, 21.
Tlhahlobo ea Glycan ke sesebelisoa se setle sa ho tsebahatsa ka botebo litlamo tsa lik'habohaedreite22. Mokhoa ona o sebelisa li-antibodies tsa monoclonal (mAbs) tse lebisitsoeng ho glycan ea lebota la sele ea semela e le li-probe ho utloisisa likamano tse rarahaneng tsa lik'habohaedreite. Ho na le li-mAb tse fetang 250 lefatšeng ka bophara, tse entsoeng khahlanong le li-oligosaccharide tse fapaneng tse otlolohileng le tse nang le makala tse sebelisang li-saccharide tse fapaneng24. Li-mAb tse 'maloa li sebelisitsoe haholo ho hlalosa sebopeho, sebopeho le liphetoho tsa lebota la sele ea semela, kaha ho na le liphapang tse kholo ho latela mofuta oa sele ea semela, setho, lilemo, mohato oa nts'etsopele le tikoloho ea kholo25,26. Haufinyane tjena, mokhoa ona o sebelisitsoe ho utloisisa bongata ba li-vesicle lits'ebetsong tsa limela le liphoofolo le mesebetsi ea tsona ho tsamaiseng glycan joalo ka ha ho khethoa ke matšoao a subcellular, mekhahlelo ea nts'etsopele, kapa lintho tse susumetsang tikoloho, le ho fumana ts'ebetso ea enzyme. Tse ling tsa dibopeho tse fapaneng tsa di-glycan le di-xylan tse hlwailweng di kenyeletsa pectin (P), xylan (X), mannan (M), xyloglucans (XylG), di-mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) le arabino-galactan (ArbG)29.
Leha ho le jwalo, ho sa tsotellehe boiteko bona bohle ba dipatlisiso, ke diphuputso tse mmalwa feela tse shebaneng le mofuta wa ho bokellana ha oligosaccharide nakong ya hydrolysis e phahameng ya di-solid load (HSL), ho kenyeletswa le ho lokollwa ha oligosaccharide, diphetoho tsa bolelele ba ketane ya oligomeric nakong ya hydrolysis, di-polymer tse fapaneng tse tlase tsa DP, le di-curve tsa tsona. Ho sa le jwalo, le hoja tlhahlobo ya glycan e ipakile e le sesebediswa se molemo bakeng sa tlhahlobo e felletseng ya sebopeho sa glycan, ho thata ho lekola di-oligosaccharide tse tlase tsa DP tse qhibilihang metsing ho sebediswa mekgwa ya di-antibody. Di-oligosaccharide tse nyane tsa DP tse nang le boima ba dimolek'hule bo ka tlase ho 5-10 kDa ha di tlamelle ho di-plate tsa ELISA 33, 34 mme di hlatsuwa pele ho eketswa di-antibody.
Mona, ka lekhetlo la pele, re bonts'a teko ea ELISA lipoleiting tse koahetsoeng ke avidin re sebelisa li-antibodies tsa monoclonal, re kopanya mokhoa oa biotinylation oa mohato o le mong bakeng sa li-oligosaccharide tse qhibilihang tse qhibilihang le tlhahlobo ea glycome. Mokhoa oa rona oa tlhahlobo ea glycome o netefalitsoe ke tlhahlobo e thehiloeng ho MALDI-TOF-MS le GC-MS ea likhokahano tse tlatsanang tsa oligosaccharide re sebelisa trimethylsilyl (TMS) derivatization ea metsoako ea tsoekere e nang le hydrolyzed. Mokhoa ona o mocha o ka ntlafatsoa e le mokhoa o phahameng oa ho hlahisa nakong e tlang 'me oa fumana ts'ebeliso e pharaletseng lipatlisisong tsa bongaka35.
Liphetoho tsa li-enzyme le li-antibodies kamora phetolelo, joalo ka glycosylation,36 li ama ts'ebetso ea tsona ea baeloji. Mohlala, liphetoho tsa glycosylation ea liprotheine tsa serum li bapala karolo ea bohlokoa ho ramatiki e ruruhang, 'me liphetoho tsa glycosylation li sebelisoa e le matšoao a tlhahlobo37. Li-glycan tse fapaneng li tlalehiloe libukeng hore li hlaha habonolo mafung a fapaneng, ho kenyeletsoa mafu a sa foleng a ho ruruha a tšilo ea lijo le sebete, tšoaetso ea vaerase, mofetše oa mae a bomme, oa matsoele le oa prostate38,39,40. Ho utloisisa sebopeho sa li-glycan ho sebelisa mekhoa ea glycan e thehiloeng ho li-antibodies ea ELISA ho tla fana ka kholiseho e eketsehileng ho fumaneng lefu ntle le ho sebelisa mekhoa e rarahaneng ea MS.
