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Iindlela ezintsha ze-immunoloji kunye ne-mass spectrometric zohlalutyo olunzima lwe-oligosaccharides eqhubekayo kwisitovu sombona esifakwe ngaphambili nge-AFEX.I-Lignocellulosic biomass yindlela ezinzileyo kumafutha efosili kwaye isetyenziswa ngokubanzi ukuphuhlisa i-biotechnologies yokuvelisa iimveliso ezifana nokutya, ukutya, amafutha kunye neekhemikhali.Undoqo kobu buchwepheshe kuphuhliso lweenkqubo zokhuphiswano lweendleko zokuguqula iicarbohydrates ezintsonkothileyo ezikhoyo kwiindonga zeeseli zesityalo zibe ziiswekile ezilula ezifana neglucose, ixylose kunye nearabinose.Ngenxa yokuba i-lignocellulosic biomass inenkani kakhulu, kufuneka ixhomekeke kunyango lwe-thermochemical (umzekelo, i-ammonia fiber exfoliation (AFEX), i-dilute acids (DA), i-ionic liquids (IL)) kunye nonyango lwe-biological (umzekelo, i-enzymatic hydrolysis kunye ne-microbial fermentation) ngokudibanisa ukufumana imveliso efunekayo..Nangona kunjalo, xa i-enzymes ye-fungi yorhwebo isetyenziselwa inkqubo ye-hydrolysis, kuphela i-75-85% yeshukela e-soluble eyenziwe yi-monosaccharides, kwaye i-15-25% eseleyo i-soluble, i-oligosaccharides engenakulinganiswa, engahlali ifumaneka kwi-microorganisms.Ngaphambili, siye sahlukanisa ngempumelelo kwaye sacoca ii-oligosaccharides ezinenkani ezinyibilikayo sisebenzisa indibanisela yekhabhoni kunye nediatomaceous ulwahlulo lomhlaba kunye nobukhulu bokungabandakanywa chromatography, kwaye siphande iipropathi zabo ze-enzyme inhibitory.Siye safumanisa ukuba i-oligosaccharides equkethe i-degree ephezulu ye-polymerization (DP) i-methylated uronic acid substitutions inzima kakhulu ukucubungula kunye neengxube ze-enzyme yorhwebo kune-DP ephantsi kunye ne-oligosaccharides engathathi hlangothi.Apha sichaza ukusetyenziswa kweendlela ezininzi ezongezelelweyo, kubandakanywa iprofayili ye-glycan usebenzisa i-monoclonal antibodies (mAbs) ecacileyo yokutyala i-biomass glycans ukuze ibonakalise i-glycan bond kwiindonga zeseli yezityalo kunye ne-enzymatic hydrolysates, i-matrix-assisted laser desorption ionization, ixesha lokuhamba kwe-flight mass- spectrometry..I-MALDI-TOF-MS) isebenzisa iincopho zokuxilonga ngesakhiwo-zolwazi olufunyenwe yi-spectroscopy emva kokubola kwesibini kweeyoni ezingezizo, i-chromatography yegesi kunye ne-mass spectrometry (GC-MS) ukubonakalisa iibhondi ze-oligosaccharide kunye nangaphandle kwe-derivatization.Ngenxa yobuncinci be-oligosaccharides (DP 4-20), ezi molekyuli zinzima ukuzisebenzisa kwi-mAb yokubopha kunye neempawu.Ukoyisa le ngxaki, sisebenzise indlela entsha ye-biotin conjugation-based oligosaccharide immobilization indlela ebhalwe ngempumelelo uninzi lwe-DP ephantsi enyibilikayo oligosaccharides kumphezulu we-microplate, eyathi ke yasetyenziswa kwinkqubo ephezulu ye-mAb yohlalutyo oluthile.Le ndlela entsha iya kuququzelela ukuphuhliswa kweendlela eziphezulu zokuvavanya i-glycome kwixesha elizayo ezingasetyenziselwa ukwahlula kunye nokubonakalisa i-oligosaccharides ekhoyo kwii-biomarkers ngeenjongo zokuxilonga.
I-Lignocellulosic biomass, eyenziwe ngezolimo, amahlathi, ingca kunye nezinto ezenziwe ngamaplanga, yinto yokutya enokubakho yokuveliswa kwemveliso ye-bio-based, kubandakanywa ukutya, ukutya, amafutha kunye ne-precursors yeekhemikhali ukuvelisa iimveliso zexabiso eliphezulu1.Iikhabhohayidrethi (ezifana neselulosi kunye ne-hemicellulose) ezikhoyo kwiindonga zeeseli zesityalo zichithwa kwi-monosaccharides ngokulungiswa kweekhemikhali kunye ne-biotransformation (efana ne-enzymatic hydrolysis kunye ne-microbial fermentation).Unyango lwangaphambili oluqhelekileyo lubandakanya ukwanda kwefayibha ye-ammonia (AFEX), i-asidi ye-dilute (DA), i-ionic liquid (IL), kunye nokuqhuma kwe-steam (SE), esebenzisa indibaniselwano yeekhemikhali kunye nokushisa ukunciphisa imveliso ye-lignocellulose ngokuvula iindonga zeseli zesityalo3,4.inkani yento, 5. I-hydrolysis ye-enzymatic iqhutyelwa kumthwalo ophezulu we-solids usebenzisa i-enzymes esebenzayo ye-carbohydrate equkethe i-enzymes (i-CAZymes) kunye ne-microbial fermentation usebenzisa i-yeasts ye-transgenic okanye ibhaktheriya ukuvelisa i-bio-based fuels kunye neekhemikhali 6.
I-CAZymes kwii-enzymes zorhwebo zenziwe ngumxube onzima wee-enzymes ezidibanisa ngokubambisana i-carbohydrate-sugar bond ukuze zenze i-monosaccharides2,7.Njengoko sixele kwangaphambili, inethiwekhi eyinkimbinkimbi ye-polymers enevumba elimnandi ye-lignin kunye ne-carbohydrates ibenza ukuba bangabikho kakhulu, nto leyo ekhokelela ekuguquleni iswekile engaphelelanga, iqokelele i-15-25% ye-oligosaccharides yesini engaveliswanga ngexesha le-enzymatic hydrolysis ye-biomass pretreated.Le yingxaki eqhelekileyo kwiindlela ezahlukeneyo zonyango lwe-biomass.Ezinye izizathu zolu thintelo lubandakanya inhibition ye-enzyme ngexesha le-hydrolysis, okanye ukungabikho okanye amanqanaba asezantsi ee-enzymes eziyimfuneko ezifunekayo ukuphula iibhondi zeswekile kwi-biomass yezityalo.Ukuqonda ukubunjwa kunye neempawu zesakhiwo zeeshukela, ezifana neebhondi zeswekile kwi-oligosaccharides, ziya kusinceda siphucule ukuguqulwa kweswekile ngexesha le-hydrolysis, ukwenza iinkqubo ze-biotechnological zikhuphisane nemveliso ye-petroleum.
