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Izindlela ezintsha ze-immunological kanye ne-mass spectrometric zokuhlaziya okuyinkimbinkimbi kwama-oligosaccharides aqhubekayo ku-corn stover aphathwe nge-AFEX. I-Lignocellulosic biomass iyindlela ehlukile eqhubekayo esikhundleni samafutha asetshenziswa kudala futhi isetshenziswa kabanzi ukuthuthukisa ubuchwepheshe be-biotechnology bokukhiqiza imikhiqizo efana nokudla, ukudla, amafutha kanye namakhemikhali. Isihluthulelo salobu buchwepheshe ukuthuthukiswa kwezinqubo ezincintisanayo zokuguqula ama-carbohydrate ayinkimbinkimbi akhona ezindongeni zamaseli ezitshalo abe ushukela olula njenge-glucose, i-xylose kanye ne-arabinose. Ngenxa yokuthi i-lignocellulosic biomass ilukhuni kakhulu, kumele iphathwe nge-thermochemical (isb., i-ammonia fiber exfoliation (AFEX), i-dilute acids (DA), i-ionic liquids (IL)) kanye nokwelashwa kwezinto eziphilayo (isb., i-enzymatic hydrolysis kanye ne-microbial fermentation) ngokuhlanganiswa ukuze kutholakale umkhiqizo owufunayo. Kodwa-ke, uma kusetshenziswa ama-enzyme esikhunta esithengiswayo enqubweni ye-hydrolysis, ama-75-85% kuphela kashukela oncibilikayo owakhiwe angama-monosaccharides, kanti ama-15-25% asele angama-oligosaccharides ancibilikayo, angalawuleki, angatholakali njalo kuma-microorganisms. Ngaphambilini, siwahlukanise ngempumelelo futhi sawahlanza ama-oligosaccharides ancibilikayo ancibilikayo sisebenzisa inhlanganisela ye-carbon kanye ne-diatomaceous earth separation kanye ne-size exclusion chromatography, futhi sahlola nezakhiwo zawo zokuvimbela ama-enzyme. Sithole ukuthi ama-oligosaccharides aqukethe izinga eliphezulu le-polymerization (DP) methylated uronic acid substitutions kunzima kakhulu ukuwacubungula ngama-enzyme blends e-commercial kune-DP ephansi kanye nama-oligosaccharides angathathi hlangothi. Lapha sibika ukusetshenziswa kwezindlela eziningana ezengeziwe, okuhlanganisa i-glycan profileing esebenzisa ama-antibodies e-monoclonal (mAbs) akhethekile kuma-glycans e-biomass ezitshalo ukuze achaze izibopho ze-glycan ezindongeni zamaseli ezitshalo kanye nama-hydrolysates e-enzymatic, i-matrix-assisted laser desorption ionization, i-time-of-flight mass-spectrometry. I-MALDI-TOF-MS) isebenzisa iziqongo zokuxilonga ezinolwazi ngesakhiwo ezitholwe yi-spectroscopy ngemuva kokubola kwesibili kwama-ion angalungile, i-gas chromatography kanye ne-mass spectrometry (GC-MS) ukuze kuchazwe izibopho ze-oligosaccharide ezine-derivatization kanye nezingenayo. Ngenxa yobukhulu obuncane bama-oligosaccharides (DP 4-20), lawa ma-molecule kunzima ukuwasebenzisa ekubopheni nasekuchazeni i-mAb. Ukuze sinqobe le nkinga, sisebenzise indlela entsha yokuvimbela i-oligosaccharide esekelwe ku-biotin conjugation eyabhala ngempumelelo iningi lama-oligosaccharides ancibilikayo e-DP aphansi ebusweni be-microplate, eyabe isisetshenziswa ohlelweni lwe-mAb olusebenzisa i-high throughput ukuze kuhlaziywe i-ligation ethile. Le ndlela entsha izosiza ekuthuthukisweni kwe-high throughput glycome assays esikhathi esizayo engasetshenziswa ukuhlukanisa nokuhlukanisa ama-oligosaccharides akhona kuma-biomarker ngezinjongo zokuxilonga.
I-Lignocellulosic biomass, eyakhiwe ngezinto zezolimo, amahlathi, utshani kanye nezinto zokhuni, iyisitsha sokudla esingaba khona sokukhiqizwa kwemikhiqizo esekelwe ku-bio, okuhlanganisa ukudla, ukudla, uphethiloli kanye nezinto ezibangela amakhemikhali ukukhiqiza imikhiqizo enenani eliphezulu1. Ama-carbohydrate (njenge-cellulose ne-hemicellulose) akhona ezindongeni zamaseli ezitshalo asuswa i-polymer abe ama-monosaccharides ngokucubungula amakhemikhali kanye nokuguqulwa kwe-biotransformation (njenge-enzymatic hydrolysis kanye nokuvutshelwa kwamagciwane). Ukwelashwa okuvamile kwangaphambi kokwelashwa kufaka phakathi ukwanda kwe-ammonia fiber (AFEX), i-dilute acid (DA), i-ionic liquid (IL), kanye nokuqhuma komusi (SE), okusebenzisa inhlanganisela yamakhemikhali nokushisa ukunciphisa ukukhiqizwa kwe-lignocellulose ngokuvula izindonga zamaseli ezitshalo3,4. ubulukhuni bezinto, 5. I-Enzymatic hydrolysis yenziwa ngomthwalo ophezulu wezinto eziqinile kusetshenziswa ama-enzyme asebenzayo aqukethe ama-carbohydrate (ama-CAZymes) kanye nokuvutshelwa kwamagciwane kusetshenziswa imvubelo ye-transgenic noma amabhaktheriya ukukhiqiza uphethiloli namakhemikhali asekelwe ku-bio6.
Ama-CAZyme kuma-enzyme ezentengiselwano akhiwa ingxube eyinkimbinkimbi yama-enzyme ahlukanisa ngokubambisana izibopho eziyinkimbinkimbi ze-carbohydrate noshukela ukuze akhe ama-monosaccharides2,7. Njengoba sibike ngaphambilini, inethiwekhi eyinkimbinkimbi yama-polymer aromatic e-lignin nama-carbohydrate iwenza angakwazi ukuxazululeka, okuholela ekuguqulweni koshukela okungaphelele, okuqoqa ama-oligosaccharides angama-15-25% angakhiqizwa ngesikhathi se-hydrolysis ye-enzyme ye-biomass ephathwe kusengaphambili. Lena inkinga evamile ngezindlela ezahlukene zokwelashwa kwe-biomass. Ezinye zezizathu zalokhu kuvimbela zihlanganisa ukuvinjelwa kwama-enzyme ngesikhathi se-hydrolysis, noma ukungabikho noma amazinga aphansi ama-enzyme abalulekile adingekayo ukuphula izibopho zikashukela ku-biomass yezitshalo. Ukuqonda ukwakheka kanye nezici zesakhiwo zikashukela, njengezibopho zikashukela kuma-oligosaccharides, kuzosisiza sithuthukise ukuguqulwa kukashukela ngesikhathi se-hydrolysis, okwenza izinqubo ze-biotechnological zincintisane nemikhiqizo etholakala ku-petroleum.
