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Iindlela ezintsha ze-immunological kunye ne-mass spectrometric zohlalutyo oluntsonkothileyo lwee-oligosaccharides eziqhubekayo kwi-corn stover ziphathwe nge-AFEX. I-Lignocellulosic biomass yindlela ezinzileyo kune-fossil fuels kwaye isetyenziswa kakhulu ekuphuhliseni i-biotechnology yokuvelisa iimveliso ezifana nokutya, ukutya, amafutha kunye neekhemikhali. Isitshixo sezi teknoloji kukuphuhliswa kweenkqubo ezikhuphisanayo neendleko zokuguqula ii-carbohydrates ezintsonkothileyo ezikhoyo eludongeni lweeseli zezityalo zibe ziishukela ezilula ezifana ne-glucose, i-xylose kunye ne-rabinose. Ngenxa yokuba i-lignocellulosic biomass inzima kakhulu, kufuneka iphathwe ngonyango lwe-thermochemical (umz., i-ammonia fiber exfoliation (AFEX), i-dilute acids (DA), i-ionic liquids (IL)) kunye nonyango lwebhayoloji (umz., i-enzymatic hydrolysis kunye ne-microbial fermentation) ngokudibeneyo ukuze kufunyanwe imveliso oyifunayo. Nangona kunjalo, xa kusetyenziswa ii-enzymes zefungus zorhwebo kwinkqubo ye-hydrolysis, yi-75-85% kuphela yeeswekile ezinyibilikayo ezenziweyo ezizii-monosaccharides, kwaye i-15-25% eseleyo zii-oligosaccharides ezinyibilikayo nezingenakunyangwa, ezingafumaneki rhoqo kwiintsholongwane. Ngaphambili, siye sahlula ngempumelelo saza sazicoca ii-oligosaccharides ezinyibilikayo nezinobuthi sisebenzisa indibaniselwano ye-carbon kunye ne-diatomaceous earth separation kunye ne-size exclusion chromatography, kwaye saphanda neepropati zazo zokuthintela i-enzyme. Sifumanise ukuba ii-oligosaccharides eziqulathe i-higher degree of polymerization (DP) methylated uronic acid substitutions kunzima ukuzicubungula ngee-enzyme blends zorhwebo kune-DP ephantsi kunye ne-neutral oligosaccharides. Apha sixela ukusetyenziswa kweendlela ezininzi ezongezelelweyo, kubandakanya i-glycan profiling kusetyenziswa ii-monoclonal antibodies (mAbs) ezithile kwi-plant biomass glycans ukuchaza ii-glycan bonds kwiindonga zeseli yesityalo kunye ne-enzymatic hydrolysates, i-matrix-assisted laser desorption ionization, i-time-of-flight mass-spectrometry. I-MALDI-TOF-MS) isebenzisa iincopho zokuxilonga ezinolwazi ngesakhiwo ezifunyenwe yi-spectroscopy emva kokubola kwesibini kwee-ions ezingalunganga, i-gas chromatography kunye ne-mass spectrometry (GC-MS) ukuchaza iibhondi ze-oligosaccharide kunye ne-derivatization kunye ne-non-derivatization. Ngenxa yobukhulu obuncinci be-oligosaccharides (DP 4–20), ezi molekyuli kunzima ukuzisebenzisa ekubopheni nasekuchazeni i-mAb. Ukuze soyise le ngxaki, sisebenzise indlela entsha ye-biotin conjugation-based oligosaccharide immobilization eyabhala ngempumelelo uninzi lwee-oligosaccharides ezinyibilikayo ze-DP ezisezantsi kumphezulu we-microplate, eyathi emva koko yasetyenziswa kwinkqubo ye-mAb ephezulu yokukhupha uhlalutyo oluthile lwe-ligation. Le ndlela intsha iya kunceda uphuhliso lwee-high throughput glycome assays eziphuculweyo kwixesha elizayo ezinokusetyenziselwa ukwahlula kunye nokuchonga ii-oligosaccharides ezikhoyo kwi-biomarkers ngeenjongo zokuxilonga.
I-Lignocellulosic biomass, eyenziwe zizinto zezolimo, amahlathi, ingca kunye nezinto ezinje ngemithi, sisixhobo esinokubakho sokuvelisa iimveliso ezisekelwe kwi-bio, kubandakanya ukutya, ukutya, amafutha kunye nezinto ezibangela iikhemikhali ukuvelisa iimveliso ezixabisa kakhulu1. Iicarbohydrate (ezifana ne-cellulose kunye ne-hemicellulose) ezikhoyo kwiindonga zeeseli zezityalo ziyasuswa kwi-polymer zibe yi-monosaccharides ngokucubungula iikhemikhali kunye nokuguqulwa kwe-biotransformation (ezifana ne-enzymatic hydrolysis kunye ne-microbial fermentation). Unyango oluqhelekileyo lwangaphambi konyango lubandakanya ukwanda kwe-ammonia fiber (AFEX), i-dilute acid (DA), i-ionic liquid (IL), kunye nokuqhuma komphunga (SE), ezisebenzisa indibaniselwano yeekhemikhali kunye nobushushu ukunciphisa imveliso ye-lignocellulose ngokuvula iindonga zeeseli zezityalo3,4. ubulukhuni bezinto, 5. I-Enzymatic hydrolysis yenziwa kumthwalo ophezulu we-solids kusetyenziswa ii-enzymes ezisebenzisa i-carbohydrate (CAZymes) kunye ne-microbial fermentation kusetyenziswa i-transgenic yeast okanye iibacteria ukuvelisa amafutha kunye neekhemikhali ezisekelwe kwi-bio6.
Ii-CAZymes kwii-enzyme zorhwebo zenziwe ngumxube ontsonkothileyo wee-enzymes ezidibanisa ngokubambisana ii-carbohydrate-sugar bonds ezintsonkothileyo ukuze zenze ii-monosaccharides2,7. Njengoko besitshilo ngaphambili, uthungelwano oluntsonkothileyo lwee-polymers ezinuka kamnandi ze-lignin kunye nee-carbohydrates luzenza zingabi namandla kakhulu, nto leyo ekhokelela ekuguqulweni kweswekile okungaphelelanga, ziqokelela i-15-25% yee-oligosaccharides zesini ezingaveliswanga ngexesha le-enzyme hydrolysis ye-biomass ephathwe kwangaphambili. Le yingxaki eqhelekileyo ngeendlela ezahlukeneyo ze-biomass pretreatment. Ezinye zezizathu zale ngxaki ziquka ukuthintela ii-enzyme ngexesha le-hydrolysis, okanye ukungabikho okanye amanqanaba aphantsi ee-enzymes ezibalulekileyo ezifunekayo ukuphula ii-sugar bonds kwi-biomass yezityalo. Ukuqonda ukwakheka kunye neempawu zesakhiwo seswekile, ezifana nee-sugar bonds kwi-oligosaccharides, kuya kusinceda siphucule ukuguqulwa kweswekile ngexesha le-hydrolysis, okwenza iinkqubo ze-biotechnology zikwazi ukukhuphisana nemveliso ephuma kwi-petroleum.