Ukumisela ulwakhiwo lwecarbohydrates lucelomngeni kwaye kufuna indibaniselwano yeendlela ezifana ne-liquid chromatography (LC)11,12, i-nuclear magnetic resonance spectroscopy (NMR)13, i-capillary electrophoresis (CE)14,15,16 kunye ne-mass spectrometry (MS)17.,Shumi elinesibhozo.Iindlela ze-MS ezifana nexesha le-flight mass spectrometry kunye ne-laser desorption kunye ne-ionization usebenzisa i-matrix (MALDI-TOF-MS) yindlela eguquguqukayo yokuchonga izakhiwo ze-carbohydrate.Kungekudala, i-Collision-Induced Dissociation (CID) i-tandem ye-MS ye-sodium ion adducts isetyenziswe ngokubanzi ukuchonga iminwe yeminwe ehambelana nezikhundla zokunamathisela i-oligosaccharide, ukucwangciswa kwe-anomeric, ukulandelelana, kunye nezikhundla ze-branching 20, 21.
Uhlalutyo lwe-Glycan sisixhobo esihle kakhulu sokuchongwa ngokunzulu kweebhondi ze-carbohydrate22.Le ndlela isebenzisa izilwa-buhlungu ze-monoclonal (mAbs) ezijoliswe ekutyaleni udonga lweseli i-glycan njengeeprobes zokuqonda unxulumano oluntsonkothileyo lwecarbohydrate.Ngaphezulu kwe-250 mAbs ekhoyo kwihlabathi jikelele, eyilwe ngokuchasene ne-oligosaccharides eyahlukeneyo kunye ne-branched kusetyenziswa ii-saccharides ezahlukeneyo24.Ama-mAbs amaninzi asetyenziswe ngokubanzi ukubonakalisa isakhiwo, ukubunjwa, kunye nokuguqulwa kodonga lweseli yesityalo, njengoko kukho ukungafani okuphawulekayo kuxhomekeke kuhlobo lweeseli zesityalo, ilungu, ubudala, isigaba sophuhliso, kunye nokukhula kwendawo25,26.Kutshanje, le ndlela isetyenziselwe ukuqonda inani le-vesicle kwiinkqubo zezityalo kunye nezilwanyana kunye nendima yazo ekuthuthweni kwe-glycan njengoko kumiselwe ngabamakishi be-subcellular, izigaba zophuhliso, okanye i-stimuli yendalo, kunye nokumisela umsebenzi we-enzymatic.Ezinye zezakhiwo ezahlukeneyo ze-glycans kunye ne-xylans eziye zachongwa ziquka i-pectin (P), i-xylan (X), i-mannan (M), i-xyloglucans (i-XylG), i-glucans edibeneyo (i-MLG), i-arabinoxylan (i-ArbX), i-galactomannan (i-GalG) , i-glucuronic acid-arabinoxy-Xblan) kunye ne-arnoxylan ye-arnoxylan (9) i-Arnoxylan (9) kunye ne-Arnoxylan (9).
Nangona kunjalo, nangona zonke ezi nzame zophando, zimbalwa izifundo eziye zagxila kubume be-oligosaccharide yokuqokelela ngexesha lomthwalo ophezulu we-solids (HSL) i-hydrolysis, kubandakanywa ukukhululwa kwe-oligosaccharide, utshintsho lwe-oligomeric chain chain ngexesha le-hydrolysis, iipolymers ezahlukeneyo ze-DP eziphantsi, kunye neengqungquthela zazo.izabelo 30,31,32.Okwangoku, nangona uhlalutyo lwe-glycan lubonakalise ukuba sisixhobo esiluncedo sohlalutyo olubanzi lwesakhiwo se-glycan, kunzima ukuvavanya i-oligosaccharides ye-DP e-soluble ephantsi kwamanzi usebenzisa iindlela ze-antibody.I-oligosaccharides ye-DP encinci ene-molecular weight engaphantsi kwe-5-10 kDa ayibopheleli kwiiplate ze-ELISA 33, i-34 kwaye ihlanjwe ngaphambi kokuba i-antibody idibanise.
Apha, ngokokuqala ngqa, sibonisa uvavanyo lwe-ELISA kwiipleyiti ezifakwe kwi-avidin usebenzisa i-antibodies ye-monoclonal, ukudibanisa inkqubo ye-biotinylation yenyathelo elinye kwi-oligosaccharides e-soluble refractory kunye nohlalutyo lwe-glycome.Indlela yethu yokuhlalutya i-glycome yaqinisekiswa yi-MALDI-TOF-MS kunye ne-GC-MS esekelwe kuhlalutyo lokudibanisa i-oligosaccharide ehambelanayo kusetyenziswa i-trimethylsilyl (TMS) yokukhutshwa kweengqungquthela zeswekile ze-hydrolyzed.Le ndlela entsha inokuphuhliswa njengendlela ephezulu yokuphumelela kwixesha elizayo kwaye ifumane ukusetyenziswa okubanzi kuphando lwe-biomedical35.
Ukuguqulwa kwe-post-translation ye-enzymes kunye ne-antibodies, njenge-glycosylation, i-36 ichaphazela umsebenzi wabo we-biological.Ngokomzekelo, utshintsho kwi-glycosylation yeeprotheni ze-serum zidlala indima ebalulekileyo kwi-arthritis evuthayo, kwaye utshintsho kwi-glycosylation lusetyenziswa njengeempawu zokuxilonga37.Ii-glycans ezahlukahlukeneyo ziye zaxelwa kwiincwadi ukuba zivele ngokukhawuleza kwizifo ezahlukahlukeneyo, kubandakanywa izifo ezivuthayo ezingapheliyo zentsholongwane yesisu kunye nesibindi, izifo zentsholongwane, i-ovarian, isifuba kunye ne-prostate cancers38,39,40.Ukuqonda isakhiwo se-glycans usebenzisa i-antibody-based based glycan ELISA iindlela ziya kunika ukuzithemba okongeziweyo ekuxilongweni kwezifo ngaphandle kokusetyenziswa kweendlela ze-MS ezinzima.
