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Izindlela ezintsha ze-immunological kanye ne-mass spectrometric zokuhlaziya okuyinkimbinkimbi kwama-oligosaccharides aphikelelayo kusitovu sommbila esenziwe ngaphambili nge-AFEX.I-Lignocellulosic biomass iyindlela esimeme kumafutha ezinto ezimbiwa phansi futhi isetshenziswa kabanzi ukuthuthukisa ubuchwepheshe bezinto eziphilayo ukuze kukhiqizwe imikhiqizo efana nokudla, okuphakelayo, izinto zokubasa namakhemikhali.Isihluthulelo salobu buchwepheshe ukuthuthukiswa kwezinqubo ezincintisanayo zokuguqula ama-carbohydrate ayinkimbinkimbi akhona ezindongeni zamaseli ezitshalo abe ushukela olula njengeglucose, i-xylose ne-arabinose.Ngenxa yokuthi i-lignocellulosic biomass inenkani kakhulu, kufanele ibe ngaphansi kokwelashwa kwe-thermochemical (isb, i-ammonia fibre exfoliation (AFEX), i-dilute acids (DA), uketshezi lwe-ionic (IL)) kanye nokwelashwa kwebhayoloji (isb, i-enzymatic hydrolysis kanye nokuvutshelwa kwe-microbial) ngokuhlangene ukuze uthole umkhiqizo owufunayo..Kodwa-ke, lapho ama-enzyme esikhunta ezentengiselwano esetshenziselwa inqubo ye-hydrolysis, kuphela i-75-85% yoshukela oncibilikayo owenziwe ama-monosaccharides, kanti i-15-25% esele iyi-oligosaccharides e-soluble, engatholakali, engatholakali njalo kuma-microorganisms.Ngaphambilini, siye sahlukanisa ngempumelelo futhi sahlanza ama-oligosaccharide anenkani ancibilikayo sisebenzisa inhlanganisela yekhabhoni ne-diatomaceous ukwehlukaniswa komhlaba kanye ne-chromatography yokukhishwa kosayizi, saphinde saphenya izakhiwo zawo ezivimbela ama-enzyme.Sithole ukuthi ama-oligosaccharides aqukethe izinga eliphezulu le-polymerization (DP) i-methylated uronic acid substitutions kunzima kakhulu ukucubungula ngezingxube ze-enzyme zezohwebo kune-DP ephansi kanye ne-oligosaccharides engathathi hlangothi.Lapha sibika ukusetshenziswa kwezindlela ezimbalwa ezengeziwe, okuhlanganisa ukuprofetha kwe-glycan kusetshenziswa amasosha omzimba e-monoclonal (mAbs) aqondene nokutshala ama-biomass glycans ukuze aveze amabhondi e-glycan ezindongeni zamaseli ezitshalo nama-enzymatic hydrolysates, i-matrix-assisted laser desorption ionization, i-time-of-flight mass- spectrometry..I-MALDI-TOF-MS) isebenzisa iziqongo zokuxilonga ezinolwazi ngesakhiwo ezitholwe i-spectroscopy ngemva kokubola kwesibili kwama-ion angemuhle, i-gas chromatography kanye ne-mass spectrometry (GC-MS) ukuze ibonise amabhondi e-oligosaccharide aphuma ngaphandle nangaphandle kokuphuma.Ngenxa yobukhulu obuncane bama-oligosaccharides (DP 4–20), lawa ma-molecule kunzima ukusebenzisela ukuhlanganisa i-mAb kanye nezinhlamvu.Ukuze sinqobe le nkinga, sisebenzise indlela entsha ye-biotin-based oligosaccharide immobilization esekelwe ku-biotin eyalebula ngempumelelo iningi lama-oligosaccharides ancibilikayo e-DP endaweni ye-microplate, eyabe isisetshenziswa ohlelweni lwe-throughput mAb ukuze kuhlaziywe intambo ethile.Le ndlela entsha izosiza ukuthuthukiswa kokuhlolwa kwe-glycome ephezulu okuthuthuke kakhulu esikhathini esizayo okungasetshenziswa ukuhlukanisa nokuhlukanisa ama-oligosaccharides akhona kuma-biomarker ngezinjongo zokuxilonga.
I-Lignocellulosic biomass, eyakhiwe ngezinto zezolimo, ezamahlathi, utshani kanye nezinkuni, iwukudla okungase kube khona ukukhiqizwa kwemikhiqizo esekelwe ku-bio, okuhlanganisa ukudla, ukudla, uphethiloli kanye nezandulela zamakhemikhali ukuze kukhiqizwe imikhiqizo enenani eliphakeme1.Ama-carbohydrate (afana ne-cellulose kanye ne-hemicellulose) akhona ezindongeni zamaseli ezitshalo akhishwa e-monosaccharides ngokucubungula amakhemikhali kanye ne-biotransformation (njenge-enzymatic hydrolysis kanye nokuvutshelwa kwe-microbial).Ukwelashwa kwangaphambili okujwayelekile kuhlanganisa ukunwetshwa kwe-ammonia fiber (AFEX), i-dilute acid (DA), i-ionic liquid (IL), nokuqhuma kwe-steam (SE), esebenzisa inhlanganisela yamakhemikhali nokushisa ukuze kuncishiswe ukukhiqizwa kwe-lignocellulose ngokuvula izindonga zamaseli ezitshalo3,4.inkani yendaba, 5. I-Enzymatic hydrolysis yenziwa ekulayishweni okuphezulu okuqinile kusetshenziswa ama-enzymes asebenzayo aqukethe ama-carbohydrate (CAZymes) kanye nokuvutshelwa kwe-microbial usebenzisa imvubelo ye-transgenic noma amabhaktheriya ukukhiqiza amafutha namakhemikhali e-bio-based 6.