Ukuchonga ulwakhiwo lweecarbohydrate kunzima 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. ,ishumi elinesibhozo. Iindlela ze-MS ezifana ne-time-of-flight mass spectrometry ene-laser desorption kunye ne-ionization kusetyenziswa i-matrix (MALDI-TOF-MS) ziyindlela eguquguqukayo yokuchonga izakhiwo ze-carbohydrate. Kutshanje, i-Collision-Induced Dissociation (CID) tandem MS ye-sodium ion adducts iye yasetyenziswa kakhulu ukuchonga iminwe ehambelana neendawo zokuncamathisela ze-oligosaccharide, ulungelelwaniso lwe-anomeric, ulandelelwano, kunye neendawo zokuhlela 20, 21.
Uhlalutyo lweGlycan sisixhobo esibalaseleyo sokuchonga ngokunzulu ii-carbohydrate bonds22. Le ndlela isebenzisa ii-antibodies ze-monoclonal (mAbs) ezijoliswe kwi-glycan eludongeni lweseli yesityalo njengee-probes ukuqonda unxibelelwano oluntsonkothileyo lwe-carbohydrate. Zingaphezulu kwama-250 mAbs ezikhoyo kwihlabathi liphela, zenzelwe ngokuchasene nee-oligosaccharides ezahlukeneyo ezithe ngqo nezinamasebe zisebenzisa ii-saccharides ezahlukeneyo24. Ii-mAbs ezininzi zisetyenziswe kakhulu ukuchaza ulwakhiwo, ukwakheka, kunye notshintsho lodonga lweseli yesityalo, njengoko kukho umahluko omkhulu ngokuxhomekeke kuhlobo lweseli yesityalo, isitho, ubudala, isigaba sophuhliso, kunye nendawo yokukhula25,26. Kutshanje, le ndlela isetyenziselwe ukuqonda amaqela e-vesicle kwiinkqubo zezityalo nezilwanyana kunye neendima zawo ekuthuthweni kwe-glycan njengoko kumiselwe ziimpawu ze-subcellular, amanqanaba ophuhliso, okanye izinto ezikhuthazayo kokusingqongileyo, kunye nokuchonga umsebenzi we-enzyme. Ezinye zezakhiwo ezahlukeneyo ze-glycans kunye ne-xylans ezichongiweyo ziquka i-pectin (P), i-xylan (X), i-mannan (M), i-xyloglucans (XylG), i-mixed bond glucans (MLG), i-arabinoxylan (ArbX), i-galactomannan (GalG), i-glucuronic acid-arabinoxylan (GArbX) kunye ne-arabino-galactan (ArbG)29.
Nangona kunjalo, nangona yonke le mizamo yophando, zimbalwa izifundo ezigxile kuhlobo lokuqokelelwa kwe-oligosaccharide ngexesha le-hydrolysis ephezulu ye-solids load (HSL), kubandakanya ukukhululwa kwe-oligosaccharide, utshintsho lobude be-oligomeric chain ngexesha le-hydrolysis, ii-polymers ezahlukeneyo ze-DP eziphantsi, kunye ne-curves zazo. Okwangoku, nangona uhlalutyo lwe-glycan lubonakalise ukuba sisixhobo esiluncedo sohlalutyo olupheleleyo lwesakhiwo se-glycan, kunzima ukuvavanya ii-oligosaccharides ze-DP eziphantsi ezinyibilikayo emanzini kusetyenziswa iindlela ze-antibody. Ii-oligosaccharides ze-DP ezincinci ezinobunzima be-molecular obungaphantsi kwe-5-10 kDa azibopheleleki kwiiplate ze-ELISA 33, 34 kwaye ziyahlanjwa ngaphambi kokufakwa kwe-antibody.
Apha, okokuqala ngqa, sibonisa uvavanyo lwe-ELISA kwiiplate ezigqunywe yi-avidin sisebenzisa ii-antibodies ze-monoclonal, sidibanisa inkqubo ye-biotinylation yenyathelo elinye yee-oligosaccharides ezinyibilikayo kunye nohlalutyo lwe-glycome. Indlela yethu yohlalutyo lwe-glycome iqinisekiswe luhlalutyo olusekelwe kwi-MALDI-TOF-MS kunye ne-GC-MS lwe-complementary oligosaccharide linkages kusetyenziswa i-trimethylsilyl (TMS) derivatization yee-hydrolyzed sugar compositions. Le ndlela intsha inokuphuhliswa njengendlela ephezulu kwixesha elizayo kwaye ifumane ukusetyenziswa okubanzi kuphando lwe-biomedical35.
Utshintsho emva kokuguqulwa kwee-enzymes kunye nee-antibodies, ezifana ne-glycosylation,36 luchaphazela umsebenzi wazo webhayoloji. Umzekelo, utshintsho kwi-glycosylation yeeproteni ze-serum ludlala indima ebalulekileyo kwi-arthritis yokuvuvukala, kwaye utshintsho kwi-glycosylation lusetyenziswa njengeempawu zokuxilonga37. Ii-glycans ezahlukeneyo ziye zaxelwa kwiincwadi ukuba zibonakala ngokulula kwizifo ezahlukeneyo, kubandakanya izifo ezingapheliyo zokuvuvukala zendlela yesisu kunye nesibindi, usulelo lwentsholongwane, umhlaza we-ovarian, webele, kunye ne-prostate38,39,40. Ukuqonda ulwakhiwo lwe-glycans kusetyenziswa iindlela ze-glycan ELISA ezisekelwe kwi-antibody kuya kubonelela ngokuzithemba okongezelelweyo ekuxilongweni kwesifo ngaphandle kokusebenzisa iindlela ezintsonkothileyo ze-MS.