Ukunquma isakhiwo sama-carbohydrate kuyinselele futhi kudinga inhlanganisela yezindlela ezifana ne-liquid chromatography (LC)11,12, i-nuclear magnetic resonance spectroscopy (NMR)13, i-capillary electrophoresis (CE)14,15,16 kanye ne-mass spectrometry (MS)17. ,ishumi nesishiyagalombili. Izindlela ze-MS ezifana ne-time-of-flight mass spectrometry ene-laser desorption kanye ne-ionization kusetshenziswa i-matrix (MALDI-TOF-MS) ziyindlela eguquguqukayo yokuhlonza izakhiwo zama-carbohydrate. Muva nje, i-Collision-Induced Dissociation (CID) tandem MS ye-sodium ion adducts isetshenziswe kakhulu ukuhlonza izigxivizo zeminwe ezihambisana nezikhundla zokunamathisela ze-oligosaccharide, ukumiswa kwe-anomeric, ukulandelana, kanye nezikhundla ze-branching 20, 21.
Ukuhlaziywa kwe-Glycan kuyithuluzi elihle kakhulu lokuhlonza ngokujulile izibopho ze-carbohydrate22. Le ndlela isebenzisa ama-antibodies e-monoclonal (ama-mAb) aqondiswe ku-glycan yodonga lweseli lesitshalo njenge-probes ukuqonda ukuxhumana okuyinkimbinkimbi kwe-carbohydrate. Kutholakala ama-mAb angaphezu kuka-250 emhlabeni wonke, aklanyelwe ngokumelene nama-oligosaccharide ahlukahlukene aqondile nangamagatsha kusetshenziswa ama-saccharide ahlukahlukene24. Ama-mAb amaningana asetshenziswe kabanzi ukuchaza isakhiwo, ukwakheka, kanye nokuguqulwa kodonga lweseli lesitshalo, njengoba kunomehluko omkhulu kuye ngohlobo lweseli lesitshalo, isitho, ubudala, isigaba sokukhula, kanye nendawo yokukhula25,26. Muva nje, le ndlela isetshenziswe ukuqonda inani lama-vesicle ezinhlelweni zezitshalo nezilwane kanye nezindima zazo ekuthuthweni kwe-glycan njengoba kunqunywa yizimpawu ezingaphansi kweseli, izigaba zokukhula, noma izinto ezikhuthaza indawo, kanye nokunquma umsebenzi we-enzyme. Ezinye zezakhiwo ezahlukene zama-glycans nama-xylan eziye zatholakala zifaka phakathi i-pectin (P), i-xylan (X), i-mannan (M), i-xyloglucans (XylG), ama-glucans ahlanganisiwe (MLG), i-arabinoxylan (ArbX), i-galactomannan (GalG), i-glucuronic acid-arabinoxylan (GArbX) kanye ne-arabino-galactan (ArbG)29.
Kodwa-ke, naphezu kwayo yonke le mizamo yocwaningo, izifundo ezimbalwa kuphela ezigxile kuhlobo lokuqongelela kwe-oligosaccharide ngesikhathi se-hydrolysis ephezulu ye-solids load (HSL), okuhlanganisa ukukhululwa kwe-oligosaccharide, izinguquko zobude be-oligomeric chain ngesikhathi se-hydrolysis, ama-polymer ahlukahlukene aphansi e-DP, kanye nama-curve awo. Okwamanje, yize ukuhlaziywa kwe-glycan kuye kwabonakala kuyithuluzi eliwusizo lokuhlaziya okuphelele kwesakhiwo se-glycan, kunzima ukuhlola ama-oligosaccharide aphansi e-DP ancibilikayo emanzini kusetshenziswa izindlela ze-antibody. Ama-oligosaccharide amancane e-DP anesisindo sama-molecule esingaphansi kuka-5-10 kDa awabophi kuma-plate e-ELISA 33, 34 futhi agezwa ngaphambi kokufakwa kwe-antibody.
Lapha, okokuqala ngqa, sibonisa ukuhlolwa kwe-ELISA kumapuleti ambozwe yi-avidin sisebenzisa ama-antibodies e-monoclonal, sihlanganisa inqubo yesinyathelo esisodwa ye-biotinylation yama-oligosaccharides ancibilikayo nokuhlaziywa kwe-glycome. Indlela yethu yokuhlaziywa kwe-glycome yaqinisekiswa ukuhlaziywa okusekelwe ku-MALDI-TOF-MS kanye ne-GC-MS kokuxhumana kwe-oligosaccharide okuhambisanayo kusetshenziswa i-trimethylsilyl (TMS) derivatization yezinhlanganisela zikashukela ezifakwe i-hydrolyzed. Le ndlela entsha ingathuthukiswa njengendlela ephezulu kakhulu esikhathini esizayo futhi ithole ukusetshenziswa okubanzi ocwaningweni lwezokwelapha35.
Ukuguqulwa kwama-enzyme nama-antibodies ngemuva kokuhumusha, njenge-glycosylation,36 kuthinta umsebenzi wawo webhayoloji. Isibonelo, izinguquko ekufakweni kwe-glycosylation kwamaprotheni e-serum zidlala indima ebalulekile ku-arthritis yokuvuvukala, futhi izinguquko ekufakweni kwe-glycosylation zisetshenziswa njengezimpawu zokuxilonga37. Ama-glycan ahlukahlukene abikwe ezincwadini ukuthi avela kalula ezifweni ezahlukahlukene, okuhlanganisa izifo ezingapheli zokuvuvukala zendlela yokugaya ukudla nesibindi, izifo ezibangelwa amagciwane, umdlavuza wesibeletho, webele, kanye nowe-prostate38,39,40. Ukuqonda isakhiwo sama-glycan kusetshenziswa izindlela ze-glycan ELISA ezisekelwe kuma-antibody kuzonikeza ukuzethemba okwengeziwe ekuxilongweni kwesifo ngaphandle kokusebenzisa izindlela eziyinkimbinkimbi ze-MS.
