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Mekhoa e mecha ea immunological le mass spectrometric bakeng sa tlhahlobo e rarahaneng ea li-oligosaccharides tse tsitsitseng ka setofong sa poone se entsoeng ka AFEX.Lignocellulosic biomass ke mokhoa o ts'oarellang ho fapana le libeso tsa khale 'me e sebelisoa haholo ho nts'etsapele theknoloji ea bioloji bakeng sa tlhahiso ea lihlahisoa tse kang lijo, phepo, libeso le lik'hemik'hale.Senotlolo sa mahlale ana ke nts'etsopele ea mekhoa ea tlholisano ea litšenyehelo bakeng sa ho fetola lik'habohaedreite tse rarahaneng tse teng maboteng a lisele tsa semela hore e be tsoekere e bonolo joalo ka glucose, xylose le arabinose.Hobane lignocellulosic biomass e manganga haholo, e tlameha ho etsoa tlas'a phekolo ea thermochemical (mohlala, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic liquids (IL)) le phekolo ea likokoana-hloko (mohlala, enzymatic hydrolysis le microbial fermentation) hammoho ho fumana sehlahisoa se lakatsehang..Leha ho le joalo, ha li-enzyme tsa fungal tsa khoebo li sebelisoa ts'ebetsong ea hydrolysis, ke 75-85% feela ea tsoekere e qhibilihang e entsoeng ke monosaccharides, 'me karolo e setseng ea 15-25% ke li-oligosaccharides tse sa tsitsang, tse sa fumaneheng kamehla ho likokoana-hloko.Nakong e fetileng, re atlehile ho arola le ho hloekisa li-oligosaccharides tse manganga tse qhibilihang ka ho sebelisa motsoako oa karohano ea lefats'e ea khabone le diatomaceous le boholo ba chromatography ea ho se kenyelle, hape ra batlisisa thepa ea bona ea ho thibela enzyme.Re fumane hore li-oligosaccharides tse nang le tekanyo e phahameng ea polymerization (DP) methylated uronic acid substitutes li thata haholo ho sebetsana le metsoako ea enzyme ea khoebo ho feta DP e tlaase le oligosaccharides e sa nke lehlakore.Mona re tlaleha ts'ebeliso ea mekhoa e 'maloa ea tlatsetso, ho kenyeletsoa profiling ea glycan e sebelisang li-antibodies tsa monoclonal (mAbs) tse tobileng ho lema biomass glycans ho tšoaea bond ea glycan maboteng a lisele tsa semela le li-enzymatic hydrolysates, matrix-assisted laser desorption ionization, nako ea ho fofa mass- spectrometry..MALDI-TOF-MS) e sebelisa litlhōrō tse nang le tsebo ea sebopeho tse fumanoang ke spectroscopy ka mor'a ho bola ha bobeli ba li-ion tse mpe, chromatography ea khase le mass spectrometry (GC-MS) ho khetholla li-oligosaccharide tse nang le li-derivatization le ntle le ho tsoa.Ka lebaka la boholo bo bonyenyane ba li-oligosaccharides (DP 4-20), limolek'hule tsena ho thata ho li sebelisa bakeng sa ho tlama le ho khetholla ha mAb.Ho hlola bothata bona, re ile ra sebelisa mokhoa o mocha oa biotin conjugation-based oligosaccharide immobilization o atlehileng ho ngola boholo ba li-oligosaccharides tse tlase tsa DP soluble holim'a microplate, tse ileng tsa sebelisoa ka mokhoa o phahameng oa throughput mAb bakeng sa tlhahlobo e khethehileng ea ligation.Mokhoa ona o mocha o tla thusa ho nts'etsopele ea litlhahlobo tse tsoetseng pele tsa glycome nakong e tlang tse ka sebelisoang ho arola le ho khetholla li-oligosaccharides tse teng ho li-biomarker bakeng sa merero ea tlhahlobo.
Lignocellulosic biomass, e entsoeng ka thepa ea temo, meru, joang le mapolanka, ke lijo tse ka sebelisoang bakeng sa tlhahiso ea lihlahisoa tse thehiloeng ho bioloji, ho kenyeletsoa lijo, lijo, mafura le li-precursors tsa lik'hemik'hale ho hlahisa lihlahisoa tsa boleng bo holimo1.Lik'habohaedreite (tse kang selulose le hemicellulose) tse teng maboteng a lisele tsa semela li theoleloa ho ba monosaccharides ka ho sebetsa ka lik'hemik'hale le biotransformation (joalo ka enzymatic hydrolysis le microbial fermentation).Mekhoa e tloaelehileng ea phekolo ea pele e kenyelletsa ammonia fiber expansion (AFEX), dilute acid (DA), ionic liquid (IL), le steam explosion (SE), e sebelisang motsoako oa lik'hemik'hale le mocheso ho fokotsa tlhahiso ea lignocellulose ka ho bula marako a lisele tsa semela3,4.manganga a taba, 5. Enzymatic hydrolysis e etsoa ka boima bo bongata bo matla ho sebelisoa lik'hemik'hale tse sebetsang tsa lik'habohaedreite tse nang le lik'habohaedreite (CAZymes) le ho belisoa ha likokoana-hloko ho sebelisa tomoso ea transgenic kapa libaktheria ho hlahisa mafura le lik'hemik'hale tse thehiloeng linthong tse phelang 6 .
Li-CAZyme tsa li-enzyme tsa khoebo li entsoe ka motsoako o rarahaneng oa li-enzyme tse kopanyang ka kopanelo li-bond tse rarahaneng tsa carbohydrate-tsoekere ho etsa monosaccharides2,7.Joalokaha re tlalehile pejana, marang-rang a rarahaneng a li-polymers tse nkhang hamonate tsa lignin tse nang le lik'habohaedreite li etsa hore li se ke tsa senyeha haholo, tse lebisang phetohong e sa fellang ea tsoekere, ho bokella 15-25% ea li-oligosaccharides tsa thobalano tse sa hlahisoang nakong ea hydrolysis ea enzymatic ea biomass e nkiloeng pele.Ena ke bothata bo atileng ka mekhoa e fapaneng ea kalafo ea biomass.Mabaka a mang a botlolo ena a kenyelletsa thibelo ea enzyme nakong ea hydrolysis, kapa ho ba sieo kapa maemo a tlase a li-enzyme tsa bohlokoa tse hlokahalang ho khaola maqhama a tsoekere ho biomass ea semela.Ho utloisisa sebopeho le litšobotsi tsa sebopeho sa tsoekere, joalo ka litlamo tsa tsoekere ho oligosaccharides, ho tla re thusa ho ntlafatsa phetoho ea tsoekere nakong ea hydrolysis, ho etsa hore lits'ebetso tsa biotechnological li qothisane lehlokoa le lihlahisoa tse entsoeng ka peterole.