Phuputso ea rona ea pejana e bontšitse hore li-oligosaccharide tse manganga li ile tsa lula li sa hloekisoa ka mor'a kalafo ea pele le hydrolysis ea enzymatic (Setšoantšo sa 1). Mosebetsing oa rona o hatisitsoeng pejana, re ntlafalitse mokhoa oa ho ntša mashala o tiileng oa mohato o sebetsang ho arola li-oligosaccharide ho tsoa ho AFEX-pretreated corn stover hydrolyzate (ACSH)8. Kamora ho ntšoa le ho aroloa ha pele, li-oligosaccharide li ile tsa aroloa ka ho eketsehileng ka boholo ba ho tlosa chromatography (SEC) 'me tsa bokelloa ka tatellano ea boima ba limolek'hule. Li-monomer tsa tsoekere le li-oligomer tse lokollotsoeng kalafong e fapaneng li ile tsa hlahlojoa ka tlhahlobo ea motsoako oa tsoekere. Ha ho bapisoa litaba tsa li-oligomer tsa tsoekere tse fumanoeng ka mekhoa e fapaneng ea kalafo ea pele, boteng ba li-oligosaccharide tse manganga ke bothata bo tloaelehileng phetohong ea biomass ho ba li-monosaccharide 'me bo ka lebisa phokotsong ea chai ea tsoekere ea bonyane 10-15% esita le ho fihlela ho 18%. US. Mokhoa ona o sebelisoa bakeng sa tlhahiso e kholo ea likaroloana tsa oligosaccharide. ACH e hlahisitsoeng le likaroloana tsa eona tse latelang tse nang le boima bo fapaneng ba limolek'hule li sebelisitsoe e le thepa ea liteko bakeng sa tlhaloso ea li-oligosaccharides mosebetsing ona.
Kamora kalafo pele ho kalafo le hydrolysis ea enzymatic, li-oligosaccharide tse sa khaotseng li ile tsa lula li sa hloekisoa. Mona (A) ke mokhoa oa karohano ea li-oligosaccharide moo li-oligosaccharide li aroloang ho AFEX-pretreated corn stover hydrolysate (ACSH) ho sebelisoa bethe e pakiloeng ea carbon e sebetsang le lefats'e la diatomaceous; (B) Mokhoa oa karohano ea li-oligosaccharide. Li-oligosaccharide li ile tsa aroloa hape ka chromatography ea boholo bo sa kenyelletsoeng (SEC); (C) Li-monomer tsa Saccharide le li-oligomer tse lokollotsoeng kalafong e fapaneng ea pele ho kalafo (asidi e hlapolotsoeng: DA, mokelikeli oa ionic: IL le AFEX). Maemo a hydrolysis ea enzyme: ho jara lintho tse tiileng tse phahameng tsa 25% (w/w) (hoo e ka bang 8% ea glucan e jarang), hydrolysis ea lihora tse 96, 20 mg/g ea enzyme ea khoebo e jarang (karolelano ea Ctec2:Htec2:MP-2:1:1) le (D) Li-monomer tsa tsoekere le li-oligomer tsa glucose, xylose le arabinose tse lokollotsoeng ho tsoa ho AFEX pre-treated corn stover (ACS).
Tlhahlobo ea Glycan e ipakile e le sesebelisoa se molemo bakeng sa tlhahlobo e felletseng ea sebopeho sa li-glycan tse ntšitsoeng ho tsoa masala a tiileng a biomass. Leha ho le joalo, li-saccharide tse qhibilihang ka metsing ha li hlahisoe hantle ho sebelisoa mokhoa ona oa setso41 hobane li-oligosaccharide tse boima bo tlase ba limolek'hule li thata ho li thibela lipoleiting tsa ELISA 'me lia hlatsuoa pele ho eketsoa li-antibody. Ka hona, bakeng sa ho tlama le ho hlalosa li-antibody, mokhoa oa biotinylation oa mohato o le mong o sebelisitsoe ho koahela li-oligosaccharide tse qhibilihang, tse sa lumellaneng le tsona lipoleiting tsa ELISA tse koahetsoeng ke avidin. Mokhoa ona o ile oa lekoa ho sebelisoa ACSH ea rona e neng e hlahisitsoe pele le karoloana e thehiloeng boima ba eona ba limolek'hule (kapa tekanyo ea polymerization, DP). Biotinylation ea mohato o le mong e sebelisitsoe ho eketsa kamano ea ho tlama ea oligosaccharide ka ho eketsa biotin-LC-hydrazide pheletsong e fokotsang ea lik'habohaedreite (Setšoantšo sa 2). Tharollong, sehlopha sa hemiacetal pheletsong e fokotsang se arabela le sehlopha sa hydrazide sa biotin-LC-hydrazide ho theha tlamo ea hydrazone. Ha ho na le sethibela-mafu NaCNBH3, tlamo ea hydrazone e fokotsoa ho ba sehlahisoa se tsitsitseng sa biotinylated. Ka phetoho ea pheletso e fokotsang tsoekere, ho tlamahana ha li-oligosaccharide tse tlase tsa DP ho lipoleiti tsa ELISA ho ile ha khoneha, 'me thutong ea rona sena se entsoe lipoleiting tse koahetsoeng ke avidin ho sebelisoa li-mAbs tse shebaneng le glycan.
Tlhahlobo ea li-antibodies tsa monoclonal tse thehiloeng ho ELISA bakeng sa li-oligosaccharide tse nang le biotinyle. Mona (A) ho kopantsoe biotinyle ea li-oligosaccharide le tlhahlobo e latelang ea ELISA ka li-mAb tse shebaneng le glycan holim'a lipoleiti tse koahetsoeng ke NeutrAvidin le (B) ho bonts'a mokhoa oa mohato o le mong oa biotinyle ea lihlahisoa tsa karabelo.