Ucwaningo lwethu lwangaphambilini lukhombisile ukuthi ama-oligosaccharide aqinile ahlala engangenisi amanzi ngemva kokwelashwa kwangaphambili kanye ne-enzyme hydrolysis (Isithombe 1). Emsebenzini wethu oshicilelwe ngaphambilini, sakha indlela yokukhipha i-charcoal solid-phase esebenze ukuze sihlukanise ama-oligosaccharide ku-AFEX-pretreated corn stover hydrolyzate (ACSH)8. Ngemva kokukhishwa kokuqala nokuhlukaniswa, ama-oligosaccharide ahlukaniswa kakhulu nge-size exclusion chromatography (SEC) futhi aqoqwa ngokulandelana kwesisindo sama-molecule. Ama-monomer kashukela nama-oligomer akhishwe ekuphathweni kwangaphambilini okuhlukahlukene ahlaziywe ngokuhlaziywa kokwakheka kukashukela. Uma kuqhathaniswa okuqukethwe kwama-oligosaccharide kashukela atholwe ngezindlela ezahlukene zokwelashwa kwangaphambilini, ukuba khona kwama-oligosaccharide aqinile kuyinkinga evamile ekuguqulweni kwe-biomass kube ama-monosaccharide futhi kungaholela ekunciphiseni isivuno sikashukela okungenani esingu-10-15% ngisho nakufika ku-18%. US. Le ndlela isetshenziselwa ukukhiqizwa okukhulu kwezingxenyana zama-oligosaccharide. I-ACH eyalandela kanye nezingxenyana zayo ezalandela ezinesisindo sama-molecule ahlukene zasetshenziswa njengezinto zokuhlola zokuchaza ama-oligosaccharides kulo msebenzi.
Ngemva kokwelashwa ngaphambi kokwelashwa kanye ne-enzyme hydrolysis, ama-oligosaccharide ahlala engafakwanga amanzi. Lapha (A) indlela yokuhlukanisa ama-oligosaccharide lapho ama-oligosaccharide ehlukaniswa khona ne-AFEX-pretreated corn stover hydrolysate (ACSH) kusetshenziswa umbhede ogcwele we-activated carbon kanye ne-diatomaceous earth; (B) Indlela yokuhlukanisa ama-oligosaccharide. Ama-oligosaccharide ahlukaniswa kabanzi nge-size exclusion chromatography (SEC); (C) Ama-Saccharide monomers kanye nama-oligomers akhishwe ekwelashweni okuhlukahlukene kwangaphambili (i-diluted acid: DA, i-ionic liquid: IL kanye ne-AFEX). Izimo ze-enzyme hydrolysis: ukulayisha okuphezulu kwe-solid okungu-25% (w/w) (cishe ukulayisha kwe-glucan okungu-8%), i-hydrolysis yamahora angu-96, ukulayisha kwe-enzyme yokuhweba okungu-20 mg/g (isilinganiso se-Ctec2:Htec2:MP-2:1:1) kanye (D) Ama-monomers kashukela nama-oligomers e-glucose, i-xylose kanye ne-arabinose akhishwe ku-AFEX pre-treated corn stover (ACS).
Ukuhlaziywa kwe-Glycan kuye kwabonakala kuyithuluzi eliwusizo lokuhlaziywa kwesakhiwo okuphelele kwama-glycans ezikhishwe ezinsaleleni ze-biomass eziqinile. Kodwa-ke, ama-saccharide ancibilikayo emanzini awavezwa kahle kusetshenziswa le ndlela yendabuko41 ngoba ama-oligosaccharide anesisindo esiphansi sama-molecule kunzima ukuwavimba kuma-plate e-ELISA futhi ahlanzwa ngaphambi kokufakwa kwama-antibody. Ngakho-ke, ukuze kuhlanganiswe futhi kuchazwe ama-antibody, kwasetshenziswa indlela yesinyathelo esisodwa ye-biotinylation ukumboza ama-oligosaccharide ancibilikayo, angahambisani nama-plate e-ELISA ambozwe yi-avidin. Le ndlela yahlolwa kusetshenziswa i-ACSH yethu ekhiqizwe ngaphambilini kanye nengxenye esekelwe esisindweni sayo sama-molecule (noma izinga le-polymerization, i-DP). I-biotinylation yesinyathelo esisodwa yasetshenziswa ukwandisa i-oligosaccharide binding affinity ngokungeza i-biotin-LC-hydrazide ekugcineni kokunciphisa kwe-carbohydrate (Isithombe 2). Esixazululweni, iqembu le-hemiacetal ekugcineni kokunciphisa lisabela neqembu le-hydrazide le-biotin-LC-hydrazide ukuze lakhe isibopho se-hydrazone. Uma kukhona i-NaCNBH3 enciphisayo, isibopho se-hydrazone sincishiswa sibe umkhiqizo wokugcina ozinzile we-biotinylated. Ngokuguqulwa kokuphela kokunciphisa ushukela, ukubopha ama-oligosaccharides aphansi e-DP kuma-plate e-ELISA kwaba nokwenzeka, futhi ocwaningweni lwethu lokhu kwenziwa kuma-plate ambozwe yi-avidin kusetshenziswa ama-mAb aqondiswe yi-glycan.
Ukuhlolwa kwama-antibodies e-monoclonal okusekelwe ku-ELISA ukuthola ama-oligosaccharide aqukethe i-biotinylated. Lapha (A) ukuhlanganiswa kwe-biotinylation yama-oligosaccharides kanye nokuhlolwa kwe-ELISA okulandelayo ngama-mAbs aqondiswe ku-glycan kuma-NeutrAvidin coated plates kanye (B) kukhombisa inqubo yesinyathelo esisodwa ye-biotinylation yemikhiqizo yokusabela.
Amapuleti ambozwe nge-Avidin anama-antibodies ahlanganiswe ne-oligosaccharide abe esengezwa kuma-antibodies ayinhloko nawesibili bese egezwa endaweni ekhanyayo nezwela isikhathi. Ngemva kokuthi ukubopha ama-antibody sekuqediwe, engeza i-substrate ye-TMB ukuze ufukamele ipuleti. Ukusabela kwagcina kumiswe nge-sulfuric acid. Amapuleti afakwe ahlaziywa kusetshenziswa i-ELISA reader ukuze kutholakale amandla okubopha e-antibody ngayinye ukuze kutholakale ukuxhumanisa okuqondile kwama-antibody. Ukuze uthole imininingwane kanye nemingcele yokuhlolwa, bheka isigaba esihambisanayo esithi “Izinto Nezindlela”.