Uphononongo lwethu lwangaphambili lubonise ukuba i-oligosaccharides enenkani yahlala i-unhydrolyzed emva kokunyangwa kwangaphambili kunye ne-enzymatic hydrolysis (Umfanekiso 1).Kumsebenzi wethu opapashwe ngaphambili, siphuhlise indlela yokukhupha i-charcoal solid-phase yokukhupha i-oligosaccharides ukusuka kwi-AFEX-pretreated corn stover hydrolyzate (ACSH)8.Emva kokutsalwa kokuqala kunye nokwahlula, i-oligosaccharides yaphinda yahlulwa ngobungakanani be-chromatography (SEC) kwaye iqokelelwe ngokomyalelo we-molecular weight.I-Sugar monomers kunye ne-oligomers ekhutshwe kwii-pretreatments ezahlukeneyo zahlalutywa ngohlalutyo lokubunjwa kweswekile.Xa kuthelekiswa umxholo we-oligomers yeswekile efunyenwe ngeendlela ezahlukeneyo zokutshatyalaliswa kwangaphambili, ubukho be-oligosaccharides enenkani yingxaki eqhelekileyo ekuguqulweni kwe-biomass kwi-monosaccharides kwaye kunokukhokelela ekunciphiseni isivuno seswekile ubuncinane kwi-10-15% kwaye ukuya kuthi ga kwi-18%.US.Le ndlela isetyenziselwa ukuqhubela phambili imveliso enkulu yamaqhezu e-oligosaccharide.Isiphumo se-ACH kunye namaqhezu alandelayo aneesindo ezahlukeneyo zeemolekyuli zasetyenziswa njengezinto zokulinga ukubonakaliswa kwe-oligosaccharides kulo msebenzi.
Emva konyango lwangaphambili kunye ne-enzymatic hydrolysis, i-oligosaccharides eqhubekayo yahlala i-unhydrolysed.Apha (A) yindlela yokwahlula i-oligosaccharide apho i-oligosaccharides ihlukanisiwe kwi-AFEX-pretreated corn stover hydrolyzate (ACSH) isebenzisa ibhedi epakishweyo ye-carbon activated kunye ne-diatomaceous earth;(B) Indlela yokwahlula i-oligosaccharides.I-oligosaccharides yaphinda yahlukana ngobungakanani be-chromatography (SEC);(C) I-Saccharide monomers kunye ne-oligomers ekhutshwe kwii-pretreatments ezahlukeneyo (i-asidi ehlanjululweyo: i-DA, i-ionic liquid: IL kunye ne-AFEX).Iimeko ze-Enzymatic hydrolysis: ukulayishwa kwezinto eziqinileyo eziphezulu ze-25% (w/w) (malunga ne-8% yokulayisha iglucan), iiyure ezingama-96 ze-hydrolysis, i-20 mg/g yokulayishwa kwe-enzyme yorhwebo (Ctec2:Htec2:MP-2:1:1 ratio) kunye (D) I-Sugar monomers kunye ne-oligomers ye-glucose, i-xyracorn EXA ikhululwe kwi-xyraafCSA-CSA ekhutshwe i-xyracorn CSA ikhululwe kwi-xybinose ye-xybinose.
Uhlalutyo lwe-Glycan lubonakalise ukuba sisixhobo esiluncedo kuhlalutyo olubanzi lwesakhiwo se-glycans kwizicatshulwa ezihlukanisiweyo kwiintsalela ze-biomass eziqinileyo.Nangona kunjalo, i-saccharides enyibilikayo yamanzi ibonakaliswe ngaphantsi kusetyenziswa le ndlela yendabuko41 kuba i-oligosaccharides ephantsi kwe-molecular weight inzima ukuyikhupha kwiipleyiti ze-ELISA kwaye ihlanjwe ngaphambi kokudibanisa i-antibody.Ke ngoko, ukubopha i-antibody kunye nokuchazwa kweempawu, indlela ye-biotinylation enenyathelo elinye yayisetyenziselwa ukwambathisa i-oligosaccharides enyibilikayo, engahambelaniyo kwiipleyiti ze-ELISA ezifakwe kwi-avidin.Le ndlela yavavanywa kusetyenziswa i-ACSH yethu eveliswe ngaphambili kunye neqhezu ngokusekelwe kubunzima bayo be-molecular (okanye i-degree of polymerization, DP).I-biotinylation yesinyathelo esisodwa yayisetyenziselwa ukunyusa i-oligosaccharide yokubopha i-affinity ngokongeza i-biotin-LC-hydrazide ekupheleni kokunciphisa i-carbohydrate (umzobo 2).Kwisisombululo, iqela le-hemiacetal ekupheleni kokunciphisa liphendula kunye neqela le-hydrazide ye-biotin-LC-hydrazide ukwenza ibhondi ye-hydrazone.Ebukhoneni be-agent yokunciphisa i-NaCNBH3, ibhondi ye-hydrazone iyancitshiswa ibe yimveliso yokuphela kwe-biotinylated ezinzileyo.Ngokuguqulwa kokuphela kokunciphisa iswekile, ukubophelela kwe-DP oligosaccharides ephantsi kwiiplate ze-ELISA kuye kwenzeka, kwaye kwisifundo sethu oku kwenziwa kwiiplate ezifakwe kwi-avidin usebenzisa i-glycan-targeted mAbs.
Ukuhlolwa kwe-antibodies ye-monoclonal esekelwe kwi-ELISA ye-oligosaccharides ye-biotinylated.Apha (A) i-biotinylation edibeneyo ye-oligosaccharides kunye nokuhlolwa kwe-ELISA okulandelayo kunye ne-glycan-targeted mAbs kwi-NeutrAvidin i-coated plates kunye (B) ibonisa inkqubo yesinyathelo esisodwa se-biotinylation yeemveliso zokuphendula.
Iiplati ezifakwe kwi-Avidin kunye ne-oligosaccharide-conjugated antibodies zongezwa kwii-antibodies eziziiprayimari kunye nezesibini kwaye zihlanjwe kwindawo yokukhanya kunye nexesha elibonakalayo.Emva kokuba kugqitywe ukudityaniswa kwe-antibody, yongeza i-TMB substrate yokufukama ipleyiti.Ukusabela kwagqitywa ekugqibeleni nge-asidi ye-sulfuric.Iipleyiti eziqanduselweyo zahlalutywa kusetyenziswa umfundi we-ELISA ukumisela amandla okubopha i-antibody nganye ukubhaqa i-antibody-specific cross-linking.Ukufumana iinkcukacha kunye neeparitha zovavanyo, jonga icandelo elihambelanayo "Izinto kunye neendlela".