Lipoleiti tse koahetsoeng ka Avidin tse nang le li-antibodies tse kopantsoeng le oligosaccharide li ile tsa eketsoa ho li-antibodies tsa mantlha le tsa bobeli 'me tsa hlatsuoa ka har'a sesebelisoa se bobebe le se utloang nako. Kamora hore ho tlama li-antibody ho phethehe, eketsa substrate ea TMB ho hotetsa poleiti. Karabelo e ile ea emisoa qetellong ka sulfuric acid. Lipoleiti tse hotelitsoeng li ile tsa hlahlojoa ho sebelisoa 'mali oa ELISA ho fumana matla a ho tlama a antibody ka 'ngoe ho lemoha ho hokahana ho ikhethileng ha antibody. Bakeng sa lintlha le liparamente tsa teko, bona karolo e tsamaellanang "Lisebelisoa le Mekhoa".
Re bonts'a thuso ea mokhoa ona o mocha bakeng sa lits'ebetso tse ikhethileng ka ho hlalosa li-oligosaccharide tse qhibilihang tse teng ho ACSH hammoho le likarolong tsa oligosaccharide tse tala le tse hloekisitsoeng tse arohantsoeng ho tsoa ho li-hydrolysates tsa lignocellulosic. Joalokaha ho bontšitsoe ho Setšoantšo sa 3, li-xylan tse atileng haholo tse nkeloang sebaka ke epitope tse fumanoeng ho ACSH ho sebelisoa mekhoa ea tlhahlobo ea glycome ea bioacylated hangata ke uronic (U) kapa methyluronic (MeU) le pectic arabinogalactans. Bongata ba tsona li fumanoe hape thutong ea rona e fetileng mabapi le tlhahlobo ea li-glycans tsa solids tse sa hydrolyzed (UHS)43.
Ho lemoha li-epitope tsa oligosaccharide tse sa fetoheng ho sebelisoa antibody ea monoclonal e lebisitsoeng ho glycan ea lebota la sele. Karoloana e "sa nke lehlakore" ke karoloana ea ACN 'me karoloana e "acidic" ke karoloana ea FA. Bofubelu bo khanyang 'mapeng oa mocheso bo bontša litaba tse phahameng tsa epitope, 'me boputsoa bo khanyang bo bontša bokamorao bo se nang letho. Litekanyetso tsa 'mala sekaleng li ipapisitse le litekanyetso tsa OD tse tala bakeng sa liforomo N=2. Li-epitope tse kholo tse lemohuoang ke li-antibodies li bontšitsoe ka letsohong le letona.
Dibopeho tsena tse seng tsa cellulose di ne di ke ke tsa arolwa ke di-cellulase le di-hemicellulase tse tlwaelehileng haholo motswakong wa di-enzyme tsa kgwebo tse lekilweng, o kenyeletsang di-enzyme tsa kgwebo tse sebediswang haholo. Ka hona, di-enzyme tse ntjha tse thusang di a hlokahala bakeng sa hydrolysis ya tsona. Ntle le di-enzyme tse hlokahalang tse thusang tse seng tsa cellulose, ditlamo tsena tse seng tsa cellulose di thibela phetoho e feletseng ho di-monosaccharides, leha di-polymer tsa tsona tsa tsoekere tse tswalwang di ka hatellwa haholo hore e be dikotwana tse kgutshwane mme tsa qhibilihiswa ho sebediswa metswako ya di-enzyme tsa kgwebo.
Phuputso e 'ngoe ea kabo ea matšoao le matla a eona a ho tlama e bontšitse hore li-epitope tse tlamang li ne li le tlase likarolong tse phahameng tsa tsoekere tsa DP (A, B, C, DP ho fihlela ho 20+) ho feta likarolong tse tlase tsa DP (D, E, F, DP) ho dimers) (Setšoantšo sa 1). Likaroloana tsa asiti li atile haholo ho li-epitope tse seng tsa cellulose ho feta likaroloana tse sa nke lehlakore. Liketsahalo tsena li tsamaellana le mohlala o bonoeng thutong ea rona e fetileng, moo likarolo tse phahameng tsa DP le acid li neng li hanela hydrolysis ea enzymatic. Ka hona, boteng ba li-epitope tse seng tsa cellulose glycan le li-substitution tsa U le MeU li ka thusa haholo botsitsong ba li-oligosaccharides. Ho lokela ho hlokomeloa hore katleho ea ho tlama le ho lemoha e ka ba bothata bakeng sa li-oligosaccharides tse tlase tsa DP, haholo-holo haeba epitope e le dimeric kapa trimeric oligosaccharide. Sena se ka lekoa ho sebelisoa li-oligosaccharides tsa khoebo tsa bolelele bo fapaneng, e 'ngoe le e 'ngoe e na le epitope e le 'ngoe feela e tlamang ho mAb e itseng.
Kahoo, tšebeliso ea li-antibodies tse ikhethileng ka sebopeho e senotse mefuta e itseng ea litlamo tse sa fetoheng. Ho latela mofuta oa li-antibody tse sebelisitsoeng, mokhoa o loketseng oa ho hokahana, le matla a lets'oao leo e le hlahisang (boholo le bo fokolang ka ho fetisisa), li-enzyme tse ncha li ka khetholloa 'me tsa eketsoa ka bongata bo lekanang le motsoako oa li-enzyme bakeng sa phetoho e felletseng ea glycoconversion. Ha re nka tlhahlobo ea li-oligosaccharides tsa ACSH e le mohlala, re ka theha database ea litlamo tsa glycan bakeng sa thepa e 'ngoe le e 'ngoe ea biomass. Ho lokela ho hlokomeloa mona hore kamano e fapaneng ea li-antibodies e lokela ho nahanoa, 'me haeba kamano ea tsona e sa tsejoe, sena se tla baka mathata a itseng ha ho bapisoa matšoao a li-antibodies tse fapaneng. Ho phaella moo, papiso ea litlamo tsa glycan e ka sebetsa hantle pakeng tsa lisampole bakeng sa antibody e tšoanang. Litlamo tsena tse manganga li ka hokahanngoa le database ea CAZyme, eo ho eona re ka khethollang li-enzyme, ra khetha li-enzyme tse khethiloeng le ho leka li-enzyme tse robang tlamo, kapa ra nts'etsapele litsamaiso tsa likokoana-hloko ho hlahisa li-enzyme tsena bakeng sa tšebeliso ea li-biorefineries44.