Sibonisa ukusetshenziswa kwale ndlela esanda kuthuthukiswa kwezicelo ezithile ngokuchaza ama-oligosaccharide ancibilikayo akhona ku-ACSH kanye nasezingxenyaneni ze-oligosaccharide ezingavuthiwe nezihlanziwe ezihlukaniswe kuma-hydrolysates e-lignocellulosic. Njengoba kuboniswe kuMfanekiso 3, ama-xylan avame kakhulu afakwe esikhundleni se-epitope atholakala ku-ACSH kusetshenziswa izindlela zokuhlola i-glycome ye-bioacylated ngokuvamile yi-uronic (U) noma i-methyluronic (MeU) kanye ne-pectic arabinogalactans. Iningi lawo litholakale ocwaningweni lwethu lwangaphambilini mayelana nokuhlaziywa kwama-glycans ama-solid angewona ama-hydrolyzed (UHS)43.
Ukutholwa kwama-recalcitrant oligosaccharide epitopes kusetshenziswa i-antibody ye-monoclonal eqondiswe ku-glycan yodonga lweseli. Ingxenye "engathathi hlangothi" yingxenyana ye-ACN kanti ingxenye "ene-acidic" yingxenyana ye-FA. Okubomvu okukhanyayo kumephu yokushisa kubonisa okuqukethwe okuphezulu kwe-epitope, kanti okuluhlaza okwesibhakabhaka okukhanyayo kubonisa ingemuva elingenalutho. Amanani ombala esikalini asekelwe kumanani e-OD aluhlaza emafomula N=2. Ama-epitopes amakhulu aqashelwa ama-antibodies aboniswe ngakwesokudla.
Lezi zakhiwo ezingezona i-cellulose azikwazanga ukuhlukaniswa yi-cellulases kanye ne-hemicellulases ezivame kakhulu engxubeni ye-enzyme yezohwebo evivinyiwe, ehlanganisa ama-enzyme ezohwebo asetshenziswa kakhulu. Ngakho-ke, ama-enzyme amasha asizayo ayadingeka ukuze kufakwe i-hydrolysis yawo. Ngaphandle kwama-enzyme adingekayo angewona i-cellulose, lawa ma-non-cellulose bonds avimbela ukuguqulwa okuphelele kube yi-monosaccharides, noma ngabe ama-polymer awo ashukela angumzali afakwa i-hydrolyzed kakhulu abe yizicucu ezimfushane futhi ancibilikiswe kusetshenziswa izingxube ze-enzyme yezohwebo.
Ucwaningo olwengeziwe lokusatshalaliswa kwesignali kanye namandla ayo okubopha lubonise ukuthi ama-epitope okubopha ayephansi kuma-fraction kashukela aphezulu e-DP (A, B, C, DP afinyelela ku-20+) kunakuma-fraction aphansi e-DP (D, E, F, DP) kuma-dimers) (Isithombe 1). Izingcezu ze-asidi zivame kakhulu kuma-epitope angewona ama-cellulose kunezingcezu ezingathathi hlangothi. Lezi zimo zihambisana nephethini ebonwe ocwaningweni lwethu lwangaphambilini, lapho ama-DP aphezulu kanye nama-acid moieties ayemelana kakhulu ne-enzyme hydrolysis. Ngakho-ke, ukuba khona kwama-non-cellulose glycan epitopes kanye nokufakwa esikhundleni kwe-U kanye ne-MeU kungasiza kakhulu ekuzinzeni kwama-oligosaccharides. Kufanele kuqashelwe ukuthi ukusebenza kahle kokubopha kanye nokuthola kungaba yinkinga kuma-oligosaccharides aphansi e-DP, ikakhulukazi uma i-epitope iyi-oligosaccharide ye-dimeric noma ye-trimeric. Lokhu kungahlolwa kusetshenziswa ama-oligosaccharides ezentengiselwano ezinobude obuhlukene, ngalinye liqukethe i-epitope eyodwa kuphela ebopha ku-mAb ethile.
Ngakho-ke, ukusetshenziswa kwama-antibodies athile ngokwesakhiwo kwembule izinhlobo ezithile zama-recalcitrant bonds. Kuye ngohlobo lwe-antibody esetshenzisiwe, iphethini efanele yokubopha, kanye namandla esignali ekhiqizayo (eningi kakhulu neyincane kakhulu), ama-enzyme amasha angabonakala futhi engezwe ngokulingana engxubeni ye-enzyme ukuze kube nokuguqulwa okuphelele kwe-glycoconversion. Uma sithatha ukuhlaziywa kwama-oligosaccharides e-ACSH njengesibonelo, singakha isizindalwazi sama-glycan bonds ezinto ngazinye ze-biomass. Kufanele kuqashelwe lapha ukuthi ukuhambisana okuhlukile kwama-antibodies kufanele kucatshangelwe, futhi uma ukuhambisana kwawo kungaziwa, lokhu kuzodala ubunzima obuthile lapho kuqhathaniswa izimpawu zama-antibodies ahlukene. Ngaphezu kwalokho, ukuqhathaniswa kwama-glycan bonds kungasebenza kangcono phakathi kwamasampula e-antibody efanayo. Lezi zibopho eziqinile zingaxhunyaniswa nesizindalwazi se-CAZyme, lapho singathola khona ama-enzyme, sikhethe ama-enzyme afanele futhi sihlole ama-enzyme aphula izibopho, noma sithuthukise izinhlelo zamagciwane ukuveza la ma-enzyme ukuze asetshenziswe kuma-biorefineries44.
Ukuze sihlole ukuthi izindlela zokwelapha umzimba zihambisana kanjani nezinye izindlela zokuhlukanisa ama-oligosaccharide anesisindo esiphansi sama-molecule akhona kuma-hydrolysates e-lignocellulosic, senze i-MALDI (Isithombe 4, S1-S8) kanye nokuhlaziywa kwama-saccharide atholakala ku-TMS asekelwe ku-GC-MS kuphaneli efanayo (Isithombe 5) ingxenye ye-oligosaccharide. I-MALDI isetshenziselwa ukuqhathanisa ukuthi ukusatshalaliswa kwesisindo sama-molecule e-oligosaccharide kufana yini nesakhiwo esihlosiwe. Ku-Fig. 4 kukhombisa i-MC yezingxenye ezingathathi hlangothi ze-ACN-A kanye ne-ACN-B. Ukuhlaziywa kwe-ACN-A kuqinisekisile uhla lukashukela we-pentose olusukela ku-DP 4–8 (Isithombe 4) kuya ku-DP 22 (Isithombe S1), osisindo sawo sihambisana nama-oligosaccharide e-MeU-xylan. Ukuhlaziywa kwe-ACN-B kuqinisekisile uchungechunge lwe-pentose kanye ne-glucoxylan nge-DP 8-15. Ezintweni ezengeziwe ezifana neSithombe S3, amamephu okusatshalaliswa kwesisindo se-FA-C acidic moiety abonisa uhla lweshukela le-pentose elifakwe esikhundleni se-(Me)U eline-DP engu-8-15 elihambisana nama-xylan afakwe esikhundleni atholakala ekuhlolweni kwe-mAb okusekelwe ku-ELISA. Ama-epitopes ayahambisana.