Ho fumana sebopeho sa lik'habohaedreite ho thata 'me ho hloka motsoako oa mekhoa e kang liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 le mass spectrometry (MS)17., leshome le metso e robeli.Mekhoa ea MS e kang ea nako ea sefofaneng mass spectrometry e nang le laser desorption le ionization sebelisa matrix (MALDI-TOF-MS) ke mokhoa o fapaneng oa ho khetholla mekhoa ea lik'habohaedreite.Haufinyane tjena, Collision-Induced Dissociation (CID) tandem MS ea sodium ion adducts e 'nile ea sebelisoa ka ho fetisisa ho khetholla menoana ea menoana e lumellanang le maemo a li-attachment tsa oligosaccharide, litlhophiso tsa anomeric, tatellano le maemo a makala a 20, 21.
Tlhahlobo ea Glycan ke sesebelisoa se setle sa ho tsebahatsa bond22 ea lik'habohaedreite.Mokhoa ona o sebelisa li-antibodies tsa monoclonal (mAbs) tse lebisitsoeng ho jala lerako la sele ea glycan e le lipatlisiso tsa ho utloisisa likamano tse rarahaneng tsa lik'habohaedreite.Ho na le li-mAbs tse fetang 250 lefatšeng ka bophara, tse etselitsoeng khahlano le li-oligosaccharides tse fapaneng tse nang le mela le makala ho sebelisoa li-saccharides tse fapaneng24.Li-mAbs tse 'maloa li sebelisitsoe haholo ho khetholla sebopeho, sebopeho, le liphetoho tsa lerako la sele ea semela, kaha ho na le phapang e kholo ho latela mofuta oa sele ea semela, setho, lilemo, sethala sa nts'etsopele, le tikoloho ea kholo25,26.Haufinyane tjena, mokhoa ona o 'nile oa sebelisoa ho utloisisa palo ea li-vesicles tsamaisong ea limela le liphoofolo le likarolo tsa tsona tse fapaneng tsa ho tsamaisa glycan joalokaha ho laetsoe ke li-subcellular marker, mekhahlelo ea tsoelo-pele, kapa tšusumetso ea tikoloho, le ho tseba hore na mosebetsi oa enzymatic ke ofe.Tse ling tsa meaho e fapaneng ea glycans le xylans e 'nileng ea khetholloa e kenyelletsa pectin (P), xylan (X), mannan (M), xyloglucans (XylG), li-glucans tse tsoakiloeng (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxy-XbGGGGG9 le2Arbinoxylan (2) ArbXbGG.
Leha ho le joalo, ho sa tsotellehe boiteko bona bohle ba lipatlisiso, ke liphuputso tse 'maloa feela tse lebisitseng tlhokomelo ea tlhaho ea oligosaccharide e bokellang nakong ea hydrolysis e phahameng ea solids load (HSL), ho kenyelletsa le tokollo ea oligosaccharide, liphetoho tsa bolelele ba ketane ea oligomeric nakong ea hydrolysis, li-polymers tse fapaneng tse tlaase tsa DP, le li-curve tsa tsona.kabo 30,31,32.Ho sa le joalo, le hoja tlhahlobo ea glycan e ipakile e le sesebelisoa se molemo bakeng sa tlhahlobo e felletseng ea sebopeho sa glycan, ho thata ho lekola li-oligosaccharides tsa DP tse sa qhibilihang ka metsing ho sebelisa mekhoa ea li-antibody.Li-oligosaccharides tse nyane tsa DP tse nang le boima ba molek'hule tse ka tlase ho 5-10 kDa ha li tlamelle lipoleiti tsa ELISA 33, 34 'me li hlatsuoa pele ho tlatsetso ea antibody.
Mona, ka lekhetlo la pele, re bonts'a tlhahlobo ea ELISA ka lipoleiti tse koahetsoeng ke avidin tse sebelisang li-antibodies tsa monoclonal, ho kopanya mokhoa o le mong oa biotinylation oa li-oligosaccharides tse qhibilihang ka har'a glycome.Mokhoa oa rona oa tlhahlobo ea glycome o netefalitsoe ke tlhahlobo e thehiloeng ho MALDI-TOF-MS le GC-MS ea likhokahano tse tlatselletsang tsa oligosaccharide tse sebelisang tlhahiso ea trimethylsilyl (TMS) ea metsoako ea tsoekere ea hydrolyzed.Mokhoa ona o mocha o ka ntlafatsoa e le mokhoa o phahameng oa katleho nakong e tlang mme oa fumana ts'ebeliso e batsi ho lipatlisiso tsa biomedical35.
Liphetoho tsa morao-rao tsa li-enzyme le li-antibodies, tse kang glycosylation,36 li ama mosebetsi oa bona oa likokoana-hloko.Ka mohlala, liphetoho tsa glycosylation ea liprotheine tsa serum li phetha karolo ea bohlokoa ho ramatiki ea ho ruruha, 'me liphetoho tsa glycosylation li sebelisoa e le matšoao a ho hlahloba37.Li-glycans tse fapa-fapaneng li 'nile tsa tlalehoa libukeng hore li ka hlaha habonolo maloetseng a sa tšoaneng, ho akarelletsa le mafu a sa foleng a ho ruruha a pampiri ea mala le sebete, tšoaetso ea kokoana-hloko, mae a bomme, matsoele le kankere ea prostate38,39,40.Ho utloisisa sebopeho sa li-glycans ho sebelisa mekhoa ea li-antibody-based glycan ELISA ho tla fana ka kholiseho e eketsehileng tabeng ea ho hlahloba mafu ntle le ho sebelisa mekhoa e rarahaneng ea MS.