Ho lekola hore na mekhoa ea immunological e tlatsana joang le mekhoa e meng ea ho khetholla li-oligosaccharide tse boima bo tlase ba limolek'hule tse teng ka har'a li-hydrolysates tsa lignocellulosic, re entse MALDI (Setšoantšo sa 4, S1-S8) le tlhahlobo ea li-saccharide tse nkiloeng ho TMS tse thehiloeng ho GC-MS phanele e le 'ngoe (Setšoantšo sa 5) karolo ea oligosaccharide. MALDI e sebelisoa ho bapisa hore na kabo ea bongata ba limolek'hule tsa oligosaccharide e lumellana le sebopeho se reriloeng. Setšoantšong sa 4 ho bontšoa MS ea likarolo tse sa nke lehlakore tsa ACN-A le ACN-B. Tlhahlobo ea ACN-A e tiisitse mefuta e fapaneng ea tsoekere ea pentose ho tloha ho DP 4-8 (Setšoantšo sa 4) ho isa ho DP 22 (Setšoantšo sa S1), eo boima ba eona bo tsamaellanang le li-oligosaccharide tsa MeU-xylan. Tlhahlobo ea ACN-B e tiisitse letoto la pentose le glucoxylan ka DP 8-15. Linthong tse tlatsetsang tse kang Setšoantšo sa S3, limmapa tsa kabo ea boima ba asiti ea FA-C li bontša mefuta e fapaneng ea tsoekere ea pentose e nketsoeng sebaka ke (Me)U e nang le DP ea 8-15 e lumellanang le li-xylan tse nketsoeng sebaka tse fumanoang tlhahlobong ea mAb e thehiloeng ho ELISA. Li-epitopes lia tsitsisa.
MALDI-MS spectrum ea li-oligosaccharide tse sa lateleng tse qhibilihang tse fumanehang ho ACS. Mona, (A) Likaroloana tsa ACN-A tse nang le methylated uronic acid (DP 4-8) li nkile sebaka sa li-oligosaccharide tsa glucuroxylan le (B) ACN-B xylan le methylated uronic acid oligosaccharides tse nketsoeng sebaka ke glucuroxylan (DP 8-15).
Tlhahlobo ea sebopeho sa masala a glycan a li-oligosaccharide tse sa sebetseng. Mona (A) Sebopeho sa saccharide sa TMS sa likaroloana tse fapaneng tsa oligosaccharide se fumanoeng ho sebelisoa tlhahlobo ea GC-MS. (B) Sebopeho sa tsoekere e fapaneng e tsoang ho TMS e teng ho li-oligosaccharide. ACN - karolo ea acetonitrile e nang le li-oligosaccharide tse sa nke lehlakore le karolo ea FA - ferulic acid e nang le li-oligosaccharide tsa acid.
Qeto e 'ngoe e khahlisang e nkiloe tlhahlobong ea LC-MS ea karoloana ea oligosaccharide, joalo ka ha ho bontšitsoe ho Setšoantšo sa S9 (mekhoa e ka bonoa thepa e tlatsetsang ea elektroniki). Likaroloana tsa lihlopha tsa hexose le -OAc li ile tsa bonoa khafetsa nakong ea ho hokela karoloana ea ACN-B. Sephetho sena ha se tiise feela karohano e bonoang tlhahlobong ea glycome le MALDI-TOF, empa hape se fana ka tlhahisoleseling e ncha mabapi le lintho tse ka bang teng tse tsoang lik'habohaedreite biomass e phekotsoeng esale pele.
Hape re entse tlhahlobo ea sebopeho sa glycan ea likaroloana tsa oligosaccharide re sebelisa TMS glycan derivatization. Re sebelisa GC-MS, re fumane sebopeho sa neural (non-derivative) le tsoekere e nang le asiti (GluA le GalA) karolong ea oligosaccharide (Setšoantšo sa 5). Asiti ea Glucuronic e fumanoa likarolong tsa asiti C le D, ha galacturonic acid e fumanoa likarolong tsa asiti A le B, tseo ka bobeli e leng likarolo tse phahameng tsa DP tsa tsoekere e nang le asiti. Liphetho tsena ha li tiise feela data ea rona ea ELISA le MALDI, empa li boetse li lumellana le lithuto tsa rona tsa pejana tsa ho bokellana ha oligosaccharide. Ka hona, re lumela hore mekhoa ea sejoale-joale ea immunological e sebelisang biotinylation ea oligosaccharides le tlhahlobo ea ELISA e latelang e lekane ho lemoha li-oligosaccharides tse qhibilihang tse qhibilihang lisampoleng tse fapaneng tsa baeloji.