I-MALDI-MS spectrum yama-oligosaccharide angahlangani ancibilikayo akhona ku-ACS. Lapha, (A) Izingxenyana ze-ACN-A ezisezingeni eliphansi eziqukethe i-methylated uronic acid (DP 4-8) zithathe indawo yama-oligosaccharide e-glucuroxylan kanye (B) ama-oligosaccharide e-ACN-B xylan kanye ne-methylated uronic acid oligosaccharides athathe indawo yama-glucuroxylan (DP 8-15).
Ukuhlaziywa kokwakheka kwe-glycan residue yama-oligosaccharide angenasici. Lapha (A) Ukwakheka kwe-TMS saccharide kwezingxenyana ezahlukene ze-oligosaccharide ezitholwe kusetshenziswa ukuhlaziywa kwe-GC-MS. (B) Izakhiwo zoshukela ezahlukene ezithathwe yi-TMS ezikhona kuma-oligosaccharide. I-ACN – ingxenye ye-acetonitrile equkethe ama-oligosaccharide angathathi hlangothi kanye nengxenye ye-FA – ingxenye ye-ferulic acid equkethe ama-oligosaccharide e-acid.
Esinye isiphetho esithakazelisayo sithathwe ekuhlaziyweni kwe-LC-MS kwengxenyana ye-oligosaccharide, njengoba kuboniswe kuMfanekiso S9 (izindlela zingabonakala ezintweni ezengeziwe ze-elekthronikhi). Izingcezu zamaqembu e-hexose kanye ne--OAc zabonwa ngokuphindaphindiwe ngesikhathi sokuhlanganiswa kwengxenyana ye-ACN-B. Lokhu okutholakele akuqinisekisi nje kuphela ukuqhekeka okubonwe ekuhlaziyweni kwe-glycome kanye ne-MALDI-TOF, kodwa futhi kunikeza ulwazi olusha mayelana nemikhiqizo engaba khona ye-carbohydrate ku-lignocellulosic biomass ephathwe ngaphambilini.
Siphinde sahlaziya ukwakheka kukashukela kwengxenye ye-oligosaccharide sisebenzisa i-TMS sugar derivatization. Sisebenzisa i-GC-MS, sithole ukwakheka kukashukela we-neural (ongasuswanga) kanye noshukela we-acid (i-GluA kanye ne-GalA) engxenyeni ye-oligosaccharide (Isithombe 5). I-Glucuronic acid itholakala ezingxenyeni ze-acidic C kanye no-D, kuyilapho i-galacturonic acid itholakala ezingxenyeni ze-acidic A kanye no-B, zombili eziyizingxenye ze-DP eziphezulu zoshukela we-acidic. Le miphumela ayiqinisekisi nje kuphela idatha yethu ye-ELISA kanye ne-MALDI, kodwa futhi iyahambisana nezifundo zethu zangaphambilini zokuqongelela kwe-oligosaccharide. Ngakho-ke, sikholelwa ukuthi izindlela zesimanje ze-immunological ezisebenzisa i-biotinylation yama-oligosaccharide kanye nokuhlolwa okulandelayo kwe-ELISA zanele ukuthola ama-oligosaccharide ancibilikayo ancibilikayo kumasampula ahlukahlukene e-biological.
Njengoba izindlela zokuhlola i-mAb ezisekelwe ku-ELISA ziqinisekiswe ngezindlela eziningana ezahlukene, besifuna ukuhlola kabanzi amandla ale ndlela entsha yokulinganisa. Ama-oligosaccharide amabili ezentengiselwano, i-xylohexasaccharide oligosaccharide (XHE) kanye ne-23-α-L-arabinofuranosyl-xylotriose (A2XX), athengwe futhi ahlolwa kusetshenziswa indlela entsha ye-mAb eqondise i-glycan yodonga lweseli. Isithombe 6 sibonisa ukuhlangana okuqondile phakathi kwesignali yokubopha ye-biotinylated kanye nokuhlushwa kwelogi kokuhlushwa kwe-oligosaccharide, okuphakamisa imodeli yokumunca okungenzeka ye-Langmuir. Phakathi kwama-mAb, i-CCRC-M137, i-CCRC-M138, i-CCRC-M147, i-CCRC-M148, kanye ne-CCRC-M151 kuhlobene ne-XHE, kanye ne-CCRC-M108, i-CCRC-M109, kanye ne-LM11 kuhlobene ne-A2XX ebangeni eliphakathi kwe-1 nm kuya ku-100 nano. Ngenxa yokutholakala okulinganiselwe kwama-antibodies ngesikhathi sokuhlolwa, kwenziwa ukuhlolwa okulinganiselwe nge-oligosaccharide ngayinye eqoqweni. Kufanele kuqashelwe lapha ukuthi amanye ama-antibodies asabela ngendlela ehlukile kakhulu kuma-oligosaccharide afanayo njenge-substrate, mhlawumbe ngoba anamathela kuma-epitopes ahlukene kancane futhi angaba nokuhlobana okuhlukile kakhulu kokubopha. Izindlela nemiphumela yokuhlonza i-epitope enembile kuzoba nzima kakhulu uma indlela entsha ye-mAb isetshenziswa kumasampula angempela.
Kusetshenziswe ama-oligosaccharide amabili ezentengiselwano ukunquma ububanzi bokuthola ama-mAb ahlukahlukene aqondisa i-glycan. Lapha, ukuhlangana okuqondile nokuhlushwa kwelogi kokuhlushwa kwe-oligosaccharide kubonisa amaphethini okumunca i-Langmuir e-(A) XHE ene-mAb kanye ne-(B) A2XX ene-mAb. Ama-epitopes ahambisanayo akhombisa izakhiwo zama-oligosaccharide ezentengiselwano asetshenziswa njengezinto ezisetshenziswayo ekuhlolweni.