Uphononongo lwethu lwangaphambili lubonise ukuba ii-oligosaccharides eziqinileyo zahlala zingabinamanzi emva konyango lwangaphambi konyango kunye ne-enzyme hydrolysis (Umfanekiso 1). Kumsebenzi wethu opapashwe ngaphambili, siphuhlise indlela yokukhupha i-charcoal solid-phase evuselelweyo ukuze sahlukanise ii-oligosaccharides kwi-AFEX-pretreated corn stover hydrolyzate (ACSH)8. Emva kokukhupha nokwahlulahlula kokuqala, ii-oligosaccharides zahlulwahlulwa ngakumbi yi-size exclusion chromatography (SEC) kwaye zaqokelelwa ngokulandelelana kobunzima be-molecular. Ii-monomers zeswekile kunye nee-oligomers ezikhutshwe kwiindlela ezahlukeneyo zonyango lwangaphambi konyango zahlalutywa ngohlalutyo lokwakheka kweswekile. Xa kuthelekiswa umxholo wee-oligomers zeswekile ezifunyenwe ngeendlela ezahlukeneyo zonyango lwangaphambi konyango, ubukho bee-oligosaccharides eziqinileyo yingxaki eqhelekileyo ekuguqulweni kwe-biomass ibe yi-monosaccharides kwaye kunokukhokelela ekunciphiseni isivuno seswekile ubuncinane nge-10-15% kwaye kude kube yi-18%. US. Le ndlela isetyenziselwa ukuvelisa ngakumbi ii-fractions ze-oligosaccharide. I-ACH ephumayo kunye neenxalenye zayo ezilandelayo ezinobunzima obahlukeneyo beemolekyuli zisetyenziswe njengezinto zovavanyo zokuchonga ii-oligosaccharides kulo msebenzi.
Emva konyango lwangaphambi konyango kunye ne-enzyme hydrolysis, ii-oligosaccharides ezihlala zingapheli. Apha (A) yindlela yokwahlula i-oligosaccharide apho ii-oligosaccharides zahlulwe kwi-AFEX-pretreated corn stover hydrolysate (ACSH) kusetyenziswa ibhedi egcweleyo ye-activated carbon kunye ne-diatomaceous earth; (B) Indlela yokwahlula ii-oligosaccharides. Ii-oligosaccharides zahlulwe ngakumbi yi-size exclusion chromatography (SEC); (C) ii-Saccharide monomers kunye nee-oligomers ezikhutshwe kwiindlela ezahlukeneyo zonyango lwangaphambi konyango (i-diluted acid: DA, i-ionic liquid: IL kunye ne-AFEX). Iimeko ze-enzyme hydrolysis: ukulayisha okuphezulu kwe-solids ye-25% (w/w) (malunga ne-8% yokulayisha i-glucan), i-hydrolysis yeeyure ezingama-96, ukulayisha i-enzyme yorhwebo ye-20 mg/g (umlinganiselo we-Ctec2:Htec2:MP-2:1:1) kunye (D) Ii-monomers zeswekile kunye nee-oligomers ze-glucose, i-xylose kunye ne-arabinose ezikhutshwa kwi-AFEX pre-treated corn stover (ACS).
Uhlalutyo lweGlycan lubonakalise ukuba sisixhobo esiluncedo kuhlalutyo olupheleleyo lwesakhiwo se-glycans kwizicatshulwa ezithathwe kwi-biomass residues eqinileyo. Nangona kunjalo, ii-saccharides ezinyibilikayo emanzini azibonakaliswa ngokwaneleyo kusetyenziswa le ndlela yendabuko41 kuba ii-oligosaccharides ezinobunzima obuphantsi be-molecular kunzima ukuzithintela kwiiplate ze-ELISA kwaye ziyahlanjwa ngaphambi kokuba kongezwe i-antibody. Ke ngoko, ukubopha kunye nokuchonga ii-antibody, indlela ye-biotinylation yenyathelo elinye isetyenzisiwe ukugquma ii-oligosaccharides ezinyibilikayo, ezingahambelaniyo nemithetho kwiiplate ze-ELISA ezigqunywe yi-avidin. Le ndlela ivavanyiwe kusetyenziswa i-ACSH yethu eveliswe ngaphambili kunye neqhezu elisekelwe kubunzima bayo be-molecular (okanye inqanaba le-polymerization, i-DP). I-biotinylation yenyathelo elinye isetyenzisiwe ukwandisa i-oligosaccharide binding affinity ngokongeza i-biotin-LC-hydrazide kwisiphelo sokunciphisa se-carbohydrate (Umzobo 2). Kwisisombululo, iqela le-hemiacetal kwisiphelo sokunciphisa lisabela neqela le-hydrazide le-biotin-LC-hydrazide ukwenza i-hydrazone bond. Xa kukho i-NaCNBH3 enciphisa i-arhente, i-hydrazone bond iyancitshiswa ibe yimveliso yokugqibela ezinzileyo ye-biotinylated. Ngokuguqulwa kwesiphelo sokunciphisa iswekile, ukubopha ii-oligosaccharides ezisezantsi ze-DP kwiiplate ze-ELISA kuye kwenzeka, kwaye kuphononongo lwethu oku kwenziwe kwiiplates ezifakwe i-avidin kusetyenziswa ii-mAbs ezijoliswe kwi-glycan.
Ukuhlolwa kwee-antibodies ze-monoclonal ezisekelwe kwi-ELISA ukuze kufunyanwe ii-oligosaccharides ezifakwe kwi-biotinylated. Apha (A) kudityaniswe i-biotinylation yee-oligosaccharides kunye nokuhlolwa okulandelayo kwe-ELISA kunye nee-mAbs ezijoliswe kwi-glycan kwiiplates ezifakwe kwi-NeutrAvidin kwaye (B) kubonisa inkqubo yenyathelo elinye ye-biotinylation yeemveliso zokusabela.
Iipleyiti ezigqunywe nge-Avidin ezine-antibody ezidityaniswe ne-oligosaccharide zongezwa kwi-antibodies eziphambili nezesibini zaza zahlanjwa kwindawo ekhanyayo nesengqiqweni. Emva kokuba ukubopha kwe-antibody kugqityiwe, yongeza i-substrate ye-TMB ukuze ufukame ipleyiti. Ekugqibeleni impendulo yamiswa nge-sulfuric acid. Iipleyiti ezigqunywe nge-incubation zahlalutywa kusetyenziswa i-ELISA reader ukufumanisa amandla okubopha kwe-antibody nganye ukuze kufunyanwe ukudibana kwe-antibody ethile. Ukuze ufumane iinkcukacha kunye nemilinganiselo yovavanyo, jonga icandelo elihambelanayo elithi “Izixhobo kunye neendlela”.