Ama-CAZymes kuma-enzyme ezentengiselwano akhiwe ingxube eyinkimbinkimbi yama-enzyme ahlukanisa ngokuhlangene amabhondi ayinkimbinkimbi e-carbohydrate-ushukela ukuze akhe ama-monosaccharides2,7.Njengoba sike sabika ngaphambili, inethiwekhi eyinkimbinkimbi yama-polymers anephunga elimnandi ye-lignin enama-carbohydrates ibenza bangabi namandla kakhulu, okuholela ekuguqulweni kukashukela okungaphelele, kuqongelela i-15-25% yama-oligosaccharides ocansi angakhiqizwa ngesikhathi se-enzymatic hydrolysis ye-biomass pretreated.Lena inkinga evamile ngezindlela ezihlukahlukene zokwelashwa kwe-biomass.Ezinye zezizathu zalokhu kuphazamiseka zihlanganisa ukuvinjelwa kwe-enzyme ngesikhathi se-hydrolysis, noma ukungabikho noma amazinga aphansi ama-enzyme abalulekile adingekayo ukuze aphule izibopho zikashukela ku-biomass yezitshalo.Ukuqonda ukwakheka kanye nezici zesakhiwo sikashukela, njengamabhondi kashukela kuma-oligosaccharides, kuzosisiza ukuthi sithuthukise ukuguqulwa kukashukela ngesikhathi se-hydrolysis, okwenza izinqubo ze-biotechnological zibe nezindleko zokuncintisana nemikhiqizo etholakala ku-petroleum.
Ukunquma ukwakheka kwamacarbohydrate 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 zamacarbohydrate.Muva nje, i-Collision-Induced Dissociation (CID) tandem ye-MS ye-sodium ion adducts isetshenziswe kakhulu ukukhomba izigxivizo zeminwe ezihambisana nezikhundla zokunamathisela i-oligosaccharide, ukulungiselelwa kwe-anomeric, ukulandelana, nezikhundla ze-branching 20, 21.
Ukuhlaziywa kwe-Glycan kuyithuluzi elihle kakhulu lokuhlonza ngokujulile amabhondi ama-carbohydrate22.Le ndlela isebenzisa amasosha omzimba e-monoclonal (mAbs) aqondiswe ekutshaleni udonga lwamaseli i-glycan njenge-probes ukuqonda ukuxhumana okuyinkimbinkimbi kwama-carbohydrate.Ngaphezu kwama-250 mAbs atholakala emhlabeni wonke, aklanyelwe ngokumelene nama-oligosaccharide ahlukene alayini kanye namagatsha kusetshenziswa ama-saccharides ahlukahlukene24.Ama-mAb amaningana asetshenziswe kabanzi ukuze abonise ukwakheka, ukwakheka, kanye nokuguqulwa kodonga lwamaseli esitshalo, njengoba kunomehluko omkhulu kuye ngohlobo lweseli yesitshalo, isitho, ubudala, isigaba sokukhula, kanye nendawo yokukhula25,26.Muva nje, le ndlela isetshenziselwe ukuqonda inani lama-vesicle ezinhlelweni zezitshalo nezilwane kanye neqhaza lazo ekuthuthweni kwe-glycan njengoba kunqunywa omaka abancane, izigaba zokuthuthuka, noma izisusa zemvelo, kanye nokunquma umsebenzi we-enzymatic.Ezinye zezinhlaka ezihlukene ze-glycans nama-xylans ezikhonjiwe zihlanganisa i-pectin (P), i-xylan (X), i-mannan (M), i-xyloglucans (XylG), i-mix bond glucans (MLG), i-arabinoxylan (ArbX), i-galactomannan (GalG) , i-glucuronic acid-arabinoxy-XbGGGD kanye ne-aarbinoxylan (9) Arbinoxylan (9) kanye ne-arbinoxylan (9).
Kodwa-ke, naphezu kwayo yonke le mizamo yocwaningo, izifundo ezimbalwa kuphela eziye zagxila esimweni sokuqoqwa kwe-oligosaccharide ngesikhathi se-hydrolysis ephezulu ye-solids (HSL), kuhlanganise nokukhululwa kwe-oligosaccharide, izinguquko ze-oligomeric chain chain ngesikhathi se-hydrolysis, ama-polymers aphansi e-DP ahlukahlukene, kanye namajika awo.izabelo 30,31,32.Phakathi naleso sikhathi, nakuba ukuhlaziywa kwe-glycan kuye kwafakazela ukuthi kuyithuluzi eliwusizo lokuhlaziywa okuphelele kwesakhiwo se-glycan, kunzima ukuhlola ama-oligosaccharides e-DP ancibilikayo aphansi kusetshenziswa izindlela zokulwa nama-antibody.Ama-oligosaccharides e-DP amancane anesisindo samangqamuzana esingaphansi kuka-5-10 kDa awabophi kumapuleti e-ELISA angu-33, 34 futhi ayagezwa ngaphambi kokufakwa kwe-antibody.
Lapha, ngokokuqala ngqa, sibonisa ukuhlolwa kwe-ELISA kumapuleti ahlanganiswe ne-avidin esebenzisa amasosha omzimba e-monoclonal, ehlanganisa inqubo ye-biotinylation yesinyathelo esisodwa ye-oligosaccharides e-soluble refractory nokuhlaziywa kwe-glycome.Indlela yethu yokuhlaziya i-glycome yaqinisekiswa ukuhlaziya okusekelwe kwe-MALDI-TOF-MS kanye ne-GC-MS yokuxhumana okuhambisanayo kwe-oligosaccharide kusetshenziswa ukukhishwa kwe-trimethylsilyl (TMS) kokuqanjwa kukashukela we-hydrolyzed.Le ndlela entsha ingathuthukiswa njengendlela yokuphumelela okuphezulu esikhathini esizayo futhi ithole ukusetshenziswa okubanzi ocwaningweni lwe-biomedical35.
Ukuguqulwa kwangemuva kokuhumusha kwama-enzyme namasosha omzimba, njenge-glycosylation,36 kuthinta umsebenzi wabo wezinto eziphilayo.Isibonelo, izinguquko ku-glycosylation yamaprotheni e-serum zidlala indima ebalulekile ku-arthritis evuthayo, futhi izinguquko ku-glycosylation zisetshenziswa njengezimpawu zokuxilonga37.Ama-glycans ahlukahlukene abikwe ezincwadini ukuze avele kalula ezifweni ezihlukahlukene, kuhlanganise nezifo ezivuthayo ezingapheli ze-gastrointestinal tract nesibindi, izifo ezibangelwa amagciwane, i-ovarian, isifuba, kanye nomdlavuza we-prostate38,39,40.Ukuqonda ukwakheka kwama-glycans kusetshenziswa izindlela ze-glycan ELISA ezisuselwa ku-antibody kuzonikeza ukuzethemba okwengeziwe ekuxilongweni kwezifo ngaphandle kokusetshenziswa kwezindlela eziyinkimbinkimbi ze-MS.