Kaha mekhoa ea tlhahlobo ea mAb e thehiloeng ho ELISA e netefalitsoe ka mekhoa e 'maloa e fapaneng, re ne re batla ho batlisisa haholoanyane bokhoni ba mokhoa ona o mocha oa bongata. Li-oligosaccharide tse peli tsa khoebo, xylohexasaccharide oligosaccharide (XHE) le 23-α-L-arabinofuranosyl-xylotriose (A2XX), li rekiloe 'me tsa lekoa ho sebelisoa mokhoa o mocha oa mAb o shebaneng le glycan ea lebota la sele. Setšoantšo sa 6 se bontša kamano e otlolohileng pakeng tsa lets'oao la ho tlama la biotinylated le khatello ea log ea khatello ea oligosaccharide, e leng se bontšang mohlala o ka bang teng oa ho monya Langmuir. Har'a mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, le CCRC-M151 li amana le XHE, 'me CCRC-M108, CCRC-M109, le LM11 li amana le A2XX holim'a mefuta e pakeng tsa 1 nm ho isa ho 100 nano. Ka lebaka la ho fumaneha ho fokolang ha li-antibodies nakong ea teko, liteko tse fokolang li entsoe ka khatello e 'ngoe le e 'ngoe ea oligosaccharide. Ho lokela ho hlokomeloa mona hore li-antibodies tse ling li arabela ka tsela e fapaneng haholo ho oligosaccharide e tšoanang le substrate, mohlomong hobane li tlama ho li-epitope tse fapaneng hanyane 'me li ka ba le likamano tse fapaneng haholo tsa ho tlama. Mekhoa le litlamorao tsa ho khetholla epitope ka nepo li tla ba thata haholo ha mokhoa o mocha oa mAb o sebelisoa lisampoleng tsa 'nete.
Ho sebelisitsoe li-oligosaccharide tse peli tsa khoebo ho fumana mefuta e fapaneng ea li-mAb tse shebaneng le glycan. Mona, likamano tse otlolohileng le mahloriso a log a mahloriso a oligosaccharide li bontša mekhoa ea ho monya Langmuir bakeng sa (A) XHE ka mAb le (B) A2XX ka mAb. Li-epitope tse tsamaellanang li bontša libopeho tsa li-oligosaccharide tsa khoebo tse sebelisoang e le li-substrate tlhahlobong.
Tšebeliso ea li-antibodies tsa monoclonal tse shebaneng le glycan (tlhahlobo ea glycocomic kapa tlhahlobo ea mAb e thehiloeng ho ELISA) ke sesebelisoa se matla sa ho hlalosa ka botebo boholo ba li-glycan tse kholo tsa lebota la sele tse etsang biomass ea limela. Leha ho le joalo, tlhahlobo ea glycan ea khale e khetholla li-glycan tse kholo tsa lebota la sele feela, kaha li-oligosaccharide tse ngata ha li thibeloe ho tsamaea ka katleho lipoleiting tsa ELISA. Thutong ena, stover ea poone e phehiloeng esale pele ea AFEX e ile ea haelloa ke enzyme ka hydrolyzed ka bongata bo phahameng ba lintho tse tiileng. Tlhahlobo ea tsoekere e sebelisitsoe ho fumana hore na lik'habohaedreite tsa lebota la sele li ka khutlisoa joang ka har'a hydrolyzate. Leha ho le joalo, tlhahlobo ea mAb ea li-oligosaccharide tse nyane ka har'a hydrolysates ha e nyenyefatsoe, 'me ho hlokahala lisebelisoa tse ling ho thibela li-oligosaccharide ka katleho lipoleiting tsa ELISA.
Mona re tlaleha mokhoa o mocha le o sebetsang oa ho thibela oligosaccharide ho sisinyeha ha lisele bakeng sa tlhahlobo ea mAb ka ho kopanya biotinylation ea oligosaccharide e lateloang ke tlhahlobo ea ELISA holim'a lipoleiti tse koahetsoeng ke NeutrAvidin™. Li-oligosaccharide tse sa sisinyeheng tse sieo li bontšitse kamano e lekaneng bakeng sa antibody ho nolofalletsa ho lemoha ka potlako le ka katleho li-oligosaccharide tse sa fetoheng. Tlhahlobo ea sebopeho sa li-oligosaccharide tsena tse manganga tse thehiloeng ho mass spectrometry e tiisitse liphetho tsa mokhoa ona o mocha oa ho hlahloba 'mele. Kahoo, lithuto tsena li bontša hore motsoako oa biotinylation ea oligosaccharide le tlhahlobo ea ELISA ka li-antibodies tsa monoclonal tse lebisitsoeng ho glycan li ka sebelisoa ho lemoha likhokahano tse kopaneng ho li-oligosaccharide 'me li ka sebelisoa haholo lithutong tse ling tsa biochemical tse hlalosang sebopeho sa li-oligosaccharide.
Mokhoa ona oa ho etsa profiling ea glycan o thehiloeng ho biotin ke tlaleho ea pele e khonang ho batlisisa litlamo tsa lik'habohaedreite tse sa fetoheng tsa li-oligosaccharide tse qhibilihang ka har'a biomass ea limela. Sena se thusa ho utloisisa hore na ke hobane'ng ha likarolo tse ling tsa biomass li manganga hakaale ha ho tluoa tlhahisong ea biofuel. Mokhoa ona o tlatsa sekheo sa bohlokoa mekhoeng ea tlhahlobo ea glycome 'me o atolosa ts'ebeliso ea ona ho mefuta e mengata ea substrates ho feta li-oligosaccharide tsa limela. Nakong e tlang, re ka sebelisa liroboto bakeng sa biotinylation 'me ra sebelisa mokhoa oo re o hlahisitseng bakeng sa tlhahlobo e phahameng ea lisampole re sebelisa ELISA.