Sibonisa ukusetyenziswa kwale ndlela intsha kwizicelo ezithile ngokuchonga ii-oligosaccharides ezinyibilikayo ezikhoyo kwi-ACSH kunye nakwii-oligosaccharide fractions ezikrwada nezicociweyo ezihlukaniswe kwi-lignocellulosic hydrolysates. Njengoko kubonisiwe kuMfanekiso 3, ii-xylans ezixhaphakileyo ezifakwe kwi-epitope ezichongiweyo kwi-ACSH kusetyenziswa iindlela ze-bioacylated glycome assay zihlala ziyi-uronic (U) okanye i-methyluronic (MeU) kunye ne-pectic arabinogalactans. Uninzi lwazo lufunyenwe nakwisifundo sethu sangaphambili malunga nohlalutyo lwe-glycans ye-non-hydrolyzed solids (UHS)43.
Ukufunyanwa kwee-recalcitrant oligosaccharide epitopes kusetyenziswa i-antibody ye-monoclonal eqondiswe kwi-glycan yodonga lweseli. I-fraction "engathathi cala" yi-ACN fraction kwaye i-fraction "e-acidic" yi-FA fraction. Imibala ebomvu ekhanyayo kwi-heatmap ibonisa umxholo ophezulu we-epitope, kwaye i-blues ekhanyayo ibonisa imvelaphi engenanto. Imilinganiselo yombala kwisikali isekelwe kwixabiso le-OD eluhlaza kwiifomyula N=2. Ii-epitopes eziphambili ezibonwa zii-antibodies ziboniswe ngasekunene.
Ezi zakhiwo zingeyiyo i-cellulose azikwazanga ukuhlukaniswa zii-cellulases kunye nee-hemicellulases eziqhelekileyo kumxube we-enzyme yorhwebo ovavanyiweyo, oquka ii-enzymes zorhwebo ezisetyenziswa kakhulu. Ke ngoko, ii-enzymes ezintsha ezincedisayo ziyafuneka kwi-hydrolysis yazo. Ngaphandle kwee-enzymes eziyimfuneko ezingezizo i-cellulose, ezi bonds ezingezizo i-cellulose zithintela ukuguqulwa ngokupheleleyo zibe yi-monosaccharides, nokuba ii-polymers zazo zeswekile zi-hydrolyzed kakhulu zibe ziingceba ezimfutshane kwaye zinyibilikiswe kusetyenziswa imixube ye-enzyme yorhwebo.
Uphononongo olongezelelweyo lokusasazwa kwesignali kunye namandla ayo okubopha lubonise ukuba ii-epitopes zokubopha zaziphantsi kwiinxalenye zeswekile ze-DP eziphezulu (A, B, C, DP ukuya kuthi ga kwi-20+) kunakwiinxalenye ze-DP eziphantsi (D, E, F, DP) kwii-dimers) (Umzobo 1). Iziqwenga ze-asidi zixhaphake kakhulu kwii-epitopes ezingezizo iiselilulose kuneziqwenga ezingathathi cala. Ezi zinto zihambelana nomzekelo obonwe kwisifundo sethu sangaphambili, apho ii-DP eziphezulu kunye nee-acid moieties zazixhathisa ngakumbi kwi-enzyme hydrolysis. Ke ngoko, ubukho bee-non-cellulose glycan epitopes kunye nokutshintshwa kwe-U kunye ne-MeU kunokunceda kakhulu ekuzinzeni kwee-oligosaccharides. Kufuneka kuqatshelwe ukuba ukusebenza kakuhle kokubopha kunye nokufumanisa kunokuba yingxaki kwii-oligosaccharides eziphantsi ze-DP, ngakumbi ukuba i-epitope yi-dimeric okanye i-trimeric oligosaccharide. Oku kunokuvavanywa kusetyenziswa ii-oligosaccharides zorhwebo zobude obahlukeneyo, nganye iqulethe i-epitope enye kuphela ebopha kwi-mAb ethile.
Ngoko ke, ukusetyenziswa kwee-antibodies ezithile kwisakhiwo kutyhile iintlobo ezithile zeebhondi eziphinda zisebenze. Ngokuxhomekeke kuhlobo lwe-antibody esetyenzisiweyo, ipateni efanelekileyo yokuligation, kunye namandla esignali eyivelisayo (ininzi nencinci kakhulu), ii-enzymes ezintsha zinokuchongwa kwaye zongezwe ngokwe-semi-quantitatively kumxube we-enzyme ukuze kubekho i-glycoconversion epheleleyo. Ukuthatha uhlalutyo lwee-oligosaccharides ze-ACSH njengomzekelo, singenza isiseko sedatha seebhondi ze-glycan kwisixhobo ngasinye se-biomass. Kufuneka kuqatshelwe apha ukuba i-affinity eyahlukileyo yee-antibodies kufuneka ithathelwe ingqalelo, kwaye ukuba i-affinity yazo ayaziwa, oku kuya kudala ubunzima obuthile xa kuthelekiswa imiqondiso yee-antibodies ezahlukeneyo. Ukongeza, uthelekiso lweebhondi ze-glycan lunokusebenza ngcono phakathi kweesampuli ze-antibody efanayo. Ezi bondi ziqinileyo zinokudityaniswa nesiseko sedatha se-CAZyme, apho sinokuchonga khona ii-enzymes, sikhethe ii-enzymes ezifanelekileyo kwaye sivavanye ii-enzymes eziphula ibhondi, okanye siphuhlise iinkqubo zeentsholongwane zokuveza ezi enzymes ukuze zisetyenziswe kwi-biorefineries44.
Ukuvavanya indlela iindlela zempilo ezihambelana ngayo nezinye iindlela zokuchonga ii-oligosaccharides ezinobunzima obuphantsi beemolekyuli ezikhoyo kwi-hydrolysates ze-lignocellulosic, senze i-MALDI (Umzobo 4, S1-S8) kunye nohlalutyo lwee-saccharides ezivela kwi-TMS ezisekwe kwi-GC-MS kwinxalenye efanayo yephaneli (Umzobo 5) ye-oligosaccharide. I-MALDI isetyenziselwa ukuthelekisa ukuba ukusasazwa ngobuninzi kweemolekyuli ze-oligosaccharide kuyahambelana na nesakhiwo esicetywayo. Kumzobo 4 ubonisa i-MC yezinto ezingathathi cala ze-ACN-A kunye ne-ACN-B. Uhlalutyo lwe-ACN-A luqinisekisile uluhlu lweeswekile ze-pentose ukusuka kwi-DP 4–8 (Umzobo 4) ukuya kwi-DP 22 (Umzobo S1), ezinobunzima obuhambelana ne-MeU-xylan oligosaccharides. Uhlalutyo lwe-ACN-B luqinisekisile uthotho lwe-pentose kunye ne-glucoxylan nge-DP 8-15. Kwizinto ezongezelelweyo ezifana noMfanekiso S3, iimaphu zokusasazwa kobunzima be-FA-C acidic moiety zibonisa uluhlu lweeshukela ze-pentose ezifakwe endaweni ye-(Me)U ezine-DP ye-8-15 ezihambelana nee-xylans ezifakwe endaweni ye-ELISA kwi-mAb screening. Ii-epitopes ziyafana.