Ucwaningo lwethu lwangaphambilini lubonise ukuthi ama-oligosaccharides anenkani ahlala e-unhydrolyzed ngemva kokwelashwa kwangaphambili kanye ne-enzymatic hydrolysis (Umfanekiso 1).Emsebenzini wethu oshicilelwe ngaphambilini, sithuthukise indlela yokukhipha i-charcoal solid-phase esebenzayo ukuze sihlukanise ama-oligosaccharides ku-AFEX-pretreated corn stover hydrolyzate (ACSH)8.Ngemuva kokukhipha nokuhlukaniswa kokuqala, ama-oligosaccharides aphinde ahlukaniswa ngosayizi we-chromatography (SEC) futhi aqoqwe ngokulandelana kwesisindo samangqamuzana.Ama-Sugar monomers nama-oligomers akhishwe ekwelapheni okuhlukahlukene ahlaziywa ngokuhlaziywa kokwakheka kukashukela.Uma uqhathanisa okuqukethwe kwe-oligomers kashukela etholwe ngezindlela ezihlukahlukene zokwelashwa, ukuba khona kwama-oligosaccharides anenkani kuyinkinga evamile ekuguqulweni kwe-biomass ibe yi-monosaccharides futhi kungaholela ekunciphiseni isivuno sikashukela okungenani i-10-15% futhi kuze kufike ku-18%.E-US.Le ndlela isetshenziselwa ukukhiqizwa okwengeziwe kwezingxenye ze-oligosaccharide.I-ACH ewumphumela kanye nezingxenyana zayo ezalandela ezinezisindo zamangqamuzana ezihlukene zasetshenziswa njengezinto zokuhlola ukuze kufanekiswe ama-oligosaccharides kulo msebenzi.
Ngemuva kokwelashwa kwangaphambili kanye ne-enzymatic hydrolysis, ama-oligosaccharides aphikelelayo ahlala e-unhydrolysis.Lapha (A) kuyindlela yokuhlukanisa i-oligosaccharide lapho ama-oligosaccharides ehlukaniswa ne-AFEX-pretreated corn stover hydrolyzate (ACSH) esebenzisa umbhede ogcwele we-activated carbon kanye nomhlaba we-diatomaceous;(B) Indlela yokuhlukaniswa kwama-oligosaccharides.Ama-oligosaccharides aphinde ahlukaniswa ngosayizi we-chromatography (SEC);(C) Ama-Saccharide monomers nama-oligomers akhishwe ekwelapheni okuhlukahlukene (i-asidi ehlanjululwe: DA, uketshezi lwe-ionic: IL kanye ne-AFEX).Izimo ze-Enzymatic hydrolysis: ukulayisha okuqinile okuphezulu okungama-25% (w/w) (cishe u-8% wokulayisha iglucan), amahora angama-96 i-hydrolysis, ukulayisha i-enzyme yokuhweba engu-20 mg/g (Ctec2:Htec2:MP-2:1:1 ratio) kanye (D) Noshukela monomers nama-oligomers eglucose, i-xyraated EXA-CSA ekhishwe i-EXACSACS ekhishwe i-xyracorn corn AFCS ekhishwe i-xylover CSA.
Ukuhlaziywa kwe-Glycan kufakazele ukuthi kuyithuluzi eliwusizo lokuhlaziywa kwesakhiwo okuphelele kwama-glycans ezikhishweni ezihlukanisiwe nezinsalela ze-biomass eziqinile.Kodwa-ke, ama-saccharide ancibilikayo emanzini awavezwa kancane kusetshenziswa le ndlela yendabuko41 ngoba ama-oligosaccharides anesisindo esiphansi se-molecular anzima ukuwafaka kumapuleti e-ELISA futhi ahlanjululwe ngaphambi kokwengezwa kwe-antibody.Ngakho-ke, ukuze kuhlanganiswe ama-antibody kanye nezimpawu, indlela yesinyathelo esisodwa se-biotinylation yasetshenziselwa ukugqoka ama-oligosaccharide ancibilikayo, angathobeli kumapuleti e-ELISA ahlanganiswe ne-avidin.Le ndlela ihlolwe kusetshenziswa i-ACSH yethu ekhiqizwe ngaphambilini kanye nengxenye esekelwe esisindweni sayo samangqamuzana (noma idigri ye-polymerization, DP).I-biotinylation yesinyathelo esisodwa yayisetshenziselwa ukwandisa i-oligosaccharide ebophezelayo ngokufaka i-biotin-LC-hydrazide ekupheleni kokunciphisa kwe-carbohydrate (Fig. 2).Esixazululweni, iqembu le-hemiacetal ekupheleni kokunciphisa lisabela neqembu le-hydrazide le-biotin-LC-hydrazide ukwakha ibhondi ye-hydrazone.Ebukhoneni be-ejenti yokunciphisa i-NaCNBH3, isibopho se-hydrazone sincishiswa kumkhiqizo wokugcina ozinzile we-biotinylated.Ngokuguqulwa kokuphela kokunciphisa ushukela, ukubopha ama-oligosaccharides e-DP aphansi kumapuleti e-ELISA kwaba nokwenzeka, futhi esifundweni sethu lokhu kwenziwa kumapuleti ane-avidin-coated kusetshenziswa ama-mAbs ahloselwe i-glycan.
Ukuhlolwa kwama-antibodies e-monoclonal asekelwe ku-ELISA kuma-oligosaccharides e-biotinylated.Lapha (A) i-biotinylation ehlanganisiwe yama-oligosaccharides kanye nokuhlolwa kwe-ELISA okulandelayo ngama-mAbs ahloselwe i-glycan kumapuleti ahlanganiswe ne-NeutrAvidin futhi (B) ibonisa inqubo yesinyathelo esisodwa se-biotinylation yemikhiqizo yokusabela.
Amapuleti ahlanganiswe ne-Avidin anama-oligosaccharide-conjugated antibodies abe engezwa kuma-antibodies ayisisekelo kanye nesesibili futhi ahlanjululwe endaweni ekhanyayo kanye nesikhathi esibucayi.Ngemva kokuqeda ukubopha amasosha omzimba, engeza i-TMB substrate ukuze ufukamele ipuleti.Ukusabela kwagcina kumiswe nge-sulfuric acid.Amapuleti afakwe incubated ahlaziywa kusetshenziswa isifundi se-ELISA ukuze kutholwe amandla okubopha amasosha omzimba ngamunye ukuze kutholwe ukuxhumanisa okukhethekile kwe-antibody.Ukuze uthole imininingwane namapharamitha wokuhlolwa, bona isigaba esihambisanayo "Izinto kanye Nezindlela".