Boithuto ba rona bo fetileng bo bonts'itse hore li-oligosaccharides tse manganga li ile tsa lula li se na hydrolyzed ka mor'a hore li sebelisoe pele le enzymatic hydrolysis (Setšoantšo sa 1).Mosebetsing oa rona o phatlalalitsoeng pejana, re thehile mokhoa oa ho ntša mashala a tiileng ho arola li-oligosaccharides ho AFEX-pretreated corn stover hydrolyzate (ACSH)8.Ka mor'a ho ntšoa le ho arohana ha pele, li-oligosaccharides li ile tsa boela tsa aroloa ka boholo ba chromatography (SEC) 'me tsa bokelloa ka tatellano ea boima ba limolek'hule.Li-monomer tsa tsoekere le li-oligomers tse lokollotsoeng ho tsoa mefuteng e fapaneng ea kalafo li ile tsa hlahlojoa ka tlhahlobo ea sebopeho sa tsoekere.Ha ho bapisoa likahare tsa li-oligomers tsa tsoekere tse fumanoeng ka mekhoa e fapaneng ea pele ho kalafo, boteng ba li-oligosaccharides tse manganga ke bothata bo tloaelehileng ba phetoho ea biomass ho monosaccharides mme bo ka lebisa ho fokotseng chai ea tsoekere bonyane 10-15% esita le ho fihla ho 18%.USMokhoa ona o sebelisoa bakeng sa tlhahiso e kholo ea likaroloana tsa oligosaccharide.Sephetho sa ACH le likaroloana tsa eona tse latelang tse nang le boima ba limolek'hule tse fapaneng li ile tsa sebelisoa e le lisebelisoa tsa liteko bakeng sa ho khetholla li-oligosaccharides mosebetsing ona.
Ka mor'a ho phekoloa pele le ho etsa hydrolysis ea enzymatic, li-oligosaccharides tse phehellang li ile tsa lula li sa sebetse.Mona (A) ke mokhoa oa ho arohana ha oligosaccharide oo li-oligosaccharides li arohaneng le AFEX-pretreated corn stover hydrolyzate (ACSH) ho sebelisa bethe e tletseng ea carbon activated le diatomaceous earth;(B) Mokhoa oa ho arola li-oligosaccharides.Li-oligosaccharides li ile tsa boela tsa aroloa ka boholo ba ho khetholla chromatography (SEC);(C) Li-saccharide monomers le li-oligomers tse lokollotsoeng ho tsoa ho li-pretreatments tse fapaneng (e hlapolotsoeng ka acid: DA, mokelikeli oa ionic: IL le AFEX).Maemo a enzymatic hydrolysis: ho jara lintho tse tiileng tse phahameng tsa 25% (w/w) (hoo e ka bang 8% ea glucan loading), lihora tse 96 hydrolysis, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 ratio) le (D) Sugar monomers le oligomers tsa glucose, xybinover EXA xylover cornAFCSA e lokollotsoeng.
Tlhahlobo ea Glycan e ipakile e le sesebelisoa se sebetsang bakeng sa tlhahlobo e felletseng ea sebopeho sa li-glycans ho li-extracts tse arohaneng le masala a tiileng a biomass.Leha ho le joalo, li-saccharide tse qhibilihang ka metsing ha li sebelisoe ka mokhoa ona oa setso41 hobane li-oligosaccharides tse boima ba limolek'hule li thata ho thibela lipoleiti tsa ELISA 'me li hlatsuoa pele ho tlatsetso ea antibody.Ka hona, bakeng sa ho tlama le ho khetholla li-antibody, mokhoa o le mong oa biotinylation o ne o sebelisoa ho apara li-oligosaccharides tse qhibilihang, tse sa lumellaneng le lipoleiti tsa ELISA tse koahetsoeng ke avidin.Mokhoa ona o ile oa lekoa ho sebelisoa ACSH ea rona e hlahisitsoeng pele le karoloana e ipapisitseng le boima ba molek'hule (kapa degree of polymerization, DP).Biotinylation ea mohato o le mong e ne e sebelisetsoa ho eketsa kamano e tlamang ea oligosaccharide ka ho eketsa biotin-LC-hydrazide ho ea qetellong ea lik'habohaedreite (setšoantšo sa 2).Ka tharollo, sehlopha sa hemiacetal qetellong ea ho fokotsa se sebetsana le sehlopha sa hydrazide sa biotin-LC-hydrazide ho theha bond ea hydrazone.Ka pel'a moemeli oa ho fokotsa NaCNBH3, tlamahano ea hydrazone e fokotsehile hore e be sehlahisoa se tsitsitseng sa ho qetela sa biotinylated.Ka phetoho ea qetello ea ho fokotsa tsoekere, ho tlama li-oligosaccharides tse tlaase tsa DP ho lipoleiti tsa ELISA ho ile ha khoneha, 'me thutong ea rona sena se entsoe ka lipoleiti tse koahetsoeng ke avidin tse sebelisang mAbs tse lebisitsoeng ho glycan.
Ho hlahlojoa ha li-antibodies tsa monoclonal tse thehiloeng ho ELISA bakeng sa li-oligosaccharides tsa biotinylated.Mona (A) biotinylation e kopantsoeng ea li-oligosaccharides le tlhahlobo ea ELISA e latelang e nang le glycan-targeted mAbs holim'a lipoleiti tse koahetsoeng ke NeutrAvidin le (B) e bonts'a mokhoa oa mohato o le mong bakeng sa biotinylation ea lihlahisoa tsa karabelo.