I-MALDI-MS spectrum yee-oligosaccharides ezingaguqukiyo ezikhoyo kwi-ACS. Apha, (A) Ii-ACN-A low weight range fractions eziqulethe i-methylated uronic acid (DP 4-8) zithathe indawo yee-glucuroxylan oligosaccharides kunye (B) ii-ACN-B xylan kunye nee-methylated uronic acid oligosaccharides ezithathe indawo yee-glucuroxylan (DP 8-15).
Uhlalutyo lokwakheka kwe-glycan residue ye-oligosaccharides enganyangekiyo. Apha (A) Ukwakheka kwe-TMS saccharide yee-fractions ezahlukeneyo ze-oligosaccharide ezifunyenwe kusetyenziswa uhlalutyo lwe-GC-MS. (B) Ulwakhiwo lweeswekile ezahlukeneyo ezivela kwi-TMS ezikhoyo kwi-oligosaccharides. I-ACN – i-acetonitrile fraction equlethe i-oligosaccharides engathathi cala kunye ne-FA – i-ferulic acid fraction equlethe i-acid oligosaccharides.
Esinye isigqibo esinomdla sithathwe kuhlalutyo lwe-LC-MS lweqhekeza le-oligosaccharide, njengoko kubonisiwe kuMfanekiso S9 (iindlela zinokubonwa kwizinto ezongezelelweyo ze-elektroniki). Iziqwenga zamaqela e-hexose kunye ne-OAc zibonwe ngokuphindaphindiweyo ngexesha lokubopha iqhekeza le-ACN-B. Olu fumaniso aluqinisekisi nje kuphela ukwahlulwahlulwa okubonwe kuhlalutyo lwe-glycome kunye ne-MALDI-TOF, kodwa lukwabonelela ngolwazi olutsha malunga ne-carbohydrate derivatives enokubakho kwi-lignocellulosic biomass ephathwe kwangaphambili.
Sikwahlalutye ukwakheka kweswekile ye-oligosaccharide fraction sisebenzisa i-TMS sugar derivatization. Sisebenzisa i-GC-MS, sifumanise ukwakheka kwe-neural (non-derivative) kunye ne-acidic sugar (GluA kunye ne-GalA) kwi-oligosaccharide fraction (Umzobo 5). I-Glucuronic acid ifumaneka kwi-acidic components C kunye ne-D, ngelixa i-galacturonic acid ifumaneka kwi-acidic components A kunye ne-B, zombini ezi zi-DP components eziphezulu ze-acidic sugar. Ezi ziphumo aziqinisekisi nje idatha yethu ye-ELISA kunye ne-MALDI, kodwa zikwahambelana nezifundo zethu zangaphambili zokuqokelelana kwe-oligosaccharide. Ke ngoko, sikholelwa ukuba iindlela zanamhlanje ze-immunological ezisebenzisa i-biotinylation ye-oligosaccharides kunye nokuhlolwa kwe-ELISA okulandelayo zanele ukubona i-soluble recalcitrant oligosaccharides kwiisampuli ezahlukeneyo zebhayoloji.
Ekubeni iindlela zokuhlola i-mAb ezisekelwe kwi-ELISA ziqinisekiswe ngeendlela ezahlukeneyo, besifuna ukuphonononga ngakumbi amandla ale ndlela intsha yokulinganisa. Ii-oligosaccharides ezimbini zorhwebo, i-xylohexasaccharide oligosaccharide (XHE) kunye ne-23-α-L-arabinofuranosyl-xylotriose (A2XX), zathengwa zaza zavavanywa kusetyenziswa indlela entsha ye-mAb ejolise kwi-glycan yodonga lweseli. Umfanekiso 6 ubonisa ulwalamano oluthe ngqo phakathi kwesignali yokubopha ye-biotinylated kunye noxinzelelo lwelog loxinzelelo lwe-oligosaccharide, nto leyo ebonisa imodeli enokwenzeka ye-Langmuir adsorption. Phakathi kwee-mAbs, i-CCRC-M137, i-CCRC-M138, i-CCRC-M147, i-CCRC-M148, kunye ne-CCRC-M151 enxulumene ne-XHE, kunye ne-CCRC-M108, i-CCRC-M109, kunye ne-LM11 enxulumene ne-A2XX kuluhlu lwe-1 nm ukuya kwi-100 nano. Ngenxa yokunqongophala kokufumaneka kwee-antibodies ngexesha lovavanyo, kwenziwe uvavanyo olulinganiselweyo nge-oligosaccharide nganye enomlinganiselo. Kufuneka kuqatshelwe apha ukuba ezinye ii-antibodies zisabela ngokwahlukileyo kakhulu kwi-oligosaccharide efanayo njenge-substrate, mhlawumbi kuba zibopha kwii-epitopes ezahlukeneyo kancinci kwaye zinokuba nee-affinities ezahlukeneyo zokubopha. Iindlela kunye neziphumo zokuchonga i-epitope ngokuchanekileyo ziya kuba nzima ngakumbi xa indlela entsha ye-mAb isetyenziswa kwiisampuli zokwenyani.
Ii-oligosaccharides ezimbini zorhwebo zisetyenzisiwe ukumisela uluhlu lokufumanisa ii-mAbs ezahlukeneyo ezijolise kwi-glycan. Apha, ulwalamano oluchanekileyo kunye noxinzelelo lwelog yoxinzelelo lwe-oligosaccharide lubonisa iipateni ze-adsorption zeLangmuir ze-(A) XHE kunye ne-mAb kunye ne-(B) A2XX kunye ne-mAb. Ii-epitopes ezifanayo zibonisa izakhiwo ze-oligosaccharides zorhwebo ezisetyenziswa njengee-substrates kuvavanyo.