Sibonisa usizo lwale ndlela esanda kuthuthukiswa yezinhlelo zokusebenza ezithile ngokuveza ama-oligosaccharides ancibilikayo akhona ku-ACSH kanye nezingxenyana ze-oligosaccharide ezingahluziwe futhi ezihlanziwe ezihlukanisiwe kuma-lignocellulosic hydrolysates.Njengoba kuboniswe kuMfanekiso 3, ama-xylans ajwayele ukushintshwa epitope akhonjwe ku-ACSH kusetshenziswa izindlela zokuhlola i-glycome ye-bioacylated ngokuvamile i-uronic (U) noma i-methyluronic (MeU) kanye ne-pectic arabinogalactans.Iningi lazo liphinde latholakala ocwaningweni lwethu lwangaphambilini ekuhlaziyeni ama-glycans of non-hydrolyzed solids (UHS)43.
Ukutholwa kwe-recalcitrant oligosaccharide epitopes kusetshenziswa i-monoclonal antibody eqondiswe odongeni lweseli glycan.Ingxenye "engathathi hlangothi" iyingxenyana ye-ACN futhi ingxenye "e-acidic" iyingxenyana ye-FA.Okubomvu okugqamile kumephu yokushisa kubonisa okuqukethwe okuphezulu kwe-epitope, futhi okuluhlaza okwesibhakabhaka okugqamile kubonisa ingemuva elingenalutho.Amanani ombala esikalini asuselwe kumanani we-OD aluhlaza okwakhiwa kwe-N=2.Ama-epitopes amakhulu abonwa amasosha omzimba aboniswa kwesokudla.
Lezi zakhiwo ezingezona i-cellulose azikwazanga ukuqhekezwa ama-cellulase nama-hemicellulase avame kakhulu kungxube ye-enzyme yokuhweba ehloliwe, ehlanganisa ama-enzyme asetshenziswa kakhulu ezentengiselwano.Ngakho-ke, ama-enzyme amasha asizayo ayadingeka ku-hydrolysis yawo.Ngaphandle kwama-enzyme adingekayo angewona ama-cellulose, lawa mabhondi angewona ama-cellulose avimbela ukuguqulwa okuphelele ku-monosaccharides, ngisho noma ama-polymers kashukela ongumzali ekhishwa kakhulu nge-hydrolyzed abe yizingcezu ezimfushane futhi ahlakazwe kusetshenziswa izingxube zama-enzyme ezentengiselwano.
Ucwaningo olwengeziwe lokusabalalisa isignali namandla ayo okubopha lubonise ukuthi ama-epitopes abophezelayo ayephansi kuma-fractions aphezulu we-DP ushukela (A, B, C, DP kuze kufike ku-20 +) kunama-fractions aphansi we-DP (D, E, F, DP) kuma-dimers) (Fig. 1).Izingcezu ze-Acid zivame kakhulu kuma-non-cellulose epitopes kunezingcezu ezingathathi hlangothi.Lezi zenzakalo zihambisana nephethini ebonwe esifundweni sethu sangaphambilini, lapho i-DP ephezulu kanye nezingxenye ze-asidi zazimelana kakhulu ne-enzymatic hydrolysis.Ngakho-ke, ukuba khona kwe-non-cellulose glycan epitopes kanye nokushintshwa kwe-U ne-MeU kungasiza kakhulu ekuzinzeni kwama-oligosaccharides.Kumele kuqashelwe ukuthi ukubopha nokubona ukusebenza kahle kungase kube inkinga kuma-oligosaccharides e-DP ephansi, ikakhulukazi uma i-epitope iyi-oligosaccharide ye-dimeric noma ye-trimeric.Lokhu kungahlolwa kusetshenziswa ama-oligosaccharide entengiso anobude obuhlukile, ngayinye iqukethe i-epitope eyodwa kuphela ebophezela ku-mAb ethile.
Ngakho-ke, ukusetshenziswa kwamasosha omzimba aqondene nesakhiwo kwembula izinhlobo ezithile zamabhondi e-recalcitrant.Ngokuya ohlotsheni lwe-antibody esetshenzisiwe, iphethini efanele yokuhlanganisa, namandla esignali ekhiqizwayo (iningi futhi incane kakhulu), ama-enzyme amasha angabonakala futhi engezwe kancane kancane kungxube ye-enzyme ukuze kuguqulwe i-glycoconversion ephelele.Sithatha ukuhlaziywa kwama-oligosaccharides e-ACSH njengesibonelo, singakha isizindalwazi samabhondi e-glycan wento ngayinye ye-biomass.Kufanele kuqashelwe lapha ukuthi ukuhlobana okuhlukene kwamasosha omzimba kufanele kucatshangelwe, futhi uma ukuhlobana kwawo kungaziwa, lokhu kuzodala ubunzima obuthile lapho kuqhathaniswa amasignali amasosha omzimba ahlukene.Ngaphezu kwalokho, ukuqhathanisa amabhondi e-glycan kungase kusebenze kangcono phakathi kwamasampula e-antibody efanayo.Lezi zibopho ezinenkani zingabe sezixhunyaniswa nesizindalwazi se-CAZyme, lapho singakwazi khona ukuhlonza ama-enzyme, sikhethe ama-enzyme akhethiwe futhi sihlole ama-enzyme anqamula isibopho, noma sithuthukise amasistimu amagciwane ukuze aveze lawa ma-enzyme ukuze asetshenziswe kuma-biorefineries44.