Sibonisa ukusetyenziswa kwale ndlela esandul 'ukuphuhliswa kwezicelo ezithile ngokubonakalisa i-oligosaccharides e-soluble ekhoyo kwi-ACSH kunye namaqhezu e-oligosaccharide angcolileyo kunye ahlambulukileyo ahlukanisiwe kwi-lignocellulosic hydrolysates.Njengoko kubonisiwe kwi-Figure 3, i-xylans eqhelekileyo ye-epitope-substituted echongiwe kwi-ACSH isebenzisa iindlela ze-bioacylated glycome assay zidla ngokuba yi-uronic (U) okanye i-methyluronic (MeU) kunye ne-pectic arabinogalactans.Uninzi lwazo lufunyenwe kwisifundo sethu sangaphambili malunga nohlalutyo lwe-glycans ye-non-hydrolyzed solids (UHS)43.
Ukufunyanwa kwe-recalcitrant oligosaccharide epitopes usebenzisa i-antibody ye-monoclonal ejoliswe kwi-cell wall glycan.Iqhezu "elingathathi hlangothi" liqhekeza le-ACN kunye neqhezu elithi "acidic" licandelo le-FA.Ubomvu obuqaqambileyo kwimaphu yobushushu bubonisa umxholo ophezulu we-epitope, kwaye iibhlowu eziqaqambileyo zibonisa imvelaphi engenanto.Amaxabiso ombala kwisikali asekwe kumaxabiso e-OD ekrwada kuqulunqo N=2.Ii-epitopes eziphambili eziqatshelwe zi-antibodies ziboniswa ngasekunene.
Ezi zakhiwo ezingenayo i-cellulose azikwazi ukucandwa ngamaseli aqhelekileyo kunye ne-hemicellulases kumxube ovavanyiweyo we-enzyme yorhwebo, equka i-enzymes yorhwebo esetyenziswa ngokuqhelekileyo.Ke ngoko, ii-enzymes ezintsha ezincedisayo ziyafuneka kwi-hydrolysis yazo.Ngaphandle kwee-enzyme eziyimfuneko ze-non-cellulose accessory enzymes, ezi zibophelelo ezingenayo i-cellulose zithintela ukuguqulwa okupheleleyo kwi-monosaccharides, nokuba i-polymers yeswekile yomzali i-hydrolyzed kakhulu ibe ngamaqhekeza amafutshane kwaye inyibilikiswe ngokusebenzisa imixube ye-enzyme yorhwebo.
Uphononongo olongezelelweyo lokusasazwa komqondiso kunye namandla alo okubopha lubonise ukuba i-epitopes ebophelelayo yayiphantsi kwi-DP ephezulu ye-sugar fractions (A, B, C, DP ukuya kwi-20 +) kunamaqhezu aphantsi e-DP (D, E, F, DP) kwi-dimers) (Umfanekiso 1).Iziqwenga ze-Acid zixhaphake kakhulu kwii-epitopes ezingenayo i-cellulose kunamaqhekeza angathathi hlangothi.Ezi ziganeko zihambelana nomzekelo obonwe kwisifundo sethu sangaphambili, apho i-DP ephezulu kunye ne-acid moieties yayixhathisa ngakumbi kwi-enzymatic hydrolysis.Ngoko ke, ubukho be-non-cellulose glycan epitopes kunye ne-U kunye ne-MeU substitution ingaba negalelo elikhulu ekuzinzeni kwe-oligosaccharides.Kufuneka kuqatshelwe ukuba ukubopha kunye nokufumanisa ukusebenza kakuhle kunokuba yingxaki kwi-DP oligosaccharides ephantsi, ngakumbi ukuba i-epitope i-dimeric okanye i-trimeric oligosaccharide.Oku kunokuvavanywa kusetyenziswa i-oligosaccharides yorhwebo yobude obuhlukeneyo, nganye iqulethe i-epitope enye ebophelela kwi-mAb ethile.
Ke ngoko, ukusetyenziswa kwezilwa-buhlungu ezithe ngqo kulwakhiwo kutyhilwe iintlobo ezithile zeebhondi ezilandulayo.Ngokuxhomekeke kuhlobo lwe-antibody esetyenzisiweyo, ipateni efanelekileyo yokudibanisa, kunye namandla omqondiso ovelisayo (ininzi kunye neyona nto incinci kakhulu), i-enzymes entsha inokuchongwa kwaye yongezwe i-semi-quantitatively kumxube we-enzyme ukwenzela ukuba i-glycoconversion epheleleyo.Ukuthatha uhlalutyo lwe-ACSH oligosaccharides njengomzekelo, sinokwenza i-database yeebhondi ze-glycan kwizinto eziphathekayo ze-biomass.Kufuneka kuqatshelwe apha ukuba ukudibanisa okuhlukeneyo kwee-antibodies kufuneka kuthathelwe ingqalelo, kwaye ukuba ukuxhamla kwabo ayaziwa, oku kuya kudala ubunzima obuthile xa kuthelekiswa nemiqondiso yee-antibodies ezahlukeneyo.Ukongeza, ukuthelekiswa kweebhondi ze-glycan kunokusebenza ngcono phakathi kweesampulu ze-antibody efanayo.Ezi zibophelelo ezinenkani zinokudityaniswa ke kwi-database ye-CAZyme, apho sinokuchonga khona i-enzymes, khetha i-enzymes yabaviwa kunye nokuvavanya i-enzymes yokuqhawula ibhondi, okanye ukuphuhlisa iinkqubo ze-microbial ukuvakalisa ezi enzymes ukuze zisetyenziswe kwi-biorefineries44.
Ukuvavanya indlela iindlela ze-immunological ezincedisa ngayo ezinye iindlela zokubonisa i-oligosaccharides ephantsi kwe-molecular weight ekhoyo kwi-lignocellulosic hydrolysates, senze i-MALDI (umzobo we-4, i-S1-S8) kunye nohlalutyo lwe-saccharides ephuma kwi-TMS esekelwe kwi-GC-MS kwiphaneli efanayo (umzobo 5) inxalenye ye-oligosaccharide.I-MALDI isetyenziselwa ukuthelekisa ukuba ukuhanjiswa kobuninzi be-oligosaccharide molecules kuhambelana nesakhiwo esijoliswe kuyo.Kwikhiwane.I-4 ibonisa i-MS yamacandelo angathathi hlangothi i-ACN-A kunye ne-ACN-B.Uhlalutyo lwe-ACN-A luqinisekisile uluhlu lweeshukela ze-pentose ukusuka kwi-DP 4-8 (umzobo 4) ukuya kwi-DP 22 (umzobo we-S1), obunzima bayo buhambelana ne-MeU-xylan oligosaccharides.Uhlalutyo lwe-ACN-B luqinisekisile uchungechunge lwe-pentose kunye ne-glucoxylan kunye ne-DP 8-15.Kwizinto ezongezelelweyo ezifana neFigure S3, FA-C acidic moiety mass distribution map zibonisa uluhlu lwe (Me) U endaweni yeswekile yepentose ene-DP ye8-15 ehambelanayo nexylans efakwe endaweni efunyenwe kuhlolo lwe-ELISA-based mAb.Ii-epitopes ziyahambelana.