Ukusetyenziswa kwee-antibodies ze-monoclonal ezijoliswe kwi-glycan (uhlalutyo lwe-glycocomic okanye uvavanyo lwe-mAb olusekwe kwi-ELISA) sisixhobo esinamandla sokuchonga ngokunzulu uninzi lwee-glycans ezinkulu zodonga lweseli ezenza i-biomass yezityalo. Nangona kunjalo, uhlalutyo lwe-glycan oluqhelekileyo lubonisa kuphela ii-glycans ezinkulu zodonga lweseli, njengoko uninzi lwee-oligosaccharides zingathintelwa ngokufanelekileyo kwiiplate ze-ELISA. Kolu phononongo, i-AFEX-pretreated corn stover yenziwe nge-enzymeally hydrolyzed kumxholo ophezulu we-solids. Uhlalutyo lweswekile lusetyenziselwe ukumisela ukwakheka kwee-carbohydrate zodonga lweseli eziphinda zisebenze kwi-hydrolyzate. Nangona kunjalo, uhlalutyo lwe-mAb lwee-oligosaccharides ezincinci kwii-hydrolysates aluthathelwa ngqalelo, kwaye kufuneka izixhobo ezongezelelweyo ukuze kuthintelwe ngokufanelekileyo ii-oligosaccharides kwiiplate ze-ELISA.
Apha sixela indlela entsha nesebenzayo yokuthintela i-oligosaccharide yokuvavanya i-mAb ngokudibanisa i-oligosaccharide biotinylation elandelwa kukuhlolwa kwe-ELISA kwiiplate ezigqunywe yi-NeutrAvidin™. Ii-oligosaccharides ezingenakushukunyiswa zibonise ukuhambelana okwaneleyo kwe-antibody ukuze kukwazi ukufunyanwa ngokukhawuleza nangokufanelekileyo kwee-oligosaccharides eziphinda ziguqulwe. Uhlalutyo lokwakheka kwezi oligosaccharides ziqinileyo ezisekelwe kwi-mass spectrometry luqinisekisile iziphumo zale ndlela intsha yokuvavanya i-immuno. Ke ngoko, ezi zifundo zibonisa ukuba indibaniselwano ye-oligosaccharide biotinylation kunye nokuhlolwa kwe-ELISA kunye nee-antibodies ze-monoclonal ezijoliswe kwi-glycan zinokusetyenziselwa ukubona ii-crosslinks kwi-oligosaccharides kwaye zingasetyenziswa kakhulu kwezinye izifundo ze-biochemical ezichaza ulwakhiwo lwee-oligosaccharides.
Le ndlela yokwenza i-glycan esekwe kwi-biotin yingxelo yokuqala ekwaziyo ukuphanda ii-recalcitrant carbohydrate bonds ze-oligosaccharides ezinyibilikayo kwi-biomass yezityalo. Oku kunceda ukuqonda ukuba kutheni ezinye iindawo ze-biomass zilukhuni kangaka xa kufikwa kwimveliso ye-biofuel. Le ndlela igcwalisa umsantsa obalulekileyo kwiindlela zohlalutyo lwe-glycome kwaye yandisa ukusetyenziswa kwayo kuluhlu olubanzi lwe-substrates ngaphaya kwe-oligosaccharides yezityalo. Kwixesha elizayo, singasebenzisa iirobhothi kwi-biotinylation kwaye sisebenzise indlela esiyiphuhlisileyo yohlalutyo oluphezulu lweesampuli sisebenzisa i-ELISA.
Ingca yombona (CS) ekhuliswe kwimbewu yePioneer 33A14 hybrid yavunwa ngo-2010 kwiKramer Farms eRay, eColorado. Ngemvume yomnini-mhlaba, le biomass ingasetyenziselwa uphando. Iisampulu zigcinwe zomile <6% ukufuma kwiingxowa ezitshixwayo zip-lock kubushushu begumbi. Iisampulu zigcinwe zomile <6% ukufuma kwiingxowa ezitshixwayo zip-lock kubushushu begumbi. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Iisampulu zigcinwe zomile kubushushu obungaphantsi kwe-6% kwiingxowa ezineziphu kubushushu begumbi.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 zigcinwa kwiingxowa ze-zipper kubushushu begumbi obunomswakama ongaphantsi kwe-6%.Olu phononongo luhambelane nezikhokelo zasekuhlaleni nezesizwe. Uhlalutyo lokwakheka lwenziwe kusetyenziswa iprotocol ye-NREL. Ukwakheka kwafunyaniswa ukuba kuqulethe i-glucan engama-31.4%, i-xylan engama-18.7%, i-arabinan engama-3.3%, i-galactan engama-1.2%, i-acetyl engama-2.2%, i-lignin engama-14.3%, iproteni engama-1.7% kunye nothuthu oluyi-13.4%.
I-Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) ngumxube ontsonkothileyo we-cellulase, i-β-glucosidase kunye ne-Cellic® HTec2 (157 mg protein/ml, lot VHN00001) evela kwiNovozymes (Franklinton, NC, USA)). I-Multifect Pectinase® (72 mg protein/mL), umxube ontsonkothileyo wee-enzymes ezitshabalalisa i-pectin, yanikelwa yiDuPont Industrial Biosciences (Palo Alto, CA, USA). Uxinzelelo lwe-enzyme protein lumiselwe ngokuqikelela umxholo weproteni (kunye nokuthabatha igalelo le-non-protein nitrogen) kusetyenziswa uhlalutyo lwe-Kjeldahl nitrogen (indlela ye-AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). I-Diatomaceous earth 545 ithengwe kwi-EMD Millipore (Billerica, MA). I-activated carbon (i-DARCO, ii-granules ezili-100 ze-mesh), i-Avicel (PH-101), i-beech xylan, kunye nazo zonke ezinye iikhemikhali zithengwe kwiSigma-Aldrich (eSt. Louis, MO).
Unyango lwangaphambi kwe-AFEX lwenziwe kwi-GLBRC (i-Biomass Conversion Research Laboratory, i-MSU, i-Lansing, i-MI, e-USA). Unyango lwangaphambi kwe-AFEX lwenziwa kwi-140° C. imizuzu eli-15. Ixesha lokuhlala eliyi-46 kwi-1:1 ratio ye-anhydrous ammonia kwi-biomass kwi-60% (w/w) yokulayisha kwi-stainless steel benchtop batch reactor (i-Parr Instruments Company). Kwathatha imizuzu engama-30. I-reactor yaziswa kwi-140°C kwaye i-ammonia yakhululwa ngokukhawuleza, okuvumela i-biomass ukuba ibuyele ngokukhawuleza kubushushu begumbi. Ulwakhiwo lwe-AFEX pre-treated corn stover (ACS) lwalufana nolwe-corn stover enganyangwanga (UT-CS).