Ukuze sihlole ukuthi izindlela ze-immunological zihambisana kanjani nezinye izindlela zokubonisa ama-oligosaccharides anesisindo esiphansi se-molecular ekhona ku-lignocellulosic hydrolysates, senze i-MALDI (Fig. 4, S1-S8) kanye nokuhlaziywa kwama-saccharides atholakala ku-TMS ngokusekelwe ku-GC-MS kuphaneli efanayo (Fig. 5) ingxenye ye-oligosaccharide.I-MALDI isetshenziselwa ukuqhathanisa ukuthi ngabe ukusatshalaliswa okukhulu kwama-oligosaccharide molecule kufana nesakhiwo esihlosiwe.Emkhiwaneni.4 ibonisa i-MC yezingxenye ezingathathi hlangothi i-ACN-A ne-ACN-B.Ukuhlaziywa kwe-ACN-A kuqinisekisile uhla loshukela we-pentose kusukela ku-DP 4-8 (Fig. 4) kuya ku-DP 22 (Fig. S1), obunzima bayo obuhambisana ne-MeU-xylan oligosaccharides.Ukuhlaziywa kwe-ACN-B kuqinisekise uchungechunge lwe-pentose ne-glucoxylan nge-DP 8-15.Kokubalulekile okungeziwe okufana nomfanekiso we-S3, amamephu okusabalalisa inqwaba ye-acidic ye-FA-C abonisa ububanzi be-(Me)U esikhundleni sikashukela we-pentose nge-DP ka-8-15 ehambisana nama-xylans afakwe esikhundleni atholakala ekuhlolweni kwe-ELISA-based mAb.Ama-epitopes ayahambisana.
I-spectrum ye-MALDI-MS yama-oligosaccharides angancibilikiyo ancibilikayo akhona ku-ACS.Lapha, (A) I-ACN-A izingxenyana zebanga lesisindo esiphansi eziqukethe i-methylated uronic acid (DP 4-8) esikhundleni se-glucuroxylan oligosaccharides kanye (B) ne-ACN-B xylan ne-methylated uronic acid oligosaccharides esikhundleni se-glucuroxylan (DP 8-15).
Ukuhlaziywa kokwakheka kwezinsalela ze-glycan zama-oligosaccharides aphikisayo.Lapha (A) ukwakheka kwe-TMS saccharide yamafrakshini ahlukahlukene e-oligosaccharide atholwe kusetshenziswa ukuhlaziywa kwe-GC-MS.(B) Izakhiwo zoshukela ezihlukahlukene ezivela ku-TMS ezitholakala kuma-oligosaccharides.I-ACN - ingxenye ye-acetonitrile equkethe i-oligosaccharides engathathi hlangothi kanye ne-FA - ingxenye ye-ferulic acid equkethe ama-oligosaccharides e-asidi.
Esinye isiphetho esithakazelisayo sithathwe ekuhlaziyweni kwe-LC-MS kwengxenye ye-oligosaccharide, njengoba kuboniswe ku-Figure S9 (izindlela zingabonakala ku-electronic supplementary material).Izingcezu ze-hexose kanye namaqembu e-OAc abonwa ngokuphindaphindiwe ngesikhathi sokuhlanganiswa kwengxenyana ye-ACN-B.Lokhu okutholakele akuqinisekisi nje kuphela ukuhlukana okubonwe ekuhlaziyweni kwe-glycome kanye ne-MALDI-TOF, kodwa futhi kunikeza ulwazi olusha mayelana nokuphuma kokunye okutholakala kuma-carbohydrate ku-pretreated lignocellulosic biomass.
Siphinde sahlaziya ukwakheka kukashukela kwengxenyana ye-oligosaccharide sisebenzisa i-TMS sugar derivatization.Sisebenzisa i-GC-MS, sinqume ukubunjwa kwe-neural (non-derivative) kanye noshukela one-acidic (i-GluA ne-Gala) engxenyeni ye-oligosaccharide (Fig. 5).I-Glucuronic acid itholakala ezingxenyeni ezine-acidic C no-D, kuyilapho i-galacturonic acid itholakala ezingxenyeni ezine-acidic A no-B, zombili okuyizingxenye eziphezulu ze-DP zikashukela one-acidic.Le miphumela ayiqinisekisi kuphela idatha yethu ye-ELISA ne-MALDI, kodwa futhi ihambisana nezifundo zethu zangaphambili zokuqoqwa kwe-oligosaccharide.Ngakho-ke, sikholelwa ukuthi izindlela zesimanje ze-immunological ezisebenzisa i-biotinylation yama-oligosaccharides kanye nokuhlolwa kwe-ELISA okwalandela zanele ukuthola ama-oligosaccharides e-recalcitrant ancibilikayo kumasampula ezinto eziphilayo.
Njengoba izindlela zokuhlola ezisuselwe ku-ELISA-mAb ziye zaqinisekiswa ngezindlela ezimbalwa ezihlukene, besifuna ukuqhubeka nokuhlola amandla ale ndlela entsha yokulinganisa.Ama-oligosaccharide amabili okuhweba, i-xylohexasaccharide oligosaccharide (XHE) ne-23-α-L-arabinofuranosyl-xylotriose (A2XX), athengwe futhi ahlolwa kusetshenziswa indlela entsha ye-mAb eqondise odongeni lweseli glycan.Umfanekiso wesi-6 ubonisa ukuhlobana komugqa phakathi kwesignali ebophezelayo ye-biotinylated kanye nokugxiliswa kwelogi lokuhlushwa kwe-oligosaccharide, okuphakamisa imodeli ye-adsorption ye-Langmuir engenzeka.Phakathi kwama-mAbs, i-CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, ne-CCRC-M151 ehlotshaniswa ne-XHE, ne-CCRC-M108, CCRC-M109, kanye ne-LM11 ehlotshaniswa ne-A2XX phezu kobubanzi obungu-100 nmno.Ngenxa yokutholakala okulinganiselwe kwama-antibodies phakathi nokuhlolwa, ukuhlolwa okulinganiselwe kwenziwa ngokugxilisa kwe-oligosaccharide ngayinye.Kufanele kuqashelwe lapha ukuthi amanye amasosha omzimba asabela ngendlela ehluke kakhulu ku-oligosaccharide efanayo njenge-substrate, mhlawumbe ngenxa yokuthi abophezela kuma-epitopes ahluke kancane futhi angaba nama-affinities ahluke kakhulu abophezelayo.Izinqubo nemithelela yokuhlonza i-epitope enembile izoba yinkimbinkimbi kakhulu lapho indlela entsha ye-mAb isetshenziswa kumasampuli wangempela.
Kwasetshenziswa ama-oligosaccharide amabili okuhweba ukuze kutholwe ububanzi bokutholwa kwama-mAbs ahloselwe i-glycan.Lapha, ukuhlobana komugqa nokugxiliswa kwelogi lokugxiliswa kwe-oligosaccharide kubonisa amaphethini e-Langmuir adsorption okuthi (A) XHE nge-mAb kanye (B) ne-A2XX ene-mAb.Ama-epitopes ahambisanayo akhombisa izakhiwo zama-oligosaccharides ezentengiselwano asetshenziswa njengama-substrates ekuhlolweni.