Lipoleiti tse nang le li-avidin tse nang le li-antibodies tsa oligosaccharide-conjugated li ile tsa kenngoa ho li-antibodies tsa mantlha le tsa bobeli 'me tsa hlatsuoa ka mokhoa o bobebe le oa nako.Ka mor'a hore ho tlamisoa ha li-antibody ho phethetsoe, eketsa substrate ea TMB ho kenya poleiti.Karabelo e ile ea qetella e emisitsoe ka asiti ea sulfuric.Lipoleiti tse kentsoeng li ile tsa hlahlobjoa ho sebelisoa sebali sa ELISA ho fumana matla a tlamang a lesole le leng le le leng ho lemoha ho hokahana ho itseng ha antibody.Bakeng sa lintlha le litekanyetso tsa teko, bona karolo e tsamaellanang ea "Lisebelisoa le Mekhoa".
Re bonts'a ts'ebeliso ea mokhoa ona o sa tsoa ntlafatsoa bakeng sa lits'ebetso tse ikhethileng ka ho khetholla li-oligosaccharides tse qhibilihang tse teng ho ACSH hammoho le likaroloana tsa oligosaccharide tse tala le tse hloekisitsoeng tse arohaneng le lignocellulosic hydrolysates.Joalo ka ha ho bonts'itsoe ho Setšoantšo sa 3, li-xylans tse atileng haholo tse nkeloang sebaka ke epitope tse khethiloeng ho ACSH li sebelisa mekhoa ea tlhahlobo ea glycome ea bioacylated hangata ke uronic (U) kapa methyluronic (MeU) le pectic arabinogalactans.Bongata ba tsona li ile tsa fumanoa hape thutong ea rona e fetileng mabapi le tlhahlobo ea li-glycans tsa lintho tse thata tse se nang hydrolyzed (UHS)43.
Ho fumanoa ha li-epitopes tsa oligosaccharide recalcitrant ho sebelisa monoclonal antibody e lebisitsoeng leboteng la sele ea glycan.Karolo ea "ho se nke lehlakore" ke karoloana ea ACN 'me karolo ea "acidic" ke karolo ea FA.Bofubelu bo khanyang 'mapeng oa mocheso bo bontša litaba tse holimo tsa epitope, 'me boputsoa bo khanyang bo bontša bokamorao bo se nang letho.Litekanyetso tsa mebala sekaleng li ipapisitse le litekanyetso tse tala tsa OD bakeng sa liforomo N=2.Li-epitopes tse kholo tse amoheloang ke li-antibodies li bonts'oa ka ho le letona.
Mehaho ena e se nang cellulose e ne e ke ke ea phunyeletsoa ke li-cellulase le li-hemicellulase tse tloaelehileng ka har'a motsoako o lekiloeng oa enzyme ea khoebo, o kenyelletsang li-enzyme tse sebelisoang ka ho fetisisa tsa khoebo.Ka hona, li-enzyme tse ncha tse thusang lia hlokahala bakeng sa hydrolysis ea tsona.Ntle le li-enzyme tse hlokahalang tse se nang cellulose, litlamo tsena tse se nang cellulose li thibela phetoho e felletseng ho li-monosaccharides, leha li-polymers tsa tsoekere ea motsoali li tšeloa haholo ka likhechana tse khutšoane ebe li qhibiliha ka ho sebelisa metsoako ea enzyme ea khoebo.
Boithuto bo eketsehileng ba kabo ea matšoao le matla a eona a tlamang bo bontšitse hore li-epitopes tse tlamang li ne li le tlase ho likarolo tse phahameng tsa tsoekere tsa DP (A, B, C, DP ho fihlela ho 20 +) ho feta likaroloana tse tlase tsa DP (D, E, F, DP) ka dimers) (setšoantšo sa 1).Likhechana tsa asiti li atile haholo li-epitopes tse se nang cellulose ho feta likhechana tse sa nke lehlakore.Liketsahalo tsena li lumellana le mokhoa o hlokometsoeng thutong ea rona e fetileng, moo likarolo tse phahameng tsa DP le acid li neng li hanyetsana le enzymatic hydrolysis.Ka hona, ho ba teng ha li-non-cellulose glycan epitopes le li-substitutes tsa U le MeU li ka kenya letsoho haholo ho tsitsitseng ha li-oligosaccharides.Hoa lokela ho hlokomeloa hore katleho ea ho tlama le ho lemoha e ka ba bothata bakeng sa li-oligosaccharides tse tlaase 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.
Ka hona, tšebeliso ea li-antibodies tse khethehileng tsa sebopeho li ile tsa senola mefuta e itseng ea litlamo tsa recalcitrant.Ho ipapisitsoe le mofuta oa antibody o sebelisitsoeng, mokhoa o nepahetseng oa ho kopanya, le matla a lets'oao leo le le hlahisang (le ngata haholo ebile le le ngata haholo), li-enzyme tse ncha li ka tsejoa 'me tsa eketsoa ka semi-quantitatively ho motsoako oa enzyme bakeng sa phetoho e felletseng ea glycoconversion.Ho nka tlhahlobo ea li-oligosaccharides tsa ACSH e le mohlala, re ka etsa database ea li-bond 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 nkoa, 'me haeba kamano ea bona e sa tsejoe, sena se tla baka mathata a itseng ha ho bapisoa lipontšo tsa li-antibodies tse fapaneng.Ho feta moo, ho bapisa li-bond tsa glycan ho ka sebetsa hantle lipakeng tsa lisampole tsa antibody e tšoanang.Joale maqhama ana a manganga a ka amahanngoa le database ea CAZyme, eo ho eona re ka khethollang li-enzyme, re khetha li-enzyme tse khethiloeng le ho hlahloba li-enzyme tse senyang maqhama, kapa ho hlahisa mekhoa ea likokoana-hloko ho hlahisa li-enzyme tsena bakeng sa tšebeliso ea biorefineries44.