Joang ba poone (CS) bo lenngoeng ho tsoa peong e kopantsoeng ea Pioneer 33A14 bo ile ba kotuloa ka 2010 ho tsoa Kramer Farms e Ray, Colorado. Ka tumello ea mong'a mobu, biomass ena e ka sebelisoa bakeng sa lipatlisiso. Disampole di bolokilwe di omme <6% mongobo ka mekotleng e notletsweng ka zip mochesong wa kamore. Disampole di bolokilwe di omme <6% mongobo ka mekotleng e notletsweng ka zip mochesong wa kamore. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Disampole di bolokilwe di omme mongobong o ka tlase ho 6% ka mekotleng e nang le zipu mochesong wa kamore.樣品在室温下以干燥< 6% 的水分储存在自封袋中。样品在室温下以干燥< 6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Disampole di bolokwa ka mekotleng ya zipper mochesong wa kamore o nang le mongobo o ka tlase ho 6%.Phuputso e latetse litataiso tsa lehae le tsa naha. Tlhahlobo ea metsoako e entsoe ho sebelisoa mokhoa oa NREL. Ho fumanoe hore motsoako o na le 31.4% ea glucan, 18.7% ea xylan, 3.3% ea arabinan, 1.2% ea galactan, 2.2% ea acetyl, 14.3% ea lignin, 1.7% ea protheine le 13.4% ea molora.
Cellic® CTec2 (138 mg protheine/ml, lot VCNI 0001) ke motsoako o rarahaneng oa cellulase, β-glucosidase le Cellic® HTec2 (157 mg protheine/ml, lot VHN00001) ho tsoa ho Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg protheine/mL), motsoako o rarahaneng oa li-enzyme tse senyang pectin, e fanoe ke DuPont Industrial Biosciences (Palo Alto, CA, USA). Maemo a protheine ea enzyme a ile a fumanoa ka ho hakanya litaba tsa protheine (le ho tlosa monehelo oa naetrojene e seng ea protheine) ho sebelisoa tlhahlobo ea naetrojene ea Kjeldahl (mokhoa oa AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 e rekiloe ho EMD Millipore (Billerica, MA). Khabone e sebetsang (DARCO, li-granule tse 100 tsa letlooeng), Avicel (PH-101), beech xylan, le lik'hemik'hale tse ling kaofela li rekiloe ho Sigma-Aldrich (St. Louis, MO).
Kalafo ea pele ho AFEX e entsoe ho GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Kalafo ea pele ho e entsoe ho 140° C. metsotso e 15. Nako ea bolulo ea 46 ka karolelano ea 1:1 ea ammonia e sa hloekang ho biomass ka 60% (w/w) e kenngoang ka har'a sesebelisoa sa tšepe e sa hloekang (Parr Instruments Company). Ho nkile metsotso e 30. Sesebelisi se ile sa tlisoa ho 140°C 'me ammonia ea lokolloa ka potlako, e leng se ileng sa lumella biomass ho khutlela mochesong oa kamore kapele. Sebopeho sa AFEX pre-treated corn stover (ACS) se ne se tšoana le sa corn stover e sa phekoloang (UT-CS).
Dintho tse tiileng tse phahameng tsa ACSH 25% (w/w) (hoo e ka bang 8% ya dextran e jarwang) di lokisitswe e le thepa ya ho qala bakeng sa tlhahiso e kgolo ya di-oligosaccharide. Hydrolysis ya enzymatic ya ACS e entswe ka ho sebedisa motswako wa enzyme ya kgwebo ho kenyeletswa Cellic® Ctec2 10 mg protein/g glucan (ka biomass e phekotsweng esale pele), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, le Multifect Pectinase (Genencor Inc, USA). ). ), 5 mg protein/g dextran. Hydrolysis ya enzymatic e entswe ka bioreactor ya dilithara tse 5 ka bophahamo bo sebetsang ba dilithara tse 3, pH 4.8, 50°C le 250 rpm. Kamora hydrolysis ka dihora tse 96, hydrolyzate e ile ya bokellwa ka centrifugation ka 6000 rpm metsotso e 30 ebe ka 14000 rpm metsotso e 30 ho tlosa dintho tse tiileng tse sa kenngwang metsi. Hydrolyzate e ile ea sefshoa e se nang mafura ka beaker ea sefe ea 0.22 mm. Hydrolyzate e sefshoang e ile ea bolokoa ka libotlolong tse se nang mafura mochesong oa 4°C. ebe e aroloa ka har'a khabone.
Tlhahlobo ea sebopeho sa lisampole tsa biomass tse thehiloeng ho qotsitsoeng ho latela mekhoa ea tlhahlobo ea laboratori ea NREL: ho lokisoa ha lisampole bakeng sa tlhahlobo ea sebopeho (NREL/TP-510-42620) le ho fumana lik'habohaedreite tsa sebopeho le lignin ka har'a biomass (NREL/TP-510 - 42618)47.