Izinto eziqinileyo eziphezulu ze-ACSH 25% (w/w) (malunga ne-8% ye-dextran loading) zalungiswa njengesixhobo sokuqala ukuveliswa okukhulu kwee-oligosaccharides. I-Enzymatic hydrolysis ye-ACS yenziwe kusetyenziswa umxube we-enzyme othengiswayo oquka i-Cellic® Ctec2 10 mg protein/g glucan (kwi-biomass elungisiweyo kwangaphambili), i-Htec2 (Novozymes, Franklinton, NC), i-5 mg protein/g glucan, kunye ne-Multifect Pectinase (Genencor Inc, USA). ). ), i-5 mg protein/g dextran. I-Enzymatic hydrolysis yenziwe kwi-bioreactor ye-5-litre enomthamo osebenzayo we-3 litres, i-pH 4.8, 50°C kunye ne-250 rpm. Emva kwe-hydrolysis iiyure ezingama-96, i-hydrolyzate yaqokelelwa nge-centrifugation kwi-6000 rpm imizuzu engama-30 kwaye emva koko kwi-14000 rpm imizuzu engama-30 ukususa izinto eziqinileyo ezingafakwanga amanzi. Emva koko i-hydrolyzate yahluzwa nge-sterile nge-0.22 mm filter beaker. I-hydrolyzate ehluziweyo yagcinwa kwiibhotile ezicocekileyo kwi-4°C. yaze yahluzwa kwi-carbon.
Uhlalutyo lokwakheka kweesampuli ze-biomass ezisekelwe kwi-extract ngokweenkqubo zohlalutyo lwelebhu ye-NREL: ukulungiswa kweesampuli zohlalutyo lokwakheka (NREL/TP-510-42620) kunye nokugqitywa kweecarbohydrates zesakhiwo kunye ne-lignin kwi-biomass (NREL/TP-510 – 42618)47.
Uhlalutyo lwe-oligosaccharide lomlambo we-hydrolyzate lwenziwe kwisikali se-2 ml kusetyenziswa indlela ye-autoclave-based acid hydrolysis. Xuba isampuli ye-hydrolyzate kunye ne-69.7 µl ye-72% ye-sulfuric acid kwityhubhu ye-screw cap ye-10 ml kwaye uyibeke i-incubate iyure e-1 kwi-benchtop kwi-121 °C, uyipholise emkhenkceni uze uyihluze kwi-high performance liquid chromatography (HPLC) vial . Uxinzelelo lwee-oligosaccharides lumiselwe ngokususa uxinzelelo lwee-monosaccharides kwisampuli engeyiyo i-hydrolyzed kwi-total sugar concentration kwisampuli ye-acid-hydrolyzed.
Uxinzelelo lweglucose, i-xylose, kunye ne-rabinose kwi-acid hydrolysed biomass luhlalutywe kusetyenziswa inkqubo yeShimadzu HPLC exhotyiswe nge-autosampler, i-column heater, i-isocratic pump, kunye ne-refractive index detector kwikholamu yeBio-Rad Aminex HPX-87H. Ikholamu igcinwe kwi-50°C kwaye yasuswa nge-0.6 ml/min ye-5 mM H2SO4 emanzini. flow.
I-hydrolyzate supernatant yaxutywa yaza yahlalutywa ukuze kufunyanwe umxholo we-monomer kunye ne-oligosaccharide. Iiswekile ze-monomeric ezifunyenwe emva kwe-enzyme hydrolysis zahlalutywa yi-HPLC exhotyiswe ngekholamu ye-Bio-Rad (Hercules, CA) Aminex HPX-87P kunye nekholamu yokukhusela uthuthu. Ubushushu bekholamu bugcinwe kwi-80°C, amanzi asetyenziswa njengesigaba esihambayo esinesantya sokuhamba se-0.6 ml/min. Ii-Oligosaccharides zichongiwe nge-hydrolysis kwi-dilute acid kwi-121°C ngokweendlela ezichazwe kwiirefs. 41, 48, 49.
Uhlalutyo lweSaccharide lwenziwe kwiintsalela ze-biomass ezingavuthwanga, ezilungisiweyo kwangaphambili ze-AFEX kunye nazo zonke iintsalela ze-biomass ezingafakwanga i-hydrolyzed (kubandakanya ukuveliswa kwee-extracts zodonga lweeseli ezilandelelanayo kunye nokuhlolwa kwazo kwe-mAb) kusetyenziswa iinkqubo ezichazwe ngaphambili 27, 43, 50, 51. Kuhlalutyo lwe-glycome, iintsalela ezinganyibilikiyo zotywala zezinto zodonga lweeseli zezityalo zilungiswa kwiintsalela ze-biomass kwaye zikhutshwe ngokulandelelana ngee-reagents ezinamandla ngakumbi ezifana ne-ammonium oxalate (50 mM), i-sodium carbonate (50 mM kunye ne-0.5% w/v), i-CON. (1M kunye ne-4M, zombini zine-1% w/v sodium borohydride) kunye ne-acid chlorite njengoko kuchaziwe ngaphambili52,53. Ezi extracts emva koko zafakwa kwi-ELISA ngokuchasene nephaneli eyinkimbinkimbi yee-mAb50s ezijoliswe kwi-glycan yodonga lweeseli, kwaye iimpendulo zokubopha ze-mAb zavezwa njengemephu yobushushu. I-mAbs ejolise kwi-plant cell wall glycan ithengwe kwiisitokhwe zelebhu (CCRC, JIM kunye ne-MAC series).
I-biotinylation yenyathelo elinye yee-oligosaccharides. Ukudibanisa ii-carbohydrates ne-biotin-LC-hydrazide kwenziwe kusetyenziswa le nkqubo ilandelayo. I-Biotin-LC-hydrazide (4.6 mg/12 μmol) yanyibilikiswa kwi-dimethyl sulfoxide (DMSO, 70 μl) ngokuyixuba ngamandla nokuyifudumeza kwi-65° C. umzuzu o-1. I-Glacial acetic acid (30 µl) yongezwa kwaye umxube wathululelwa kwi-sodium cyanoborohydride (6.4 mg/100 µmol) waza wanyibilika ngokupheleleyo emva kokufudumeza kwi-65° C. umzuzu o-1. Emva koko, ukusuka kwi-5 ukuya kwi-8 μl yomxube wempendulo kongezwa kwi-oligosaccharide eyomileyo (1-100 nmol) ukuze kufunyanwe i-molar excess ephindwe kalishumi okanye ngaphezulu kweleyibhile ngaphezulu kwesiphelo sokunciphisa. I-reaction yenziwa kwi-65°C iiyure ezi-2, emva koko iisampulu zahlanjwa ngoko nangoko. Akukho sodium cyanoborohydride esetyenzisiweyo kwizilingo zokulebhelisha ngaphandle kokunciphisa, kwaye iisampulu zaphendulwa kwi-65°C. kangangeeyure eziyi-2.5.