Ho lekola hore na mekhoa ea immunology e tlatsana joang le mekhoa e meng ea ho khetholla li-oligosaccharides tse tlaase tsa molek'hule tse teng ka lignocellulosic hydrolysates, re ile ra etsa MALDI (setšoantšo sa 4, S1-S8) le tlhahlobo ea li-saccharides tse entsoeng ka TMS tse thehiloeng ho GC-MS ka phanele e le 'ngoe (Setšoantšo sa 5) karolo ea oligosaccharide.MALDI e sebelisetsoa ho bapisa hore na kabo ea bongata ea limolek'hule tsa oligosaccharide e lumellana le sebopeho se reriloeng.Ka feiga.4 e bontša MC ea likarolo tse sa nke lehlakore ACN-A le ACN-B.Tlhahlobo ea ACN-A e netefalitse mefuta e mengata ea tsoekere ea pentose ho tloha DP 4-8 (setšoantšo sa 4) ho ea ho DP 22 (setšoantšo sa S1), eo boima ba eona bo lumellanang le li-oligosaccharides tsa MeU-xylan.Tlhahlobo ea ACN-B e netefalitse letoto la pentose le glucoxylan le DP 8-15.Linthong tsa tlatsetso tse kang Figure S3, FA-C acidic moiety mass distribution limmapa li bonts'a mefuta e mengata ea (Me) U e nkileng sebaka sa tsoekere ea pentose ka DP ea 8-15 e tsamaellanang le li-xylans tse nketsoeng sebaka tse fumanehang tlhahlobong ea ELISA-based mAb.Li-epitopes lia lumellana.
MALDI-MS spectrum ea li-oligosaccharides tse qhibilihang tse sa lumellaneng tse teng ho ACS.Mona, (A) ACN-A likaroloana tse fokolang tsa boima bo tlaase tse nang le methylated uronic acid (DP 4-8) li nkela sebaka sa glucuroxylan oligosaccharides le (B) ACN-B xylan le methylated uronic acid oligosaccharides e nkeloang sebaka ke glucuroxylan (DP 8-15).
Tlhahlobo ea sebopeho sa masala a glycan a li-oligosaccharides tsa refractory.Mona (A) sebopeho sa saccharide sa TMS sa likaroloana tse fapaneng tsa oligosaccharide tse fumanoeng ho sebelisoa tlhahlobo ea GC-MS.(B) Mehaho ea mefuta e fapaneng ea tsoekere e tsoang ho TMS e teng ka li-oligosaccharides.ACN - karoloana ea acetonitrile e nang le li-oligosaccharides tse sa nke lehlakore le FA - karoloana ea ferulic acid e nang le oligosaccharides ea acid.
Qeto e 'ngoe e thahasellisang e ile ea etsoa ho tsoa tlhahlobisong ea LC-MS ea karoloana ea oligosaccharide, joalokaha ho bontšoa ho Setšoantšo sa S9 (mekhoa e ka bonoa ka thepa ea tlatsetso ea elektronike).Likaroloana tsa lihlopha tsa hexose le -OAc li ile tsa hlokomeloa khafetsa nakong ea ligation ea karoloana ea ACN-B.Se fumanoeng sena ha se tiise feela karohano e hlokometsoeng tlhahlobisong ea glycome le MALDI-TOF, empa hape e fana ka leseli le lecha mabapi le lintho tse ka bang teng tsa lik'habohaedreite ho biomass ea lignocellulosic.
Re boetse re hlahlobile sebopeho sa tsoekere sa karolo ea oligosaccharide re sebelisa ts'ebetso ea tsoekere ea TMS.Re sebelisa GC-MS, re ile ra etsa qeto ea ho hlophisoa ha neural (non-derivative) le tsoekere ea acidic (GluA le GalA) karolong ea oligosaccharide (setšoantšo sa 5).Asiti ea Glucuronic e fumanoa likarolong tse nang le asiti C le D, ha galacturonic acid e fumanoa likarolong tse nang le asiti A le B, tseo ka bobeli e leng likarolo tse phahameng tsa DP tsa tsoekere ea asiti.Liphetho tsena ha li tiise data ea rona ea ELISA le MALDI feela, empa hape li lumellana le lithuto tsa rona tse fetileng tsa ho bokella 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 bona li-oligosaccharides tsa soluble recalcitrant ka lisampole tse fapaneng tsa baeloji.
Kaha mekhoa ea tlhahlobo ea mAb e thehiloeng ho ELISA e netefalitsoe ke mekhoa e mengata e fapaneng, re ne re batla ho tsoela pele ho hlahloba bokhoni ba mokhoa ona o mocha oa palo.Li-oligosaccharides tse peli tsa khoebo, xylohexasaccharide oligosaccharide (XHE) le 23-α-L-arabinofuranosyl-xylotriose (A2XX), li ile tsa rekoa 'me tsa lekoa ka mokhoa o mocha oa mAb o lebisang leboteng la sele ea glycan.Setšoantšo sa 6 se bonts'a khokahano e lipakeng tsa lets'oao le tlamang la biotinylated le lets'oao la li-oligosaccharide, le fana ka maikutlo a hore ho na le mohlala oa adsorption oa Langmuir.Har'a li-mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, le CCRC-M151 e amanang le XHE, le CCRC-M108, CCRC-M109, le LM11 e amanang le A2XX ho feta 100 nmno.Ka lebaka la ho fumaneha ho fokolang ha li-antibodies nakong ea teko, liteko tse fokolang li ile tsa etsoa ka mahloriso a mang le a mang a oligosaccharide.Hoa lokela ho hlokomeloa mona hore li-antibodies tse ling li itšoara ka tsela e fapaneng haholo ho oligosaccharide e tšoanang le substrate, mohlomong hobane li tlama li-epitopes tse fapaneng hanyenyane 'me li ka ba le likamano tse fapaneng haholo tse tlamang.Mekhoa le litlamorao tsa boitsebahatso bo nepahetseng ba epitope li tla ba thata haholo ha mokhoa o mocha oa mAb o sebelisoa ho lisampole tsa 'nete.
Li-oligosaccharides tse peli tsa khoebo li ile tsa sebelisoa ho fumana hore na ho na le mefuta e fapaneng ea glycan-targeting mAbs.Mona, likhokahano tsa linear le log concentration tsa oligosaccharide li bonts'a lipaterone tsa Langmuir adsorption bakeng sa (A) XHE e nang le mAb le (B) A2XX e nang le mAb.Li-epitopes tse lumellanang li bonts'a meaho ea li-oligosaccharides tsa khoebo tse sebelisoang e le li-substrates tlhahlobong.