I-MALDI-MS i-spectrum ye-oligosaccharides enyibilikayo engahambelaniyo ekhoyo kwi-ACS.Apha, (A) i-ACN-A amaqhezu aphantsi obunzima obune-methylated uronic acid (DP 4-8) ithathelwe indawo ye-glucuroxylan oligosaccharides kunye (B) ne-ACN-B i-xylan kunye ne-methylated uronic acid oligosaccharides efakwe endaweni ye-glucuroxylan (DP 8-15).
Uhlalutyo lokubunjwa kwe-glycan intsalela ye-oligosaccharides ephikisayo.Apha (A) ukubunjwa kwe-TMS saccharide yeentlobo ezahlukeneyo ze-oligosaccharide ezifunyenwe kusetyenziswa uhlalutyo lwe-GC-MS.(B) Izakhiwo zeeshukela ezahlukeneyo ezivela kwi-TMS ezikhoyo kwiioligosaccharides.I-ACN - iqhezu le-acetonitrile eliqukethe i-oligosaccharides engathathi hlangothi kunye ne-FA - i-ferulic acid fraction equkethe i-oligosaccharides ye-asidi.
Esinye isiphetho esinomdla sithathwe kuhlalutyo lwe-LC-MS lweqhekeza le-oligosaccharide, njengoko kuboniswe kuMfanekiso S9 (iindlela zingabonwa kwi-electronics supplementary material).Amacandelo e-hexose kunye namaqela e-OAc aye abonwa ngokuphindaphindiweyo ngexesha lokuxutywa kweqhekeza le-ACN-B.Oku kufunyaniswayo akuqinisekisi nje ukuqhekeka okubonwe kwi-glycome kunye nohlalutyo lwe-MALDI-TOF, kodwa lubonelela ngolwazi olutsha malunga nezinto ezinokuthi zenzeke kwi-carbohydrate kwi-pretreated lignocellulosic biomass.
Siphinde senza uhlalutyo lwe-glycan yokwakheka kwamaqhezu e-oligosaccharide usebenzisa i-TMS glycan derivatization.Ukusebenzisa i-GC-MS, sinqume ukubunjwa kwe-neural (non-derivative) kunye ne-sugar acidic (i-GluA kunye ne-Gala) kwiqhekeza le-oligosaccharide (umzobo 5).I-asidi ye-Glucuronic ifumaneka kwi-acidic components C kunye ne-D, ngelixa i-galacturonic acid ifumaneka kumacandelo e-acidic A kunye ne-B, zombini ezo ziyi-DP eziphezulu ze-sugar acidic.Ezi ziphumo aziqinisekisi kuphela idatha yethu ye-ELISA kunye ne-MALDI, kodwa iyahambelana nezifundo zethu zangaphambili zokuqokelela i-oligosaccharide.Ngoko ke, sikholelwa ukuba iindlela zanamhlanje ze-immunology zisebenzisa i-biotinylation ye-oligosaccharides kunye nokuhlolwa kwe-ELISA okulandelayo kwanele ukufumanisa i-oligosaccharides e-soluble recalcitrant kwiisampuli zebhayoloji ezahlukeneyo.
Kuba iindlela zokuhlola ezisekwe kwi-ELISA-mAb ziye zaqinisekiswa ngeendlela ezininzi ezahlukeneyo, besifuna ukuphonononga ngakumbi ukubanakho kwale ndlela intsha yobungakanani.I-oligosaccharides ezimbini zorhwebo, i-xylohexasaccharide oligosaccharide (XHE) kunye ne-23-α-L-arabinofuranosyl-xylotriose (A2XX), zathengwa kwaye zavavanywa ngokusebenzisa indlela entsha ye-mAb ejolise kwi-cell wall glycan.Umzobo we-6 ubonisa ulungelelwaniso lomgca phakathi kwesignali yokubopha i-biotinylated kunye ne-log concentration ye-oligosaccharide concentration, ephakamisa imodeli ye-adsorption ye-Langmuir enokwenzeka.Phakathi kwe-mAbs, i-CCRC-M137, i-CCRC-M138, i-CCRC-M147, i-CCRC-M148, kunye ne-CCRC-M151 ehambelana ne-XHE, kunye ne-CCRC-M108, i-CCRC-M109, kunye ne-LM11 ehambelana ne-A2XX phezu koluhlu lwe-100 nmno.Ngenxa yokufumaneka okulinganiselweyo kwee-antibodies ngexesha lovavanyo, iimvavanyo ezilinganiselwe zenziwa nge-concentration nganye ye-oligosaccharide.Kufuneka kuqatshelwe apha ukuba ezinye izilwa-buhlungu zisabela ngokwahlukileyo kwi-oligosaccharide enye njenge-substrate, mhlawumbi ngenxa yokuba zibophelela kwii-epitopes ezahlukileyo kwaye zinokuba ne-affinities ebophelelayo eyahlukileyo kakhulu.Iindlela kunye neziphumo zokuchongwa kwe-epitope ezichanekileyo ziya kuba nzima ngakumbi xa indlela entsha ye-mAb isetyenziswa kwiisampuli zangempela.
Ii-oligosaccharides ezimbini zorhwebo zisetyenziselwe ukumisela uluhlu lokufumanisa i-glycan-targeting mAbs ezahlukeneyo.Apha, ulungelelwaniso lomgca kunye ne-log concentration ye-oligosaccharide yoxinaniso lubonisa iipatheni ze-adsorption ze-Langmuir ze (A) XHE kunye ne-mAb kunye (B) ne-A2XX ene-mAb.Ii-epitopes ezihambelanayo zibonisa izakhiwo ze-oligosaccharides zorhwebo ezisetyenziswe njenge-substrates kwi-assay.