Ukusetshenziswa kwama-antibodies e-monoclonal aqondiswe ku-glycan (ukuhlaziywa kwe-glycocomic noma ukuhlolwa kwe-mAb okusekelwe ku-ELISA) kuyithuluzi elinamandla lokuchaza ngokujulile iningi lama-glycans amakhulu odongeni lwamaseli akha i-biomass yezitshalo. Kodwa-ke, ukuhlaziywa kwe-glycan yakudala kuveza kuphela ama-glycans amakhulu odongeni lwamaseli, njengoba ama-oligosaccharide amaningi engavimbeki kahle kumapuleti e-ELISA. Kulolu cwaningo, i-AFEX-pretreated corn stover yafakwa i-enzymeally hydrolyzed ngokuqukethwe okuphezulu kwe-solids. Ukuhlaziywa kukashukela kwasetshenziswa ukunquma ukwakheka kwama-carbohydrate odongeni lwamaseli aphindekayo ku-hydrolyzate. Kodwa-ke, ukuhlaziywa kwe-mAb kwama-oligosaccharide amancane kuma-hydrolysates akunakwa, futhi kudingeka amathuluzi engeziwe ukuze kuvimbeki kahle ama-oligosaccharide kumapuleti e-ELISA.
Lapha sibika indlela entsha nephumelelayo yokuvimbela i-oligosaccharide yokuhlolwa kwe-mAb ngokuhlanganisa i-oligosaccharide biotinylation elandelwa ukuhlolwa kwe-ELISA kuma-NeutrAvidin™ coated plates. Ama-oligosaccharide angenakunyakaziswa abonise ukuhambisana okwanele kwe-antibody ukuze kuvunyelwe ukutholakala okusheshayo nokuphumelelayo kwama-oligosaccharide aphindaphindayo. Ukuhlaziywa kokwakheka kwala ma-oligosaccharide aqinile okusekelwe ku-mass spectrometry kuqinisekisile imiphumela yale ndlela entsha yokuhlolwa kwe-immuno. Ngakho-ke, lezi zifundo zibonisa ukuthi inhlanganisela ye-oligosaccharide biotinylation kanye nokuhlolwa kwe-ELISA nama-antibodies e-monoclonal aqondiswe yi-glycan ingasetshenziswa ukuthola izixhumanisi ezixubile kuma-oligosaccharides futhi ingasetshenziswa kabanzi kwezinye izifundo ze-biochemical ezichaza isakhiwo sama-oligosaccharides.
Le ndlela yokwenza iphrofayili ye-glycan esekelwe kwi-biotin ingumbiko wokuqala okwazi ukuphenya izibopho ze-carbohydrate eziphindaphindayo zama-oligosaccharide ancibilikayo ku-biomass yezitshalo. Lokhu kusiza ukuqonda ukuthi kungani ezinye izingxenye ze-biomass zilukhuni kangaka uma kukhulunywa ngokukhiqizwa kwe-biofuel. Le ndlela igcwalisa igebe elibalulekile ezindleleni zokuhlaziya i-glycome futhi yandisa ukusetshenziswa kwayo ebangeni elibanzi lezinto ezingaphansi kwe-oligosaccharide yezitshalo. Esikhathini esizayo, singasebenzisa amarobhothi ukuze sisebenzise i-biotinylation futhi sisebenzise indlela esiyisungulile yokuhlaziya okuphezulu kwamasampula sisebenzisa i-ELISA.
Utshani bommbila (CS) obukhuliswe ngembewu exubile yePioneer 33A14 buvunwe ngo-2010 eKramer Farms eRay, eColorado. Ngemvume yomnikazi womhlaba, le biomass ingasetshenziswa ocwaningweni. Amasampula agcinwe omile <6% umswakama ezikhwameni ezikhiywayo ezivaliwe ekushiseni kwegumbi. Amasampula agcinwe omile <6% umswakama ezikhwameni ezikhiywayo ezivaliwe ekushiseni kwegumbi. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Amasampula agcinwe omile ngaphansi kuka-6% womswakama ezikhwameni ezineziphu ekushiseni kwegumbi.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%. Amasampula agcinwa ezikhwameni ze-zipper ekamelweni elinomswakama ongaphansi kuka-6%.Ucwaningo luhambisane neziqondiso zendawo nezesizwe. Ukuhlaziywa kokwakheka kwenziwa kusetshenziswa iphrothokholi ye-NREL. Ukwakheka kutholakale ukuthi kuqukethe i-glucan engu-31.4%, i-xylan engu-18.7%, i-arabinan engu-3.3%, i-galactan engu-1.2%, i-acetyl engu-2.2%, i-lignin engu-14.3%, iphrotheni engu-1.7% kanye nomlotha ongu-13.4%.
I-Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) iyinhlanganisela eyinkimbinkimbi ye-cellulase, i-β-glucosidase kanye ne-Cellic® HTec2 (157 mg protein/ml, lot VHN00001) evela ku-Novozymes (Franklinton, NC, USA)). I-Multifect Pectinase® (72 mg protein/mL), ingxube eyinkimbinkimbi yama-enzyme abhubhisa i-pectin, yanikelwa yi-DuPont Industrial Biosciences (Palo Alto, CA, USA). Ukuhlushwa kwamaprotheni e-enzyme kunqunywe ngokulinganisa okuqukethwe kwamaprotheni (nokususa umnikelo we-non-protein nitrogen) kusetshenziswa i-Kjeldahl nitrogen analysis (indlela ye-AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). I-Diatomaceous earth 545 ithengwe ku-EMD Millipore (Billerica, MA). I-activated carbon (DARCO, ama-granule angu-100 ane-mesh), i-Avicel (PH-101), i-beech xylan, kanye nawo wonke amanye amakhemikhali athengwe kwa-Sigma-Aldrich (St. Louis, MO).
Ukwelashwa kwangaphambili kwe-AFEX kwenziwe e-GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Ukwelashwa kwangaphambili kwenziwe ku-140° C. imizuzu eyi-15. Isikhathi sokuhlala esingu-46 ngesilinganiso esingu-1:1 se-ammonia engamanzi kuya ku-biomass ku-60% (w/w) umthwalo ku-reactor yensimbi engagqwali (Parr Instruments Company). Kwathatha imizuzu engama-30. I-reactor ilethwe ku-140°C futhi i-ammonia yakhululwa ngokushesha, okuvumela i-biomass ukuthi ibuyele ngokushesha ekushiseni kwegumbi. Ukwakheka kwe-AFEX pre-treated corn stover (ACS) kwakufana nokwe-corn stover engaphathwanga (UT-CS).