Tšebeliso ea glycan-targeted monoclonal antibodies (glycocomic analysis kapa ELISA-based mAb screening) ke sesebelisoa se matla sa ho khetholla ka botebo boholo ba lisele tse kholo tsa lerako la glycans tse etsang limela tsa limela.Leha ho le joalo, tlhahlobo ea glycan ea khale e bontša feela li-glycans tse kholoanyane tsa lisele, kaha li-oligosaccharides tse ngata ha li sebetse hantle lipoleiting tsa ELISA.Thutong ena, AFEX-pretreated corn stover e ne e le enzymatic hydrolyzed ka har'a lintho tse tiileng tse phahameng.Ho ile ha sebelisoa tlhahlobo ea tsoekere ho fumana hore na lik'habohaedreite tsa lerako la recalcitrant li entsoe joang ka har'a hydrolyzate.Leha ho le joalo, tlhahlobo ea mAb ea li-oligosaccharides tse nyenyane ka har'a hydrolysates ha e lekane, 'me ho hlokahala lisebelisoa tse eketsehileng bakeng sa ho thibela li-oligosaccharides ka katleho lipoleiti tsa ELISA.
Mona re tlaleha mokhoa o mocha le o sebetsang oa oligosaccharide immobilization bakeng sa tlhahlobo ea mAb ka ho kopanya oligosaccharide biotinylation e lateloang ke tlhahlobo ea ELISA ho lipoleiti tse koahetsoeng tsa NeutrAvidin™.Li-oligosaccharides tsa immobilized biotinylated li bontšitse kamano e lekaneng bakeng sa antibody ho thusa ho lemoha ka potlako le ka katleho li-oligosaccharides tsa recalcitrant.Tlhahlobo ea ho hlophisoa ha li-oligosaccharides tsena tse manganga tse thehiloeng holim'a spectrometry ea boima li netefalitse liphello tsa mokhoa ona o mocha oa ho itšireletsa mafung.Ka hona, liphuputso tsena li bontša hore motsoako oa oligosaccharide biotinylation le ELISA screening ka glycan-targeted monoclonal antibodies e ka sebelisoa ho lemoha li-crosslink tsa oligosaccharides 'me e ka sebelisoa haholo lithutong tse ling tsa biochemical tse khethollang sebopeho sa oligosaccharides.
Mokhoa ona oa profiling oa glycan o thehiloeng ho biotin ke tlaleho ea pele e khonang ho batlisisa litlamo tsa recalcitrant carbohydrate tsa oligosaccharides tse qhibilihang ka har'a biomass ea semela.Sena se thusa ho utloisisa hore na ke hobane'ng ha likarolo tse ling tsa biomass li le manganga ha ho tluoa tabeng ea tlhahiso ea biofuel.Mokhoa ona o tlatsa lekhalo la bohlokoa mekhoeng ea ho hlahloba glycome le ho atolosa tšebeliso ea eona ho mefuta e mengata ea li-substrates ho feta li-oligosaccharides tsa limela.Nakong e tlang, re ka sebelisa liroboto bakeng sa biotinylation le ho sebelisa mokhoa oo re o entseng bakeng sa tlhahlobo e phahameng ea lisampole re sebelisa ELISA.
Corn straw (CS) e jetsoeng ho tsoa ho peo e nyalisitsoeng ea Pioneer 33A14 e ile ea kotuloa ka 2010 ho tsoa Kramer Farms e Ray, Colorado.Ka tumello ea mong'a mobu, biomass ena e ka sebelisoa bakeng sa lipatlisiso. Mehlala e ne e bolokoa e omme <6% mongobo ka mekotleng ea zip-lock ka mocheso oa kamore. Mehlala e ne e bolokoa e omme <6% mongobo ka mekotleng ea zip-lock ka mocheso oa kamore. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Mehlala e ne e bolokoa e omme ho <6% mongobo ka mekotleng e kentsoeng ka zippering mocheso oa 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%. Mehlala e bolokoa ka mekotleng ea zipper mocheso oa kamore e nang le mongobo <6%.Boithuto bona bo ipapisitse le litataiso tsa lehae le tsa naha.Tlhahlobo ea comppositional e ile ea etsoa ho sebelisoa protocol ea NREL.Sebopeho se fumanoe se na le 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protheine le 13. 4% molora.
Cellic® CTec2 (138 mg protein / ml, lot VCNI 0001) ke motsoako o rarahaneng oa cellulase, β-glucosidase le Cellic® HTec2 (157 mg protein / ml, lot VHN00001) ho tloha Novozymes (Franklinton, NC, USA) .Multifect Pectinase® (72 mg protein/mL), motsoako o rarahaneng oa li-enzyme tse senyang pectin, li fanoe ke DuPont Industrial Biosciences (Palo Alto, CA, USA).Maemo a protheine ea enzyme a ile a khethoa ka ho hakanya litaba tsa protheine (le ho tlosa tlatsetso ea naetrojene e seng protheine) ho sebelisoa tlhahlobo ea nitrogen ea Kjeldahl (mokhoa oa AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Diatomaceous earth 545 e ile ea rekoa ho EMD Millipore (Billerica, MA).Activated carbon (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, le lik'hemik'hale tse ling kaofela li ile tsa rekoa Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment e entsoe GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Kalafo ea pele e ile ea etsoa ka 140 ° C. metsotso e 15.46 nako ea ho lula sebakeng sa 1:1 karo-karolelano ea anhydrous ammonia ho biomass ho 60% (w/w) e kenang ka har'a mochini o kopanyang oa tšepe o sa hloekang oa benche (Parr Instruments Company).Ho nkile metsotso e 30.Reactor e ile ea tlisoa ho 140 ° C mme ammonia e ile ea lokolloa ka potlako, e lumella biomass hore e khutlele ka potlako mocheso oa kamore.Sebopeho sa AFEX pre-treated corn stover (ACS) se ne se tšoana le setofo sa poone se sa sebetsoang (UT-CS).