Ukusetshenziswa kwe-glycan-targeted monoclonal antibodies (ukuhlaziywa kwe-glycocomic noma ukuhlolwa kwe-mAb okusekelwe ku-ELISA) iyithuluzi elinamandla lokuhlukanisa ngokujulile ama-glycans amaningi odonga lwamaseli akha i-biomass yezitshalo.Kodwa-ke, ukuhlaziywa kwe-glycan ye-classical kubonisa kuphela ama-glycans amakhulu odonga lwamaseli, njengoba ama-oligosaccharides amaningi engasebenzi kahle kumapuleti e-ELISA.Kulolu cwaningo, i-AFEX-pretreated corn stover ifakwe i-hydrolyzed nge-enzyme kokuqukethwe okuqinile okuphakeme.Ukuhlaziywa kukashukela kwasetshenziselwa ukunquma ukwakheka kwe-recalcitrant cell wall carbohydrates ku-hydrolyzate.Kodwa-ke, ukuhlaziywa kwe-mAb kwama-oligosaccharides amancane kuma-hydrolysates acatshangelwa, futhi amathuluzi engeziwe ayadingeka ukuze ngokuphumelelayo immobilize ama-oligosaccharides kumapuleti e-ELISA.
Sibika lapha indlela esebenza kahle ye-oligosaccharide immobilization yokuhlolwa kwe-mAb ngokuhlanganisa i-oligosaccharide biotinylation elandelwa ukuhlolwa kwe-ELISA kumapuleti ahlanganiswe ne-NeutrAvidin™.Ama-oligosaccharides e-biotinylated immobilized abonisa ukuhambisana okwanele kwe-antibody ukuze kutholakale ngokushesha nangempumelelo ama-oligosaccharides e-recalcitrant.Ukuhlaziywa kokwakheka kwalawa ma-oligosaccharides anenkani okusekelwe ku-mass spectrometry kuqinisekisile imiphumela yale ndlela entsha yokuhlolwa kokuzivikela komzimba.Ngakho-ke, lezi zifundo zibonisa ukuthi inhlanganisela ye-oligosaccharide biotinylation kanye nokuhlolwa kwe-ELISA nge-glycan-targeted monoclonal antibodies kungasetshenziswa ukuthola ama-crosslink kuma-oligosaccharides futhi ingasetshenziswa kabanzi kwezinye izifundo ze-biochemical ezibonisa isakhiwo se-oligosaccharides.
Le ndlela yephrofayili ye-glycan esekwe ku-biotin ingumbiko wokuqala okwazi ukuphenya amabhondi e-carbohydrate e-recalcitrant ama-oligosaccharides ancibilikayo ku-biomass yezitshalo.Lokhu kusiza ukuqonda ukuthi kungani ezinye izingxenye ze-biomass zinenkani kangaka uma kuziwa ekukhiqizweni kwe-biofuel.Le ndlela igcwalisa igebe elibalulekile ezindleleni zokuhlaziya i-glycome futhi inweba ukusetshenziswa kwayo kuhlu olubanzi lwama-substrates ngaphandle kwe-oligosaccharides yezitshalo.Ngokuzayo, singasebenzisa amarobhothi ukwenza i-biotinylation futhi sisebenzise indlela esiyithuthukisile ukuze sihlaziye amasampula aphezulu sisebenzisa i-ELISA.
I-Corn straw (CS) etshalwe ku-Pioneer 33A14 hybrid imbewu yavunwa ngo-2010 eKramer Farms eRay, Colorado.Ngemvume yomnikazi womhlaba, le biomass ingasetshenziselwa ucwaningo. Amasampula agcinwe omile umswakama ongu-< 6% ezikhwameni zokukhiya u-zip ekamelweni lokushisa. Amasampula agcinwe omile umswakama ongu-< 6% ezikhwameni zokukhiya u-zip ekamelweni lokushisa. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Amasampula agcinwe omile kumswakama ongu-<6% ezikhwameni ezifakwe uziphu ekamelweni 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% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Amasampula agcinwa ezikhwameni zoziphu ekamelweni lokushisa elinomswakama ongu- <6%.Ucwaningo luhambisane nemihlahlandlela yendawo kanye nekazwelonke.Ukuhlaziywa kokuqamba kwenziwa kusetshenziswa iphrothokholi ye-NREL.Ukwakheka kutholakale ukuthi kuqukethe u-31.4% we-glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% wamaprotheni kanye no-13. 4% womlotha.
I-Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) iyingxube eyinkimbinkimbi ye-cellulase, 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), inhlanganisela eyinkimbinkimbi yama-enzyme ehlisa isithunzi ye-pectin, inikelwe i-DuPont Industrial Biosciences (Palo Alto, CA, USA).Ukugxila kwamaprotheni e-enzyme kwanqunywa ngokulinganisa okuqukethwe kwamaprotheni (kanye nokukhipha umnikelo we-nitrogen engeyona iphrotheni) kusetshenziswa ukuhlaziywa kwe-Kjeldahl nitrogen (indlela ye-AOAC 2001.11, i-Dairy One Cooperative Inc., Ithaca, NY, USA).I-Diatomaceous Earth 545 yathengwa kwa-EMD Millipore (Billerica, MA).I-activated carbon (DARCO, 100 mesh granules), i-Avicel (PH-101), i-beech xylan, nawo wonke amanye amakhemikhali athengwe kwa-Sigma-Aldrich (St. Louis, MO).
Ukwelashwa kwangaphambili kwe-AFEX kwenziwa e-GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Ukwelashwa kwangaphambili kwenziwa ku-140 ° C. imizuzu engu-15.46 isikhathi sokuhlala esilinganisweni esingu-1:1 se-ammonia enamanzi ukuya ku-biomass ku-60% (w/w) ilayishwa ku-reactor yebhentshi yensimbi engagqwali (Parr Instruments Company).Kuthathe imizuzu engama-30.I-reactor ilethwa ku-140 ° C futhi i-ammonia yakhululwa ngokushesha, okuvumela i-biomass ukuthi ibuyele ngokushesha ekamelweni lokushisa.Ukwakhiwa kwe-AFEX pre-treated corn stover (ACS) kwakufana nalokho kwesitovu sommbila esingalashiwe (UT-CS).