I-ACSH 25% (w/w) (cishe ukulayisha kwe-dextran okungu-8%) yalungiswa njengento yokuqala yokukhiqizwa okukhulu kwama-oligosaccharides. I-Enzymatic hydrolysis ye-ACS yenziwa kusetshenziswa ingxube ye-enzyme yezentengiselwano ehlanganisa i-Cellic® Ctec2 10 mg protein/g glucan (ku-biomass elungisiwe ngaphambilini), i-Htec2 (Novozymes, Franklinton, NC), i-5 mg protein/g glucan, kanye ne-Multifect Pectinase (Genencor Inc, USA). ). ), i-5 mg protein/g dextran. I-Enzymatic hydrolysis yenziwa ku-bioreactor engamalitha ama-5 enomthamo osebenzayo wamalitha ama-3, i-pH 4.8, 50°C kanye ne-250 rpm. Ngemva kwe-hydrolysis amahora angama-96, i-hydrolyzate yaqoqwa nge-centrifugation ku-6000 rpm imizuzu engama-30 bese kuba ku-14000 rpm imizuzu engama-30 ukususa i-nonhydrolyzed. I-hydrolyzate yabe isihlungwa nge-beaker yesihlungi engu-0.22 mm. I-hydrolyzate ehlungiwe yagcinwa emabhodleleni ahlanzekile ku-4°C. yabe isihlukaniswa ku-carbon.
Ukuhlaziywa kokwakheka kwamasampula e-biomass asekelwe ekukhishweni ngokwezinqubo zokuhlaziya zelebhu ye-NREL: ukulungiswa kwamasampula okuhlaziywa kokwakheka (NREL/TP-510-42620) kanye nokunqunywa kwama-carbohydrate esakhiwo kanye ne-lignin ku-biomass (NREL/TP-510 - 42618)47.
Ukuhlaziywa kwe-oligosaccharide komfudlana we-hydrolyzate kwenziwe esikalini esingu-2 ml kusetshenziswa indlela ye-autoclave-based acid hydrolysis. Hlanganisa isampula ye-hydrolyzate ne-69.7 µl ye-72% sulfuric acid ku-10 ml screw cap culture tube bese uyibeka encuba ihora eli-1 ebhentshini ku-121 °C, uyipholise eqhweni bese uyihlunga ku-high performance liquid chromatography (HPLC) vial . Ukuhlushwa kwama-oligosaccharide kunqunywe ngokususa ukuhlushwa kwama-monosaccharide kusampula engahlushwanga kusuka ekuhlushweni okuphelele kukashukela kusampula ehlushwa yi-acid.
Ukuhlushwa kwe-glucose, i-xylose, kanye ne-rabinose ku-acid hydrolysed biomass kuhlaziywe kusetshenziswa uhlelo lwe-Shimadzu HPLC oluhlonyiswe nge-autosampler, i-column heater, i-isocratic pump, kanye ne-refractive index detector kukholomu ye-Bio-Rad Aminex HPX-87H. Ikholomu igcinwe ku-50°C futhi yasuswa nge-0.6 ml/min 5 mM H2SO4 ekugelezeni kwamanzi.
I-hydrolyzate supernatant yancishiswa futhi yahlaziywa ukuze kutholakale okuqukethwe yi-monomer kanye ne-oligosaccharide. Ushukela we-monomeric otholwe ngemuva kwe-enzyme hydrolysis wahlaziywa yi-HPLC efakwe ikholomu ye-Bio-Rad (Hercules, CA) Aminex HPX-87P kanye nekholomu yokuvikela umlotha. Izinga lokushisa lekholomu lagcinwa ku-80°C, amanzi asetshenziswa njengesigaba sokuhamba ngesivinini sokugeleza esingu-0.6 ml/min. Ama-oligosaccharide anqunywa yi-hydrolysis ku-dilute acid ku-121°C ngokwezindlela ezichazwe ku-refs. 41, 48, 49.
Ukuhlaziywa kwe-Saccharide kwenziwe ezinsaleleni ze-biomass ezingavuthiwe, ezilungisiwe kusengaphambili ze-AFEX kanye nazo zonke ezingafakwanga i-hydrolyzed (kufaka phakathi ukukhiqizwa kwezingcezu zodonga lwamaseli alandelanayo kanye nokuhlolwa kwazo kwe-mAb) kusetshenziswa izinqubo ezichazwe ngaphambilini 27, 43, 50, 51. Ekuhlaziyweni kwe-glycome, izingcezu ezingancibiliki zotshwala zezinto zodonga lwamaseli esitshalo zilungiswa kusuka ezinsaleleni ze-biomass futhi zikhishwa ngokulandelana ngama-reagents anolaka kakhulu njenge-ammonium oxalate (50 mM), i-sodium carbonate (50 mM kanye ne-0.5% w/v), i-CON. (1M kanye ne-4M, zombili ezine-1% w/v sodium borohydride) kanye ne-acid chlorite njengoba kuchaziwe ngaphambilini52,53. Izingcezu zabe sezifakwa ku-ELISA ngokumelene nephaneli eyinkimbinkimbi yama-mAb50 aqondiswe ku-glycan yodonga lwamaseli, kanti ukusabela kokubopha kwe-mAb kwethulwe njengemephu yokushisa. I-mAbs eqondisa i-glycan yodonga lwamaseli esitshalo ithengwe ezitokweni zelebhu (uchungechunge lwe-CCRC, JIM kanye ne-MAC).
I-biotinylation yesinyathelo esisodwa yama-oligosaccharide. Ukuhlanganiswa kwama-carbohydrate ne-biotin-LC-hydrazide kwenziwa kusetshenziswa inqubo elandelayo. I-Biotin-LC-hydrazide (4.6 mg/12 μmol) yancibilikiswa ku-dimethyl sulfoxide (DMSO, 70 μl) ngokuxubha ngamandla nokushisa ku-65° C. umzuzu o-1. I-Glacial acetic acid (30 µl) yanezelwa futhi ingxube yathelwa ku-sodium cyanoborohydride (6.4 mg/100 µmol) yancibilikiswa ngokuphelele ngemva kokushisa ku-65° C. cishe umzuzu o-1. Ngemuva kwalokho, kusukela ku-5 kuya ku-8 μl yengxube yokusabela kwanezelwa ku-oligosaccharide eyomisiwe (1-100 nmol) ukuze kutholakale ukweqisa kwe-molar okuphindwe kayishumi noma ngaphezulu kwelebula ngaphezulu kokuphela kokunciphisa. Ukusabela kwenziwa ku-65°C amahora ama-2, ngemva kwalokho amasampula ahlanzwa ngokushesha. Akukho sodium cyanoborohydride esetshenziswe ekuhlolweni kokulebula ngaphandle kokunciphisa, futhi amasampula asabela ku-65°C. amahora angu-2.5.