Tlhahlobo ea oligosaccharide ea molapo oa hydrolyzate e entsoe ka sekala sa 2 ml ho sebelisoa mokhoa oa hydrolysis ea asiti e thehiloeng ho autoclave. Kopanya sampole ea hydrolyzate le 69.7 µl ea 72% sulfuric acid ka har'a tube ea culture ea screw cap ea 10 ml 'me u e futhumale hora e le' ngoe holim'a benchtop ho 121 °C, u e pholise leqhoeng 'me u e sefe ka har'a vial ea liquid chromatography (HPLC) e sebetsang hantle. Khakanyo ea oligosaccharides e ile ea fumanoa ka ho tlosa khakanyo ea li-monosaccharides ka har'a sampole e sa hiriloeng ho tsoa khakanyong eohle ea tsoekere ka har'a sampole e hiriloeng ke asiti.
Maqhubu a tsoekere, xylose, le arabinose ka har'a acid hydrolyzed biomass a ile a hlahlojoa ho sebelisoa sistimi ea Shimadzu HPLC e nang le autosampler, column heater, isocratic pump, le refractive index detector kholomong ea Bio-Rad Aminex HPX-87H. Kholomo e ile ea bolokoa ho 50°C 'me ea tlosoa ka 0.6 ml/min 5 mM H2SO4 ka har'a metsi. phallo.
Supernatant ea hydrolyzate e ile ea hlapolloa 'me ea hlahlojoa bakeng sa litaba tsa monomer le oligosaccharide. Tsoekere ea monomeric e fumanoeng kamora hydrolysis ea enzymatic e ile ea hlahlojoa ke HPLC e nang le kholomo ea Bio-Rad (Hercules, CA) Aminex HPX-87P le kholomo ea molora. Mocheso oa kholomo o ile oa bolokoa ho 80°C, metsi a ile a sebelisoa e le mohato oa ho tsamaea ka sekhahla sa phallo sa 0.6 ml/min. Li-oligosaccharide li ile tsa khethoa ka hydrolysis ka asiti e hlapollotsoeng ho 121°C ho latela mekhoa e hlalositsoeng ho litšupiso. 41, 48, 49.
Tlhahlobo ea Saccharide e entsoe holim'a masala a biomass a tala, a phekotsoeng esale pele a AFEX le a sa kenngoeng ka metsi (ho kenyeletsoa tlhahiso ea li-extracts tsa lebota la sele tse latellanang le tlhahlobo ea tsona ea mAb) ho sebelisoa mekhoa e hlalositsoeng pele 27, 43, 50, 51. Bakeng sa tlhahlobo ea glycome, masala a sa qhibiliheng a thepa ea lebota la sele ea limela a lokisoa ho tsoa masala a biomass 'me a ntšoa ka tatellano ka li-reagent tse matla tse kang ammonium oxalate (50 mM), sodium carbonate (50 mM le 0.5% w/v), CON. (1M le 4M, ka bobeli ka 1% w/v sodium borohydride) le acid chlorite joalo ka ha ho hlalositsoe pele52,53. Li-extracts li ile tsa kenngoa ELISA khahlanong le phanele e rarahaneng ea mAb50s e lebisitsoeng ho glycan ea lebota la sele, 'me likarabelo tsa ho tlama tsa mAb li ile tsa hlahisoa e le 'mapa oa mocheso. glycan ea lebota la sele ea semela e shebaneng le mAb e rekiloe ho tsoa li-stock tsa laboratori (CCRC, JIM le MAC series).
Mohato o le mong oa biotinylation ea li-oligosaccharide. Ho kopanngoa ha lik'habohaedreite le biotin-LC-hydrazide ho entsoe ho sebelisoa mokhoa o latelang. Biotin-LC-hydrazide (4.6 mg/12 μmol) e qhibilihisitsoe ka har'a dimethyl sulfoxide (DMSO, 70 μl) ka ho hlohlelletsa le ho futhumatsa ka matla ho 65° C. ka motsotso o le mong. Glacial acetic acid (30 µl) e ile ea eketsoa 'me motsoako o ile oa tšeloa holim'a sodium cyanoborohydride (6.4 mg/100 µmol) 'me oa qhibiliha ka botlalo ka mor'a ho futhumatsa ho 65° C. ka motsotso o le mong. Ebe ho tloha ho 5 ho isa ho 8 μl ea motsoako oa karabelo ho oligosaccharide e omisitsoeng (1-100 nmol) ho fumana molar excess ea leibole ka makhetlo a 10 kapa ho feta holim'a pheletso e fokotsang. Karabelo e ile ea etsoa ho 65°C ka lihora tse 2, ka mor'a moo lisampole li ile tsa hloekisoa hang-hang. Ha ho sodium cyanoborohydride e sebedisitsweng ditekong tsa ho ngola mabitso ntle le phokotso, mme disampole di ile tsa arajwa ka mocheso wa 65°C. ka dihora tse 2.5.
Ho koahela le ho hlatsoa lisa lisakerete tsa oligosaccharide tse nang le biotinyle. 25 μl ea lisakerete tse nang le biotinyle (100 μl ea sampole ka 'ngoe e kopantsoeng e tsoakiloeng ka 5 ml ea tharollo ea Tris buffer ea 0.1 M (TBS)) e ile ea eketsoa selibeng se seng le se seng sa poleiti e koahetsoeng ke avidin. Liliba tsa taolo li ile tsa koaheloa ka 50 μl ea biotin ka tekanyo ea 10 μg/ml ho 0.1 M TBS. Metsi a hloekisitsoeng a ile a sebelisoa e le sekoahelo bakeng sa litekanyo tse se nang letho. Letlapa le ile la beoa ka mochesong oa kamore lihora tse 2 lefifing. Hlatsoa poleiti ka makhetlo a 3 ka lebese le tlotsitsoeng ka mafura la 0.1% ho 0.1 M TBS ho sebelisoa lenaneo la nomoro ea 11 bakeng sa Grenier flat 3A.