Okuqinile okuphezulu I-ACSH 25% (w/w) (cishe u-8% wokulayisha i-dextran) yalungiselelwa njengento yokuqala yokukhiqizwa kwe-oligosaccharides ngezinga elikhulu.I-Enzymatic hydrolysis ye-ACS yenziwa kusetshenziswa ingxube ye-enzyme yezentengiselwano ehlanganisa i-Cellic® Ctec2 10 mg protein/g glucan (ku-biomass pretreated), i-Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, kanye ne-Multifect Pectinase (Genencor Inc, USA).).), 5 mg amaprotheni/g dextran.I-Enzymatic hydrolysis yenziwa ku-5-litre bioreactor enevolumu yokusebenza engamalitha ama-3, i-pH 4.8, 50 ° C kanye ne-250 rpm.Ngemuva kwe-hydrolysis amahora angama-96, i-hydrolyzate yaqoqwa nge-centrifugation ku-6000 rpm imizuzu engama-30 bese kuthi ngo-14000 rpm imizuzu engama-30 ukuze kukhishwe izinto eziqinile ezingahlanjululwa ngamanzi.I-hydrolyzate yabe isifakwa ngaphansi kokuhlunga okubulalayo ngebhika yesihlungi engu-0.22 mm.I-hydrolyzate ehlungiwe yagcinwa emabhodleleni angenalutho ku-4° C. yase ihlukaniswa kukhabhoni.
Ukuhlaziywa kokwakhiwa kwamasampula e-biomass asekelwe ku-extract ngokuya ngezinqubo zokuhlaziya ilabhorethri ye-NREL: ukulungiswa kwamasampula okuhlaziya ukwakheka (NREL/TP-510-42620) nokunqunywa kwama-carbohydrate esakhiwo kanye ne-lignin ku-biomass (NREL/TP-510 - 42618)47.
Ukuhlaziywa kwe-oligosaccharide yomfudlana we-hydrolyzate kwenziwa esikalini se-2 ml kusetshenziswa indlela ye-autoclave-based acid hydrolysis.Xuba isampula ye-hydrolyzate no-69.7 µl ka-72% we-sulfuric acid ku-10 ml screw cap culture tube futhi ufukamele ihora elingu-1 ebhentshini elingu-121 °C, uphole eqhweni bese uhlunga kuvial ye-liquid chromatography (HPLC) esebenza kahle kakhulu .Ukugxiliswa kwama-oligosaccharides kwanqunywa ngokukhipha ukugcwala kwama-monosaccharides kusampula engeyona i-hydrolyzed kusukela kushukela eliphelele kusampula ye-asidi-hydrolyzed.
Ukugxila kweGlucose, i-xylose, ne-arabinose ku-acid hydrolysed biomass kwahlaziywa kusetshenziswa isistimu ye-Shimadzu HPLC efakwe i-autosampler, i-heater heater, iphampu ye-isocratic, kanye nomtshina wenkomba we-refractive kukholomu ye-Bio-Rad Aminex HPX-87H.Ikholomu iye yagcinwa ku-50°C futhi yakhishwa ngo-0.6 ml/min 5 mM H2SO4 emanzini.ukugeleza.
I-hydrolyzate supernatant yahlanjululwa futhi yahlaziywa ukuze kutholwe okuqukethwe kwe-monomer ne-oligosaccharide.Ushukela we-Monomeric otholwe ngemva kwe-enzymatic hydrolysis wahlaziywa yi-HPLC efakwe ikholomu ye-Bio-Rad (Hercules, CA) Aminex HPX-87P kanye nekholomu yonogada bomlotha.Ukushisa kwekholomu kwagcinwa ku-80 ° C, amanzi asetshenziswa njengesigaba esihambayo ngesilinganiso sokugeleza kwe-0.6 ml / min.Ama-oligosaccharides anqunywa i-hydrolysis ku-asidi e-dilute ku-121 ° C ngokuvumelana nezindlela ezichazwe kuma-refs.41, 48, 49.
Ukuhlaziywa kwe-Saccharide kwenziwa kokuluhlaza, kwe-AFEX yokwelashwa kwangaphambili kanye nazo zonke izinsalela ze-biomass ezingenayo i-hydrolysed (kuhlanganise nokukhiqizwa kwe-serial cell wall extracts kanye nokuhlolwa kwazo kwe-mAb) kusetshenziswa izinqubo ezichazwe ngaphambilini 27, 43, 50, 51.Ukuze kuhlaziywe i-glycome, izinsalela ezingancibiliki ngotshwala zensimbi yodonga lwamaseli ezitshalo zilungiswa kusuka ezinsalela ze-biomass futhi zingaphansi kokukhishwa kwe-serial ngama-reagents anolaka akhulayo njenge-ammonium oxalate (50 mM), i-sodium carbonate (50 mM kanye no-0.5% w/v), CON.(I-1M ne-4M, kokubili nge-1% w/v sodium borohydride) kanye ne-acid chlorite njengoba kuchazwe ngaphambilini52,53.Okukhishiwe kube sekungaphansi kwe-ELISA ngokumelene nephaneli eyinkimbinkimbi ye-mAb50s eqondiswe ku-cell wall glycan, futhi ukusabela okubophayo kwe-mAb kwethulwa njengemephu yokushisa.Ama-mAbs aqondise i-plant cell wall glycan athengwe esitokweni saselabhorethri (CCRC, JIM kanye nochungechunge lwe-MAC).
Isinyathelo esisodwa se-biotinylation yama-oligosaccharides.Ukuhlanganiswa kwama-carbohydrate nge-biotin-LC-hydrazide kwenziwa kusetshenziswa le nqubo elandelayo.I-Biotin-LC-hydrazide (4.6 mg/12 μmol) yancibilika ku-dimethyl sulfoxide (DMSO, 70 μl) ngokushukumisa okunamandla nokushisa ku-65° C. i-1 min.I-glacial acetic acid (30 µl) yengeziwe futhi ingxube yathululelwa ku-sodium cyanoborohydride (6.4 mg/100 µmol) futhi yancibilika ngokuphelele ngemva kokushisisa ku-65° C. cishe iminithi elingu-1.Khona-ke, kusukela ku-5 kuya ku-8 μl wengxube yokusabela yengezwe ku-oligosaccharide eyomisiwe (1-100 nmol) ukuze kutholwe i-10-fold noma ngaphezulu kwe-molar yelebula phezu kokuphela kokunciphisa.Ukusabela kwenziwa ku-65 ° C amahora angu-2, ngemva kwalokho amasampula ahlanzwa ngokushesha.Ayikho i-sodium cyanoborohydride esetshenzisiwe ekuhlolweni kokulebula ngaphandle kokunciphisa, futhi amasampuli asabela ku-65 ° C. amahora angu-2.5.