Lintho tse tiileng tse phahameng ACSH 25% (w/w) (hoo e ka bang 8% dextran loading) e ile ea lokisoa e le thepa ea ho qala bakeng sa tlhahiso e kholo ea li-oligosaccharides.Enzymatic hydrolysis ea ACS e ile ea etsoa ho sebelisoa motsoako oa enzyme ea khoebo ho akarelletsa le Cellic® Ctec2 10 mg protheine / g glucan (ka biomass pretreated), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, le Multifect Pectinase (Genencor Inc, USA).).), 5 mg protheine/g dextran.Enzymatic hydrolysis e ne e etsoa ka bioreactor ea 5-liter e nang le molumo o sebetsang oa lilithara tse 3, pH 4.8, 50 ° C le 250 rpm.Ka mor'a hydrolysis bakeng sa lihora tse 96, hydrolyzate e ile ea bokelloa ka centrifugation ka 6000 rpm bakeng sa metsotso ea 30 ebe ka 14000 rpm bakeng sa metsotso ea 30 ho tlosa lintho tse tiileng tse se nang metsi.Hydrolyzate e ile ea kenngoa ts'ebetsong e sa hloekang ka beakere ea filthara ea 0.22 mm.Hydrolyzate e tlhotliloeng e ne e bolokoa ka libotlolong tse hloekisitsoeng ho 4° C. ebe e aroloa ka carbon.
Tlhahlobo ea ho hlophisoa ha disampole tse thehiloeng ho biomass ho latela mekhoa ea tlhahlobo ea laboratori ea NREL: ho lokisoa ha lisampole bakeng sa tlhahlobo ea sebopeho (NREL/TP-510-42620) le qeto ea sebopeho sa lik'habohaedreite le lignin ka biomass (NREL/TP-510 - 42618)47.
Tlhahlobo ea oligosaccharide ea phallo ea hydrolyzate e entsoe ka tekanyo ea 2 ml ho sebelisa mokhoa oa autoclave-based acid hydrolysis.Kopanya sampole ea hydrolyzate le 69.7 µl ea 72% sulfuric acid ka har'a 10 ml screw cap culture tube 'me u e kenye hora e le 'ngoe holim'a benche holim'a 121 °C, u pholile holim'a leqhoa ebe u sefa ka har'a vial ea high performance liquid chromatography (HPLC) .Motsoako oa li-oligosaccharides o ile oa khethoa ka ho tlosa bongata ba li-monosaccharides ka har'a sampole e se nang hydrolyzed ho tsoa ho kakaretso ea tsoekere ea sampole ea acid-hydrolyzed.
Glucose, xylose, le arabinose concentrations ho acid hydrolysed biomass li ile tsa hlahlobjoa ho sebelisoa Simadzu HPLC system e nang le autosampler, column heater, isocratic pump, le refractive index detector holim'a kholomo ea Bio-Rad Aminex HPX-87H.Kholomo e ne e hlokometsoe ho 50 ° C 'me e e-na le 0.6 ml / min 5 mM H2SO4 ka metsing.phalla.
Hydrolyzate supernatant e ile ea hlapolloa le ho hlahlojoa bakeng sa monomer le oligosaccharide content.Tsoekere ea monomeric e fumanoeng ka mor'a enzymatic hydrolysis e ile ea hlahlojoa ke HPLC e nang le mohala oa Bio-Rad (Hercules, CA) Aminex HPX-87P le kholomo ea molebeli oa molora.Mocheso oa mocheso o ne o bolokiloe ho 80 ° C, metsi a ne a sebelisoa e le mohato oa selefouno ka tekanyo ea phallo ea 0.6 ml / min.Li-oligosaccharides li ne li khethoa ke hydrolysis ka dilute acid ho 121 ° C ho ea ka mekhoa e hlalositsoeng ho refs.41, 48, 49.
Tlhahlobo ea li-saccharide e ile ea etsoa ka lihlahisoa tse tala, tsa AFEX tse phekotsoeng pele le masala ohle a se nang hydrolysed biomass (ho kenyeletsoa tlhahiso ea li-extracts tsa serial cell wall le mAb screening ea tsona) ho sebelisoa mekhoa e hlalositsoeng pele ea 27, 43, 50, 51.Bakeng sa tlhahlobo ea glycome, masala a ke keng a qhibiliha a joala a thepa ea lerako la lisele tsa semela a lokisoa ho tloha masalla a biomass 'me a kenngoa ka har'a serial e ntšoa ka li-reagents tse ntseng li eketseha 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 joalokaha ho hlalositsoe pele52,53.Lits'oants'o li ile tsa beoa ELISA khahlano le phanele e rarahaneng ea mAb50s e lebisitsoeng leboteng la sele ea glycan, mme karabelo e tlamang ea mAb e ile ea hlahisoa e le 'mapa oa mocheso.mAbs e shebaneng le semela sa cell wall glycan e ile ea rekoa ho tsoa litsing tsa laboratori (CCRC, JIM le MAC series).
Mohato o le mong oa biotinylation ea oligosaccharides.Ho kopanngoa ha lik'habohaedreite ka biotin-LC-hydrazide ho ile ha etsoa ka mokhoa o latelang.Biotin-LC-hydrazide (4.6 mg / 12 μmol) e ile ea qhibiliha ka dimethyl sulfoxide (DMSO, 70 μl) ka ho susumetsa ka matla le ho futhumatsa ka 65 ° C. bakeng sa 1 min.Glacial acetic acid (30 µl) e ile ea eketsoa 'me motsoako oa tšeloa holim'a sodium cyanoborohydride (6.4 mg/100 µmol) 'me ea qhibiliha ka ho feletseng ka mor'a ho futhumatsa ka 65 ° C. nako e ka bang motsotso o le mong.Joale, ho tloha ho 5 ho ea ho 8 μl ea motsoako oa karabelo o ile oa kenngoa ho oligosaccharide e omisitsoeng (1-100 nmol) ho fumana 10-fold or more molar over the label over the fokotsa bofelo.Karabelo e ile ea etsoa ka 65 ° C bakeng sa 2 h, ka mor'a moo disampole li ile tsa hloekisoa hang-hang.Ha ho na sodium cyanoborohydride e sebelisitsoeng litekong tsa ho ngolla ntle le ho fokotseha, 'me mehlala e ile ea itšoara ka 65 ° C. bakeng sa lihora tsa 2.5.