Ho eketsa le ho hlatsoa li-antibodies tsa mantlha. Kenya 40 µl ea antibody ea mantlha selibeng se seng le se seng. Beha microplate ka hora e le 'ngoe mochesong oa kamore lefifing. Lipoleiti li ile tsa hlatsuoa ka makhetlo a 3 ka lebese la 0.1% ka har'a 0.1M TBS ho sebelisoa lenaneo la ho hlatsoa #11 bakeng sa Grenier Flat 3A.
Kenya antibody ea bobeli 'me u hlatsoe. Kenya 50 µl ea antibody ea bobeli ea toeba/rat (e hlapolotsoeng ka 1:5000 ka lebese la 0.1% ka har'a 0.1 M TBS) selibeng se seng le se seng. Beha microplate ka hora e le 'ngoe mochesong oa kamore lefifing. Li-microplate li ile tsa hlatsuoa ka makhetlo a 5 ka lebese la 0.1% ka har'a 0.1 M TBS ho sebelisoa lenaneo la ho hlatsoa lipoleiti la Grenier Flat 5A #12.
Ho eketsa substrate. Kenya 50 µl ea 3,3′, 5,5′-tetramethylbenzidine (TMB) ho substrate ea motheo (ka ho eketsa marotholi a 2 a buffer, marotholi a 3 a TMB, marotholi a 2 a hydrogen peroxide ho 15 ml ea metsi a hloekisitsoeng). Lokisa substrate ea TMB. le vortex pele u e sebelisa). Futhumatsa microplate mochesong oa kamore metsotso e 30. Lefifing.
Qetella mohato 'me u bale letlapa. Kenya 50 µl ea 1 N sulfuric acid selibeng se seng le se seng 'me u tlalehe monyelo ho tloha ho 450 ho isa ho 655 nm u sebelisa sesebelisoa sa ho bala sa ELISA.
Lokisetsa ditharollo tsa 1 mg/ml tsa di-analyte tsena ka metsing a se nang ion: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (tekanyetso ya kahare). Maemo a mabedi a lokisitsoe ka ho eketsa ditharollo tsa 1 mg/mL tsa tswekere tse bontshitsweng ho Tafole ya 1. Disampole di hoamiswa mme di lyophilized ho -80° C. ho fihlela metsi ohle a tloswa (hangata hoo e ka bang dihora tse 12-18).
Kenya 100–500 µg ea sampole ho li-screw cap tubes ka tekanyo ea tlhahlobo. Ngola bongata bo ekelitsoeng. Ho molemo ho qhala sampole ka har'a motsoako o itseng oa solvent ebe u e eketsa ka har'a tube e le metsi a mangata. Sebelisa 20 µl ea 1 mg/ml inositol e le tekanyetso ea kahare bakeng sa tube ka 'ngoe ea sampole. Bongata ba tekanyetso ea kahare e ekelitsoeng sampoleng e tlameha ho lekana le bongata ba tekanyetso ea kahare e ekelitsoeng ka har'a tube e tloaelehileng.
Kenya 8 ml ea methanol e sa hloekang ka har'a sekwahelo sa sekurufu. Ebe u tšela 4 ml ea tharollo ea 3 N. methanolic HCl, e koahetsoeng ebe e sisinngoa. Ts'ebetso ena ha e sebelise metsi.
Kenya 500 µl ea tharollo ea methanol ea 1 M HCl ho lisampole tsa oligosaccharide le lipeipi tse tloaelehileng tsa TMS. Lisampole li ile tsa beoa bosiu bohle (lihora tse 168) ho 80° C. ka har'a block ea thermal. Omisa sehlahisoa sa methanolysis mochesong oa kamore u sebelisa manifold ea ho omisa. Kenya 200 µl MeOH 'me u omise hape. Ts'ebetso ena e phetoa habeli. Kenya 200 µl ea methanol, 100 µl ea pyridine le 100 µl ea acetic anhydride ho sampole 'me u kopanye hantle. Kenya lisampole mochesong oa kamore metsotso e 30. ebe u omisa. Kenya 200 µl ea methanol ebe u omisa hape.
Kenya 200 µl ea Tri-Sil 'me u chese tube e koahetsoeng ka mocheso oa 80°C, ebe u e pholisa mochesong oa kamore. Sebelisa sesebelisoa sa ho omisa ho omisa sampole ho fihlela e le bophahamo ba hoo e ka bang 50 µl. Ho bohlokoa ho hlokomela hore ha rea ​​​​ka ra lumella lisampole ho oma ka botlalo.
Kenya 2 ml ea hexane 'me u kopanye hantle ka ho phunya metsi. Tlatsa lintlha tsa li-pipettes tsa Pasteur (5-8 mm) ka sengoathoana sa ulu ea khalase ka ho kenya ulu ea khalase holim'a pipette ea bophara ba 5-3/4 inch. Lisampole li ile tsa tšeloa ka setering ho 3000 g metsotso e 2. Masalla afe kapa afe a sa qhibiliheng aa koaheloa. Omisa sampole ho fihlela ho 100-150 µl. Bongata ba hoo e ka bang 1 μl bo ile ba kenngoa ka har'a GC-MS mochesong oa pele oa 80 °C le nako ea pele ea metsotso e 2.0 (Tafole ea 2).