Ukusetyenziswa kwe-glycan-targeted monoclonal antibodies (uhlalutyo lwe-glycocomic okanye i-ELISA-based mAb screening) sisixhobo esinamandla sokubonakalisa ubunzulu beempawu ezininzi ze-glycans ezinkulu zodonga ezenza i-biomass yezityalo.Nangona kunjalo, uhlalutyo lwe-glycan ye-classical lubonisa kuphela i-glycans enkulu yodonga lweseli, njengoko uninzi lwe-oligosaccharides lungasebenzi kakuhle kwiipleyiti ze-ELISA.Kolu phononongo, i-AFEX-pretreated corn stover yenziwe nge-hydrolyzed nge-hydrolyzed kumxholo we-solids ephezulu.Uhlalutyo lweswekile lwalusetyenziselwa ukumisela ukubunjwa kwe-carbohydrates eludongeni lweseli ye-recalcitrant kwi-hydrolyzate.Nangona kunjalo, uhlalutyo lwe-mAb yee-oligosaccharides ezincinci kwii-hydrolysates zincinci, kwaye izixhobo ezongezelelweyo ziyafuneka ukuze kulungiswe ngokufanelekileyo i-oligosaccharides kwiiplate ze-ELISA.
Sinika ingxelo apha ngenoveli kunye nendlela esebenzayo ye-oligosaccharide immobilization yokuhlolwa kwe-mAb ngokudibanisa i-oligosaccharide biotinylation elandelwa kukuhlolwa kwe-ELISA kwiipleyiti ezigqunyiweyo ze-NeutrAvidin™.I-oligosaccharides ye-biotinylated immobilized ibonise ukuhambelana okwaneleyo kwi-antibody ukwenzela ukuba kubonwe ngokukhawuleza kunye nokusebenza kakuhle kwe-oligosaccharides ye-recalcitrant.Uhlalutyo lokubunjwa kwezi oligosaccharides ezinenkani ezisekelwe kwi-mass spectrometry ziqinisekisile iziphumo zale ndlela entsha yokukhusela umzimba.Ngaloo ndlela, ezi zifundo zibonisa ukuba ukudibanisa i-oligosaccharide biotinylation kunye nokuhlolwa kwe-ELISA kunye ne-glycan-targeted monoclonal antibodies ingasetyenziselwa ukufumanisa i-crosslinks kwi-oligosaccharides kwaye ingasetyenziswa ngokubanzi kwezinye izifundo ze-biochemical ezibonisa isakhiwo se-oligosaccharides.
Le ndlela ye-biotin-based glycan profiling yingxelo yokuqala ekwaziyo ukuphanda iibhondi ze-carbohydrate ze-recalcitrant ze-oligosaccharides ezinyibilikayo kwi-biomass yezityalo.Oku kunceda ukuqonda ukuba kutheni ezinye iindawo ze-biomass zinenkani xa kuziwa kwimveliso ye-biofuel.Le ndlela igcwalisa isithuba esibalulekileyo kwiindlela zokuhlalutya i-glycome kwaye yandisa isicelo sayo kuluhlu olubanzi lwe-substrates ngaphaya kwe-oligosaccharides yezityalo.Kwixesha elizayo, sinokusebenzisa iirobhothi ze-biotinylation kwaye sisebenzise indlela esiyiphuhlisele uhlalutyo oluphezulu lweesampulu usebenzisa i-ELISA.
I-Corn straw (CS) ekhuliswe kwimbewu yePioneer 33A14 engumhlanganiselwa yavunwa ngo-2010 kwiiFama zaseKramer eRay, eColorado.Ngemvume yomnini-mhlaba, le biomass ingasetyenziselwa uphando. Iisampuli zigcinwe zomile < 6% umswakama kwiingxowa ze-zip-lock kwiqondo lokushisa. Iisampuli zigcinwe zomile < 6% umswakama kwiingxowa ze-zip-lock kwiqondo lokushisa. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Iisampulu zigcinwe zomile kwi-<6% yomswakama kwiingxowa ezifakwe kwi-zippered kwiqondo lokushisa.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% Inkcazelo ngokuthe gabalala в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Iisampulu zigcinwe kwiingxowa ze-zipper kwindawo yokushisa kunye nomswakama <6%.Uphononongo luhambelana nezikhokelo zasekhaya nezesizwe.Uhlalutyo lokubumba lwenziwa kusetyenziswa i-NREL protocol.Ukubunjwa kwafunyanwa kuqulethe i-31.4% ye-glucan, i-18.7% ye-xylan, i-3.3% ye-arabinan, i-1.2% ye-galactan, i-2.2% ye-acetyl, i-14.3% ye-lignin, i-1.7% yeprotheni kunye ne-13. 4% uthuthu.
I-Cellic® CTec2 (138 mg protein / ml, lot VCNI 0001) ngumxube onzima we-cellulase, i-β-glucosidase kunye ne-Cellic® HTec2 (157 mg protein / ml, i-VHN00001 eninzi) evela kwi-Novozymes (i-Franklinton, NC, USA) .I-Multifect Pectinase® (i-72 mg protein / mL), i-complex blend of pectin degrading enzymes, yanikelwa yiDuPont Industrial Biosciences (Palo Alto, CA, USA).Uxinzelelo lweprotheyini ye-Enzyme yamiselwa ngokuqikelela umxholo weprotheyini (kunye nokususa igalelo le-nitrogen engeyiyo iprotheni) kusetyenziswa uhlalutyo lwe-Kjeldahl nitrogen (indlela ye-AOAC 2001.11, iDairy One Cooperative Inc., Ithaca, NY, USA).I-Diatomaceous earth 545 yathengwa kwi-EMD Millipore (Billerica, MA).Ikhabhoni esebenzayo (i-DARCO, i-100 mesh granules), i-Avicel (PH-101), i-beech xylan, kunye nazo zonke ezinye iikhemikhali zathengwa eSigma-Aldrich (eSt. Louis, MO).
I-AFEX pretreatment yenziwa kwi-GLBRC (i-Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Unyango lwangaphambili lwenziwa kwi-140 ° C. imizuzu eyi-15.Ixesha lokuhlala le-46 kumlinganiselo we-1:1 we-ammonia ene-anhydrous ukuya kwi-biomass kuma-60% (w/w) ukulayisha kwibhetshi yebhetshi yentsimbi engenastainless (Parr Instruments Company).Kuthathe imizuzu engama-30.I-reactor yaziswa kwi-140 ° C kwaye i-ammonia yakhululwa ngokukhawuleza, ivumela i-biomass ukuba ibuyele ngokukhawuleza kwiqondo lokushisa.Ukubunjwa kwe-AFEX ye-corn stover (ACS) efunyenwe ngaphambili (i-ACS) yayifana ne-corn stover engaphendulwanga (UT-CS).