Ukumbozwa kwe-ELISA nokugeza amasampula ama-oligosaccharides e-biotinylated.U-25 μl wamasampuli e-biotinylated (i-100 μl yesampula ngayinye egxilile ehlanjululwe ku-5 ml wesisombululo sebhafa se-0.1 M Tris (TBS)) engezwe emthonjeni ngamunye wepuleti eline-avidin.Imithombo yokulawula yayimbozwe nge-50 μl ye-biotin ekuhlanganiseni okungu-10 μg/ml ku-0.1 M TBS.Amanzi acwengekile asetshenziswa njengokwemboza izilinganiso ezingenalutho.Ithebhulethi yafakwa amahora angu-2 ekamelweni lokushisa ebumnyameni.Geza ipuleti izikhathi ezi-3 ngobisi olukhihliziwe ongu-0.1% ku-0.1 M TBS usebenzisa inombolo yohlelo.11 weGrenier flat 3A.
Ukwengezwa nokugeza amasosha omzimba okuqala.Engeza u-40 µl we-antibody eyinhloko emthonjeni ngamunye.Faka i-microplate ihora elingu-1 ekamelweni lokushisa ebumnyameni.Amapuleti abe esewashwa izikhathi ezi-3 ngobisi oluyi-0.1% ku-0.1M TBS kusetshenziswa uhlelo lokuwasha #11 lwe-Grenier Flat 3A.
Faka amasosha omzimba wesibili bese ugeza.Engeza u-50 µl we-antibody yesibili yegundane/igundane (exutshwe u-1:5000 obisini elingu-0.1% ngo-0.1 M TBS) emthonjeni ngamunye.Faka i-microplate ihora elingu-1 ekamelweni lokushisa ebumnyameni.Ama-microplates abe esewashwa izikhathi ezi-5 ngobisi olu-0.1% ku-0.1 M TBS kusetshenziswa uhlelo lwe-Grenier Flat 5A lokuwasha amapuleti #12.
Ukwengeza i-substrate.Engeza u-50 µl we-3,3′,5,5′-tetramethylbenzidine (TMB) ku-substrate eyisisekelo (ngokwengeza amaconsi ama-2 esibhafa, amathonsi ama-3 e-TMB, amaconsi ama-2 e-hydrogen peroxide ku-15 ml wamanzi a-deionized).Lungiselela i-substrate ye-TMB.kanye ne-vortex ngaphambi kokusetshenziswa).Faka i-microplate ekamelweni lokushisa imizuzu engu-30.Ebumnyameni.
Qedela isinyathelo futhi ufunde ithebhulethi.Engeza u-50 µl we-1 N sulfuric acid emthonjeni ngamunye futhi urekhode ukumunca kusuka ku-450 kuya ku-655 nm usebenzisa isifundi se-ELISA.
Lungiselela izixazululo ezingu-1 mg/ml zalezi zihlaziyi emanzini akhishwe nge-deionized: 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 (manNAcglucoace), i-N-coamine.(glcNAc), N-acetylgalactosamine (galNAc), inositol (izinga langaphakathi).Izindinganiso ezimbili zalungiswa ngokungeza izixazululo zikashukela ezingu-1 mg/mL eziboniswe kuThebula 1. Amasampula aqandisiwe futhi afakwe i-lyophilized ku--80 ° C. kuze kube yilapho wonke amanzi ekhishwa (imvamisa cishe amahora angu-12-18).
Engeza u-100–500 µg wesampula kumashubhu we-screw cap kubhalansi yokuhlaziya.Rekhoda inani elingeziwe.Kungcono kakhulu ukuncibilikisa isampula ekuhlanganiseni okuthile kwe-solvent bese uyengeza kushubhu njenge-aliquot yoketshezi.Sebenzisa u-20 µl ka-1 mg/ml inositol njengezinga langaphakathi leshubhu ngayinye yesampula.Inani lezinga langaphakathi elingezwe kusampula kufanele lilingane nenani lezinga langaphakathi elingezwe kushubhu evamile.
Engeza u-8 ml we-methanol engenalutho ku-screw cap vial.Bese kuthi u-4 ml wesisombululo se-3 N. methanolic HCl, simbozwe futhi sinyakaziswe.Le nqubo ayisebenzisi amanzi.
Engeza u-500 µl wesisombululo se-methanol esingu-1 M HCl kumasampuli we-oligosaccharide namashubhu ajwayelekile e-TMS.Amasampula afukanyelwa ubusuku bonke (amahora angu-168) ku-80° C. endaweni eshisayo.Yomisa umkhiqizo we-methanolysis ekamelweni lokushisa usebenzisa ukoma okuningana.Engeza u-200 µl MeOH bese womisa futhi.Le nqubo iphindwe kabili.Engeza u-200 µl we-methanol, u-100 µl we-pyridine no-100 µl we-acetic anhydride kusampula bese uxuba kahle.Amasampula afakwa endaweni yokushisa yasekamelweni imizuzu engama-30.futhi omisiwe.Engeza u-200 µl we-methanol bese womisa futhi.
Engeza u-200 µl we-Tri-Sil bese ushisa ithubhu elifakwe isivalo imizuzu engu-20.80°C, bese ipholiswa ekamelweni lokushisa.Sebenzisa i-manifold yokumisa ukuze uqhubeke usule isampula ibe yivolumu elinganiselwa ku-50 µl.Kubalulekile ukuqaphela ukuthi asizange sivumele amasampula ome ngokuphelele.
Engeza u-2 ml we-hexane bese uxuba kahle nge-vortexing.Gcwalisa amathiphu e-Pasteur pipettes (5-8 mm) ngocezu lwengilazi yoboya ngokufaka uboya bengilazi phezu kwepipette yobubanzi obuyi-intshi engu-5-3/4.Amasampula ayenziwe nge-centrifuged ku-3000 g imizuzu emi-2.Noma yiziphi izinsalela ezingancibiliki ziyawa.Yomisa isampula ku-100-150 µl.Ivolumu ecishe ibe ngu-1 μl yajovwa ku-GC-MS ekushiseni kokuqala okungu-80 °C nesikhathi sokuqala esiyimizuzu engu-2.0 (Ithebula 2).