Ukugqunywa kwe-ELISA kunye nokuhlanjwa kweesampuli ze-oligosaccharides ezifakwe kwi-biotinylated. Ii-25 μl zeesampuli ezifakwe kwi-biotinylated (100 μl yesampuli nganye exutywe kwi-5 ml yesisombululo se-0.1 M Tris buffer (TBS)) zongezwa kwiqula ngalinye lepleyiti efakwe kwi-avidin. Ii-control wells zigqunywe nge-50 μl ye-biotin kuxinzelelo lwe-10 μg/ml kwi-0.1 M TBS. Amanzi axutyiweyo asetyenziswe njenge-coating yokulinganisa okungenanto. Ithebhulethi yafakwa kwi-incubator iiyure ezi-2 kubushushu begumbi ebumnyameni. Hlamba ipleyiti izihlandlo ezi-3 ngobisi oluncitshisiweyo lwe-0.1% kwi-0.1 M TBS usebenzisa inkqubo inombolo 11 yeGrenier flat 3A.
Ukongeza nokuhlanjwa kwee-antibodies eziphambili. Yongeza i-40 µl ye-antibody ephambili emthonjeni ngamnye. Faka i-microplate iyure e-1 kubushushu begumbi ebumnyameni. Emva koko iiplate zahlanjwa kathathu ngobisi lwe-0.1% kwi-0.1M TBS usebenzisa inkqubo yokuhlamba #11 yeGrenier Flat 3A.
Yongeza i-antibody yesibini uze uhlambe. Yongeza i-50 µl ye-antibody yesibini yegundane/yempuku (exutywe ne-1:5000 kubisi lwe-0.1% kwi-0.1 M TBS) kwiqula ngalinye. Faka i-microplate iyure e-1 kubushushu begumbi ebumnyameni. Emva koko ii-microplates zahlanjwa izihlandlo ezi-5 ngobisi lwe-0.1% kwi-0.1 M TBS usebenzisa inkqubo yokuhlamba iipleyiti yeGrenier Flat 5A #12.
Ukongeza i-substrate. Yongeza i-50 µl ye-3,3′,5,5′-tetramethylbenzidine (TMB) kwi-substrate esisiseko (ngokongeza amathontsi ama-2 e-buffer, amathontsi ama-3 e-TMB, amathontsi ama-2 e-hydrogen peroxide kwi-15 ml yamanzi acocekileyo). Lungisa i-substrate ye-TMB. kunye ne-vortex ngaphambi kokusebenzisa). Faka i-microplate kubushushu begumbi imizuzu engama-30. Ebumnyameni.
Gqibezela inyathelo uze ufunde ithebhulethi. Yongeza i-50 µl ye-1 N sulfuric acid kwiqula ngalinye uze ubhale phantsi ukufunxwa kwayo ukusuka kwi-450 ukuya kwi-655 nm usebenzisa isixhobo sokufunda i-ELISA.
Lungisa izisombululo ze-1 mg/ml zezi analytes emanzini acocekileyo: i-arabinose, i-rhamnose, i-fucose, i-xylose, i-galacturonic acid (GalA), i-glucuronic acid (GlcA), i-mannose, i-glucose, i-galactose, i-lactose, i-N-acetylmannosamine (manNAc), i-N-acetylglucosamine. (glcNAc), i-N-acetylgalactosamine (galNAc), i-inositol (umgangatho wangaphakathi). Imigangatho emibini yalungiswa ngokongeza izisombululo zeswekile ze-1 mg/mL eziboniswe kwiTheyibhile 1. Iisampulu ziyakhenkcezwa kwaye zifakwe i-lyophil kwi--80° C. de kube kususwe onke amanzi (ngesiqhelo malunga neeyure ezili-12-18).
Yongeza i-100–500 µg yesampulu kwiityhubhu zesikrufu kwibhalansi yohlalutyo. Bhala phantsi ubungakanani obongeziweyo. Kungcono ukuyinyibilikisa isampuli kwingxinano ethile yesinyibilikisi uze uyifake kwityhubhu njenge-aliquot yolwelo. Sebenzisa i-20 µl ye-1 mg/ml ye-inositol njengomgangatho wangaphakathi kwityhubhu nganye yesampulu. Ubungakanani bomgangatho wangaphakathi obongeziweyo kwisampuli kufuneka bufane nobungakanani bomgangatho wangaphakathi obongeziweyo kwityhubhu eqhelekileyo.
Yongeza i-8 ml ye-methanol engenamanzi kwi-screw cap vial. Emva koko faka i-4 ml yesisombululo se-3 N. methanolic HCl, usivale uze usishukumise. Le nkqubo ayisebenzisi manzi.
Yongeza i-500 µl yesisombululo se-1 M HCl methanol kwiisampuli ze-oligosaccharide kunye neetyhubhu ze-TMS eziqhelekileyo. Iisampuli zafakwa e-incubation ubusuku bonke (iiyure ezili-168) kwi-80° C. kwi-thermal block. Yomisa imveliso ye-methanolysis kubushushu begumbi usebenzisa i-manifold yokomisa. Yongeza i-200 µl MeOH kwaye yomise kwakhona. Le nkqubo iphindaphindwa kabini. Yongeza i-200 µl ye-methanol, i-100 µl ye-pyridine kunye ne-100 µl ye-acetic anhydride kwisampuli kwaye udibanise kakuhle. Iisampuli zafakwa e-incubation kubushushu begumbi imizuzu engama-30. kwaye zomiswe. Yongeza i-200 µl ye-methanol kwaye yomise kwakhona.
Yongeza i-200 µl yeTri-Sil uze uyifudumeze ngetyhubhu egqunyiweyo imizuzu engama-20. 80°C, uze uyipholise ukuya kubushushu begumbi. Sebenzisa i-manifold yokomisa ukuze womise isampuli ukuya kutsho kwi-50 µl. Kubalulekile ukuqaphela ukuba asizange sivumele iisampulu ukuba zome ngokupheleleyo.
Yongeza i-2 ml ye-hexane uze udibanise kakuhle ngokuyivortex. Zalisa iincam zee-Pasteur pipettes (5-8 mm) ngesiqwenga soboya beglasi ngokufaka uboya beglasi phezu kwe-pipette enobubanzi obuyi-5-3/4 intshi. Iisampulu zifakwe kwi-centrifuge kwi-3000 g imizuzu emi-2. Naziphi na iintsalela ezinganyibilikiyo ziyavuthuluka. Yomisa isampuli ukuya kwi-100-150 µl. Umthamo omalunga ne-1 μl ufakwe kwi-GC-MS kubushushu bokuqala obuyi-80 °C kunye nexesha lokuqala lemizuzu eyi-2.0 (Itheyibhile 2).