Izinto eziqinileyo eziphezulu I-ACSH 25% (w/w) (malunga ne-8% yokulayisha i-dextran) yalungiswa njengesixhobo sokuqala kwimveliso emikhulu ye-oligosaccharides.I-Enzymatic hydrolysis ye-ACS yenziwa kusetyenziswa umxube we-enzyme yorhwebo kuquka i-Cellic® Ctec2 10 mg protein / g glucan (kwi-biomass yangaphambili), i-Htec2 (i-Novozymes, i-Franklinton, i-NC), i-5 mg protein / g glucan, kunye ne-Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein / g dextran.I-hydrolysis ye-enzymatic yenziwa kwi-5-litre bioreactor kunye nomthamo osebenzayo weelitha ezi-3, i-pH 4.8, i-50 ° C kunye ne-250 rpm.Emva kwe-hydrolysis yeeyure ze-96, i-hydrolyzate yaqokelelwa nge-centrifugation kwi-6000 rpm imizuzu engama-30 kwaye emva kwe-14000 rpm imizuzu engama-30 ukususa izinto eziqinileyo ezingenahydrolyzed.I-hydrolyzate iye yaphantsi kohluzo olunyumba ngebeaker yokucoca eyi-0.22 mm.I-hydrolyzate ehluziweyo yayigcinwe kwiibhotile ezingenazintsholongwane kwi-4 ° C. kwaye emva koko yahlulwe kwikhabhoni.
Uhlalutyo lokubunjwa kweesampuli ze-biomass ezisekelwe kwisicatshulwa ngokwemigaqo ye-NREL yohlalutyo lwebhubhoratri: ukulungiswa kweesampuli zohlalutyo lokubunjwa (NREL / TP-510-42620) kunye nokuzimisela kwe-carbohydrates yesakhiwo kunye ne-lignin kwi-biomass (NREL / TP-510 - 42618)47.
Uhlalutyo lwe-oligosaccharide yomlambo we-hydrolyzate lwenziwa kwisikali se-2 ml usebenzisa i-autoclave-based based acid hydrolysis method.Xuba isampulu yehydrolyzate kunye ne-69.7 µl ye-72% ye-asidi yesulfuric kwi-10 ml ye-screw cap yetyhubhu yenkcubeko kwaye ufukamele iyure e-1 kwi-benchtop nge-121 °C, uphole emkhenkceni kwaye uhluze kwivial yolwelo olusebenza kakhulu lwekhromatografi (HPLC) .Ukuxinwa kwe-oligosaccharides kunqunywe ngokukhupha i-concentration ye-monosaccharides kwi-sampulu ye-non-hydrolyzed kwi-sugar concentration kwi-acid-hydrolyzed sample.
I-Glucose, xylose, kunye ne-arabinose concentrations kwi-acid hydrolysed biomass yahlaziywa kusetyenziswa inkqubo ye-Shimadzu HPLC exhotywe nge-autosampler, i-heater column, i-isocratic pump, kunye ne-refractive index detector kwikholamu ye-Bio-Rad Aminex HPX-87H.Ikholomu igcinwe kwi-50 ° C kwaye ikhutshwe nge-0.6 ml / min 5 mM H2SO4 emanzini.ukuhamba.
I-hydrolyzate supernatant yahlanjululwa kwaye yahlalutya umxholo we-monomer kunye ne-oligosaccharide.Iishukela ze-Monomeric ezifunyenwe emva kwe-enzymatic hydrolysis zahlalutywa yi-HPLC exhotywe nge-Bio-Rad (Hercules, CA) ikholomu ye-Aminex HPX-87P kunye nekholomu yomlotha.Ukushisa kwekholomu kugcinwe kwi-80 ° C, amanzi asetyenziswe njengesigaba esihambahambayo kunye nesantya sokuhamba kwe-0.6 ml / min.I-Oligosaccharides inqunywe yi-hydrolysis kwi-asidi ye-dilute kwi-121 ° C ngokweendlela ezichazwe kwii-refs.41, 48, 49.
Uhlalutyo lwe-Saccharide lwenziwa kwi-raw, i-AFEX yonyango kwangaphambili kunye nayo yonke i-non-hydrolysed biomass residues (kubandakanywa nokuveliswa kwe-serial cell extracts eludongeni kunye nokuhlolwa kwe-mAb yabo) usebenzisa iinkqubo ezichazwe ngaphambili 27, 43, 50, 51.Uhlalutyo lwe-glycome, i-alcohol-insoluble i-alcohol-insoluble ye-plant cell wall material ilungiswa ukusuka kwiintsalela ze-biomass kwaye ixhomekeke kwi-serial extraction kunye ne-reagents enobundlobongela obukhulayo njenge-ammonium oxalate (50 mM), i-sodium carbonate (50 mM kunye ne-0.5% w / v), i-CON.(I-1M kunye ne-4M, zombini kunye ne-1% w / v sodium borohydride) kunye ne-acid chlorite njengoko kuchazwe ngaphambili52,53.Izicatshulwa zabe ziphantsi kwe-ELISA ngokubhekiselele kwiphaneli eyinkimbinkimbi ye-mAb50s ejoliswe kwi-cell wall glycan, kunye neempendulo ezibophezelayo ze-mAb zaboniswa njengemephu yokushisa.I-mAbs ejolise kwisityalo sodonga lweseli ye-glycan yathengwa kwizitokhwe zaselabhoratri (CCRC, JIM kunye ne-MAC series).
Inyathelo elinye le-biotinylation ye-oligosaccharides.Ukudibanisa i-carbohydrates kunye ne-biotin-LC-hydrazide yenziwa ngokusebenzisa le nkqubo ilandelayo.I-Biotin-LC-hydrazide (4.6 mg / 12 μmol) yachithwa kwi-dimethyl sulfoxide (DMSO, 70 μl) ngokuvuselela ngamandla kunye nokufudumala kwi-65 ° C. kwi-1 min.Iglacial acetic acid (30 µl) yongezwa kwaye umxube wagalelwa kwi-sodium cyanoborohydride (6.4 mg/100 µmol) kwaye inyibilika ngokupheleleyo emva kokufudumeza kwi-65 ° C. malunga nomzuzu omnye.Emva koko, ukusuka kwi-5 ukuya kwi-8 μl yomxube wokuphendula yongezwa kwi-oligosaccharide eyomileyo (i-1-100 nmol) ukufumana i-10-fold or more molar excess of the label over the reduction end.Ukuphendula kwenziwa kwi-65 ° C kwi-2 h, emva koko iisampuli zahlanjululwa ngokukhawuleza.Akukho cyanoborohydride ye-sodium esetyenziswe kwiimvavanyo zokulebula ngaphandle kokunciphisa, kwaye iisampuli zaphendulwa kwi-65 ° C. kwiiyure ze-2.5.