Ukumbozwa kwe-ELISA nokugezwa kwamasampula e-oligosaccharides e-biotinylated. Ama-25 μl amasampula e-biotinylated (100 μl yesampula ngayinye egxilile exutshwe ku-5 ml yesisombululo se-Tris buffer esingu-0.1 M (TBS)) afakwe emthonjeni ngamunye wepuleti elimbozwe yi-avidin. Ama-control wells ambozwe nge-50 μl ye-biotin ekugxilweni okungu-10 μg/ml ku-0.1 M TBS. Amanzi ahlanjululwe asetshenziswa njengengubo yokulinganisa okungenalutho. Ithebhulethi yafakwa e-incubator amahora ama-2 ekushiseni kwegumbi ebumnyameni. Geza ipuleti izikhathi ezintathu ngobisi olukhishwe ubisi oluyi-0.1% ku-0.1 M TBS usebenzisa uhlelo lwenombolo 11 lwe-Grenier flat 3A.
Ukwengeza nokugeza ama-antibodies ayisisekelo. Engeza ama-µl angu-40 e-antibody ayisisekelo emthonjeni ngamunye. Faka i-microplate ihora eli-1 ekushiseni kwegumbi ebumnyameni. Amapuleti abe esegezwa izikhathi ezintathu ngobisi oluyi-0.1% ku-0.1M TBS kusetshenziswa uhlelo lokugeza #11 lwe-Grenier Flat 3A.
Faka i-antibody yesibili bese ugeza. Engeza i-50 µl ye-antibody yesibili yegundane/igundane (exutshwe i-1:5000 kubisi oluyi-0.1% ku-0.1 M TBS) emthonjeni ngamunye. Faka i-microplate ihora eli-1 ekushiseni kwegumbi ebumnyameni. Ama-microplate abe esegezwa izikhathi ezi-5 ngobisi oluyi-0.1% ku-0.1 M TBS usebenzisa uhlelo lokugeza amapuleti lwe-Grenier Flat 5A #12.
Ukwengeza i-substrate. Engeza u-50 µl we-3,3′, 5,5′-tetramethylbenzidine (TMB) ku-substrate eyisisekelo (ngokwengeza amaconsi amabili e-buffer, amaconsi amathathu e-TMB, amaconsi amabili e-hydrogen peroxide ku-15 ml wamanzi ahlanzekile). Lungisa i-substrate ye-TMB. kanye ne-vortex ngaphambi kokusebenzisa). Faka i-microplate ekushiseni kwegumbi imizuzu engama-30. Ebumnyameni.
Qedela isinyathelo bese ufunda ithebhulethi. Engeza u-50 µl we-1 N sulfuric acid emthonjeni ngamunye bese uqopha ukumuncwa kusuka ku-450 kuya ku-655 nm usebenzisa i-ELISA reader.
Lungisa izixazululo ezingu-1 mg/ml zalezi zihlaziyi emanzini acwengekile: 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 (izinga langaphakathi). Izindinganiso ezimbili zalungiswa ngokungeza izixazululo zikashukela ezingu-1 mg/mL eziboniswe kuThebula 1. Amasampula ayaqandiswa futhi afakwe i-lyophil ku--80° C. kuze kube yilapho wonke amanzi esusiwe (ngokuvamile cishe amahora angu-12-18).
Engeza isampula engu-100–500 µg kumashubhu esivikelo esilinganisweni sokuhlaziya. Bhala phansi inani elingeziwe. Kungcono ukuncibilikisa isampula ekugxilweni okuthile kwe-solvent bese uyifaka eshubhuni njenge-aliquot yoketshezi. Sebenzisa i-inositol engu-20 µl engu-1 mg/ml njengendinganiso yangaphakathi yeshubhu ngayinye yesampula. Inani lendinganiso yangaphakathi engezwe eshubhuni kumele lifane nenani lendinganiso yangaphakathi engezwe eshubhuni elijwayelekile.
Faka u-8 ml we-methanol engamanzi ebhodleleni lesivalo sesikulufo. Bese ufaka u-4 ml wesisombululo se-3 N. methanolic HCl, uvale bese unyakaziswa. Le nqubo ayisebenzisi amanzi.
Engeza isixazululo se-methanol esingu-500 µl esingu-1 M HCl kumasampula e-oligosaccharide kanye namashubhu ajwayelekile e-TMS. Amasampula afakwa e-incubation ubusuku bonke (amahora angu-168) ku-80° C. ku-thermal block. Yomisa umkhiqizo we-methanolysis ekushiseni kwegumbi usebenzisa i-manifold yokomisa. Engeza i-200 µl MeOH bese womisa futhi. Le nqubo iphindaphindwa kabili. Engeza i-200 µl ye-methanol, i-100 µl ye-pyridine kanye ne-100 µl ye-acetic anhydride kusampula bese uxuba kahle. Amasampula afakwa e-incubation ekushiseni kwegumbi imizuzu engama-30 bese womiswa. Engeza i-200 µl ye-methanol bese womisa futhi.
Engeza u-200 µl we-Tri-Sil bese ufudumeza ishubhu elimboziwe imizuzu engama-20. 80°C, bese uphola ufike ezingeni lokushisa legumbi. Sebenzisa i-manifold yokomisa ukuze womise isampula ibe yivolumu engaba ngu-50 µl. Kubalulekile ukuqaphela ukuthi asizange sivumele amasampula ukuba ome ngokuphelele.
Engeza u-2 ml we-hexane bese uxuba kahle ngokufaka umoya. Gcwalisa iziqongo zama-pipettes e-Pasteur (5-8 mm) ngengcezu yoboya bengilazi ngokufaka uboya bengilazi phezu kwe-pipette enobubanzi obungu-5-3/4 intshi. Amasampula afakwa ku-centrifuge ku-3000 g imizuzu emi-2. Noma yiziphi izinsalela ezinganyibiliki ziyavuvukala. Yomisa isampula ibe ngu-100-150 µl. Umthamo ongaba ngu-1 μl wafakwa ku-GC-MS ekushiseni kokuqala okungu-80 °C kanye nesikhathi sokuqala semizuzu engu-2.0 (Ithebula 2).