ELISA ho roala le ho hlatsoa lisampole tsa li-oligosaccharides tsa biotinylated.25 μl ea disampole tsa biotinylated (100 μl ea sampuli e 'ngoe le e' ngoe e teteaneng e hlapolotsoeng ka 5 ml ea 0.1 M Tris buffer solution (TBS)) e ile ea kenngoa selibeng se seng le se seng sa poleiti e koahetsoeng ke avidin.Liliba tsa taolo li ne li koahetsoe ka 50 μl ea biotin sebakeng sa 10 μg / ml ho 0.1 M TBS.Metsi a hloekisitsoeng a ne a sebelisoa e le ho roala litekanyo tse se nang letho.Letlapa le ile la kenngoa ka lihora tse 2 mocheso oa kamore lefifing.Hlatsoa poleiti ka makhetlo a 3 ka 0.1% lebese la skimmed ka 0.1 M TBS u sebelisa lenaneo la no.11 bakeng sa Grenier flat 3A.
Ho eketsoa le ho hlatsoa ha li-antibodies tsa mantlha.Kenya 40 µl ea antibody ea mantlha selibeng ka seng.Eketsa microplate bakeng sa hora e 1 mocheso oa kamore lefifing.Joale lipoleiti li ne li hlatsuoa ka makhetlo a 3 ka lebese la 0.1% ho 0.1M TBS ho sebelisoa lenaneo la ho hlatsoa #11 bakeng sa Grenier Flat 3A.
Kenya sesole sa 'mele sa bobeli 'me u hlatsoe.Eketsa 50 µl ya selwantsha-mogare sa legotlo/tweba (e hlapollotswe 1:5000 ka lebeseng la 0.1% ho 0.1 M TBS) selibeng ka seng.Eketsa microplate bakeng sa hora e 1 mocheso oa kamore lefifing.Joale li-microplates li ile tsa hlatsuoa ka makhetlo a 5 ka lebese la 0.1% ho 0.1 M TBS ho sebelisoa lenaneo la Grenier Flat 5A la ho hlatsoa lijana #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 deionized).Lokisetsa substrate ea TMB.le vortex pele o sebelisoa).Kenya microplate ka mocheso oa kamore ka 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 rekote monko ho tloha ho 450 ho isa ho 655 nm u sebelisa sebali sa ELISA.
Lokisetsa litharollo tsa 1 mg/ml tsa li-analyte tsena metsing a hloekisitsoeng: arabinose, rhamnose, fucose, xylose, galacturonic acid (GlA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc) N-coamine .(glcNAc), N-acetylgalactosamine (galNAc), inositol (tekanyetso ea ka hare).Ho ile ha lokisetsoa litekanyetso tse peli ka ho eketsa tharollo ea tsoekere ea 1 mg / mL e bontšitsoeng ho Lethathamo la 1. Mehlala e leqhoa le lyophilized ho -80 ° C. ho fihlela metsi ohle a tlosoa (hangata hoo e ka bang lihora tse 12-18).
Kenya 100–500 µg ea sampole ho screw cap tubes ka tekanyo ea tlhahlobo.Ngola chelete e kentsoeng.Ho molemo ho qhaqha sampole ka har'a motsoako o itseng oa solvent ebe o e kenya ka har'a tube e le aliquot ea mokelikeli.Sebelisa 20 µl ea 1 mg/ml inositol e le tekanyetso ea ka hare bakeng sa tube e 'ngoe le e 'ngoe ea sampuli.Palo ea maemo a ka hare a kenyelletsoeng sampuling e tlameha ho lekana le palo ea tekanyo ea ka hare e kenyelletsoeng ho tube e tloaelehileng.
Kenya 8 ml ea methanol ea anhydrous ho vial cap cap.Ebe 4 ml ea tharollo ea 3 N. methanolic HCl, e koahetsoeng le ho sisinyeha.Mokhoa ona ha o sebelise metsi.
Kenya 500 µl ea 1 M HCl tharollo ea methanol ho lisampole tsa oligosaccharide le li-tubes tse tloaelehileng tsa TMS.Mehlala e ile ea kenngoa ka bosiu bo le bong (lihora tse 168) ho 80 ° C. sebakeng sa mocheso oa mocheso.Omisa sehlahisoa sa methanolysis mocheso oa kamore u sebelisa mefuta e mengata ea ho omisa.Kenya 200 µl MeOH 'me u omelle hape.Ts'ebetso ena e phetoa habeli.Kenya 200 µl ea methanol, 100 µl ea pyridine le 100 µl ea acetic anhydride ho sampole ebe u kopanya hantle.Mehlala e ile ea kenngoa ka mocheso oa kamore ka metsotso e 30.le ho omella.Kenya 200 µl ea methanol 'me u omelle hape.
Kenya 200 µl ea Tri-Sil le tube e koahetsoeng ka mocheso metsotso e 20.80 ° C, ebe e pholile mocheso oa kamore.Sebelisa hangata ho omisa ho tsoela pele ho omisa sampole ho bophahamo ba hoo e ka bang 50 µl.Ke habohlokoa ho hlokomela hore ha rea ​​ka ra lumella lisampole hore li ome ka ho feletseng.
Kenya 2 ml ea hexane 'me u kopanye hantle ka ho vortexing.Tlatsa malebela a Pasteur pipettes (5-8 mm) ka sengoathoana sa boea ba khalase ka ho kenya boea ba khalase holim'a pipette ea bophara ba 5-3 / 4.Mehlala e ne e le centrifuged ho 3000 g bakeng sa metsotso e 2.Mesaletsa efe kapa efe e sa qhibiliheng e ea putsoa.Omisa mohlala ho 100-150 μl.Molumo oa hoo e ka bang 1 μl o ile oa kenngoa ho GC-MS ka mocheso oa pele oa 80 ° C le nako ea pele ea metsotso ea 2.0 (Letlapa la 2).