I-ELISA yokugqoka kunye nokuhlamba iisampuli ze-oligosaccharides ze-biotinylated.I-25 μl yeesampulu ze-biotinylated (i-100 μl yesampuli nganye egxininisiweyo exutywe kwi-5 ml ye-0.1 M yesisombululo se-Tris buffer (TBS)) yongezwa kwiqula ngalinye le-avidin-coated plate.Amaqula okulawula afakwe kwi-50 μl ye-biotin kwi-concentration ye-10 μg / ml kwi-0.1 M TBS.Amanzi adiyiniweyo asetyenziswa njengokwaleka kwimilinganiselo engenanto.Ithebhulethi ifakwe kwiiyure ze-2 kwindawo yokushisa kwegumbi ebumnyameni.Geza ipleyiti ngamaxesha e-3 nge-0.1% yobisi oluncitshisiweyo kwi-0.1 M TBS usebenzisa iprogram ye-no.I-11 ye-Grenier flat 3A.
Ukongezwa kunye nokuhlanjwa kwee-antibodies eziphambili.Yongeza i-40 µl ye-antibody yokuqala kwiqula ngalinye.Faka i-microplate ngeyure eli-1 kwindawo yokushisa kwegumbi ebumnyameni.Iipleyiti zaye zahlanjwa amaxesha ama-3 nge-0.1% yobisi kwi-0.1M TBS kusetyenziswa inkqubo yokuhlamba #11 ye-Grenier Flat 3A.
Yongeza i-antibody yesibini kwaye uhlambe.Yongeza i-50 µl ye-antibody yesibini yempuku/impuku (ixutywe i-1:5000 kwi-0.1% yobisi kwi-0.1 M TBS) kwiqula ngalinye.Faka i-microplate ngeyure eli-1 kwindawo yokushisa kwegumbi ebumnyameni.Ii-microplates zaye zahlanjwa ngamaxesha e-5 nge-0.1% yobisi kwi-0.1 M TBS usebenzisa i-Grenier Flat 5A inkqubo yokuhlamba ipleyiti #12.
Ukongeza i-substrate.Yongeza i-50 µl ye-3,3′,5,5′-tetramethylbenzidine (TMB) kwisiseko se-substrate (ngokongeza amathontsi ama-2 e-buffer, amathontsi ama-3 e-TMB, amathontsi ama-2 e-hydrogen peroxide kwi-15 ml yamanzi adiyiniweyo).Lungiselela i-substrate ye-TMB.kunye ne-vortex ngaphambi kokusetyenziswa).Gcoba i-microplate kwindawo yokushisa kwemizuzu engama-30.Ebumnyameni.
Gqibezela inyathelo kwaye ufunde ithebhulethi.Yongeza i-50 µl ye-1 N sulfuric acid kwiqula ngalinye kwaye urekhode ukufunxa ukusuka kwi-450 ukuya kwi-655 nm usebenzisa umfundi we-ELISA.
Lungiselela i-1 mg / ml izisombululo zala ma-analytes kumanzi adibeneyo: i-arabinose, i-rhamnose, i-fucose, i-xylose, i-galacturonic acid (i-GalA), i-glucuronic acid (i-GlcA), i-mannose, i-glucose, i-galactose, i-lactose, i-N-acetylmannosamine (manNAc), i-N-coaminetylglu.(glcNAc), N-acetylgalactosamine (galNAc), inositol (umgangatho wangaphakathi).Imigangatho emibini yalungiswa ngokudibanisa i-1 mg / mL izisombululo zeswekile eziboniswe kwiThebhile 1. Iisampulu zifakwe ekhenkceni kwaye i-lyophilized kwi-80 ° C. de kube onke amanzi asuswe (ngokuqhelekileyo malunga neeyure ze-12-18).
Yongeza i-100–500 µg yesampulu kwityhubhu ye-screw cap kwibhalansi yohlalutyo.Rekhoda imali eyongeziweyo.Kungcono ukunyibilikisa isampuli kugxininiso oluthile lwe-solvent kwaye uyongeze kwi-tube njenge-aliquot yolwelo.Sebenzisa i-20 µl ye-1 mg / ml inositol njengomgangatho wangaphakathi kwityhubhu nganye yesampuli.Isixa somgangatho wangaphakathi wongezwa kwisampuli kufuneka silingane nesixa somgangatho wangaphakathi wongezwa kwi-tube eqhelekileyo.
Yongeza i-8 ml ye-anhydrous methanol kwi-screw cap vial.Emva koko i-4 ml ye-3 N. ye-methanolic HCl isisombululo, ifakwe kwaye ishukunyiswe.Le nkqubo ayisebenzisi amanzi.
Yongeza i-500 µl ye-1 M HCl isisombululo se-methanol kwiisampuli ze-oligosaccharide kunye neetyhubhu eziqhelekileyo ze-TMS.Iisampulu zafukanywa ngobusuku (iiyure ezingama-168) kwi-80 ° C. kwibhloko ye-thermal.Yomisa imveliso ye-methanolysis kwiqondo lobushushu begumbi usebenzisa i-manifold yokomisa.Yongeza i-200 µl MeOH kwaye womise kwakhona.Le nkqubo iphinda kabini.Yongeza i-200 µl ye-methanol, i-100 µl yepyridine kunye ne-100 µl ye-acetic anhydride kwisampulu kwaye udibanise kakuhle.Faka iisampuli kwiqondo lobushushu begumbi imizuzu engama-30.kwaye yomiswa.Yongeza i-200 µl yemethanol kwaye womise kwakhona.
Yongeza i-200 µl ye-Tri-Sil kunye netyhubhu evaliweyo yobushushu imizuzu engama-20.I-80 ° C, emva koko ipholile kwiqondo lokushisa.Sebenzisa ukuphindaphinda ukomisa ukomisa ngakumbi isampulu ukuya kumthamo omalunga nama-50 µl.Kubalulekile ukuba siqaphele ukuba asizange sivumele iisampuli ukuba zome ngokupheleleyo.
Yongeza i-2 ml ye-hexane kwaye udibanise kakuhle nge-vortexing.Gcwalisa iingcebiso zePasteur pipettes (5-8 mm) ngeqhekeza leglasi yoboya ngokufaka uboya beglasi phezu kwepipette ye-intshi eyi-5-3/4 yobubanzi.Iisampulu zaziyi-centrifuged kwi-3000 g yemizuzu emi-2.Naziphi na iintsalela ezinganyibilikiyo ziyavuthuluka.Yomisa isampuli ukuya kwi-100-150 µl.Umthamo omalunga ne-1 μl ufakwe kwi-GC-MS kwiqondo lokushisa lokuqala lama-80 ° C kunye nexesha lokuqala lemizuzu ye-2.0 (Itheyibhile 2).