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Ngā tikanga hou mō te tātari matatini o ngā oligosaccharides pumau i roto i te oumu kānga kua tukatukahia ki te AFEX. Ko te koiora lignocellulosic he huarahi pumau hei whakakapi i ngā wahie pūmua, ā, e whakamahia whānuitia ana hei whakawhanake i ngā hangarau koiora mō te whakaputa hua pēnei i te kai, te whāngai kararehe, ngā wahie me ngā matū. Ko te mea nui ki ēnei hangarau ko te whakawhanake i ngā tukanga whakataetae-utu mō te huri i ngā warowaihā matatini kei roto i ngā pakitara pūtau tipu hei huka māmā pēnei i te glucose, te xylose me te arabinose. Nā te mea he tino uaua te koiora lignocellulosic, me tukuna ki ngā maimoatanga thermochemical (hei tauira, te tango i te muka ammonia (AFEX), ngā waikawa waimeha (DA), ngā wai ionic (IL)) me ngā maimoatanga koiora (hei tauira, te hydrolysis enzymatic me te fermentation microbial) hei whiwhi i te hua e hiahiatia ana. . Heoi, ina whakamahia ngā whākōkī harore arumoni i roto i te tukanga hydrolysis, ko te 75-85% anake o ngā huka wairewa i hangaia he monosaccharides, ā, ko te toenga 15-25% he oligosaccharides wairewa, uaua, kāore e wātea tonu ki ngā moroiti. I mua, kua angitu mātou ki te wehe me te pure i ngā oligosaccharides uaua wairewa mā te whakamahi i te huinga o te wehenga waro me te whenua diatomaceous me te chromatography whakakore rahi, ā, i tirotirohia hoki ō rātou āhuatanga aukati whākōkī. Kua kitea e mātou he uaua ake te tukatuka i ngā oligosaccharides kei roto te taumata teitei o te polymerization (DP) methylated uronic acid substitutions me ngā ranunga whākōkī arumoni i ngā oligosaccharides DP iti me te koretake. I konei ka ripoatahia e mātou te whakamahinga o ētahi atu tikanga, tae atu ki te whakatauira glycan mā te whakamahi i ngā paturopi monoclonal (mAbs) motuhake ki ngā glycans koiora tipu hei whakaahua i ngā here glycan i roto i ngā pakitara pūtau tipu me ngā hydrolysates enzymatic, te ionization desorption laser āwhina-matrix, te spectrometry papatipu wā-o-te-rere. . MALDI-TOF-MS) e whakamahi ana i ngā tihi tātaritanga mōhiohio-hanganga i whiwhihia mā te spectroscopy i muri i te pirau tuarua o ngā katote kino, te chromatography hau me te spectrometry papatipu (GC-MS) hei whakaahua i ngā here oligosaccharide me te kore derivatization. Nā te iti o ngā oligosaccharides (DP 4–20), he uaua te whakamahi i ēnei ngota hei here me te tautuhi i te mAb. Hei whakakore i tēnei raruraru, i whakamahia e mātou he tikanga whakaū oligosaccharide hou e hangai ana ki te whakakotahitanga biotin i tapaina angitu ai te nuinga o ngā oligosaccharides wairewa iti-DP i runga i te mata o te pereti iti, i whakamahia i roto i te pūnaha mAb tere-rahi mō te tātaritanga here motuhake. Mā tēnei tikanga hou ka āwhina i te whanaketanga o ngā whakamātautau glycome tere-rahi matatau ake ā muri ake nei ka taea te whakamahi hei wehe me te tautuhi i ngā oligosaccharides kei roto i ngā tohu koiora mō ngā kaupapa tātaritanga.
Ko te koiora lignocellulosic, he mea tito mai i ngā rauemi ahuwhenua, ngahere, tarutaru me ngā rauemi rakau, he rauemi pea mō te whakaputa hua koiora, tae atu ki te kai, te whāngai kararehe, te wahie me ngā matū matū hei whakaputa hua nui ake te uara1. Ko ngā warowaihā (pēnei i te cellulose me te hemicellulose) kei roto i ngā pakitara pūtau tipu ka tangohia te polymerization hei monosaccharides mā te tukatuka matū me te biotransformation (pēnei i te hydrolysis enzymatic me te fermentation microbial). Ko ngā maimoatanga-mua noa ko te whakawhānui muka ammonia (AFEX), te waikawa waimeha (DA), te wai ionic (IL), me te pahūtanga mamaoa (SE), e whakamahi ana i te huinga o ngā matū me te wera hei whakaiti i te whakaputanga lignocellulose mā te whakatuwhera i ngā pakitara pūtau tipu3,4. te uaua o te matū, 5. Ka mahia te hydrolysis enzymatic i te kawenga totoka teitei mā te whakamahi i ngā whākōkī kei roto te warowaihā hohe arumoni (CAZymes) me te fermentation microbial mā te whakamahi i ngā rewena transgenic, i ngā huakita rānei hei whakaputa wahie me ngā matū koiora6.
Ko ngā CAZyme i roto i ngā whākōkī arumoni he mea tito mai i tētahi ranunga matatini o ngā whākōkī e hono tahi ana i ngā hononga warowaihā-huka matatini hei hanga i ngā monosaccharides2,7. E ai ki tā mātou i kōrero ai i mua ake nei, ko te whatunga matatini o ngā pūhui kakara o te lignin me ngā warowaihā ka tino uaua te whakakore, ka hua ake ai te huringa huka kāore i oti, ka kohia te 15-25% o ngā oligosaccharides ira kāore i hangaia i te wā o te waikura whākōkī o te koiora kua tukatukahia i mua. He raruraru noa tēnei me ngā tikanga maimoatanga koiora maha. Ko ētahi o ngā take mō tēnei arai ko te aukati whākōkī i te wā o te waikura, te kore rānei, te iti rānei o ngā taumata o ngā whākōkī matua e hiahiatia ana hei wawahi i ngā hononga huka i roto i te koiora tipu. Mā te mārama ki te hanganga me ngā āhuatanga hanganga o ngā huka, pērā i ngā hononga huka i roto i ngā oligosaccharides, ka āwhina i a tātou ki te whakapai ake i te huringa huka i te wā o te waikura, ka whakataetae te utu o ngā tukanga hangarau koiora ki ngā hua i ahu mai i te hinu.
He uaua te whakatau i te hanganga o ngā warowaihā, ā, me whakamahi i te huinga o ngā tikanga pēnei i te chromatography wai (LC)11,12, te ine irahiko aukume karihi (NMR)13, te electrophoresis capillary (CE)14,15,16 me te ine ira papatipu (MS)17. ,tekau mā waru. Ko ngā tikanga MS pēnei i te ine ira papatipu wā-o-te-rere me te tango laser me te ionization mā te whakamahi i te matrix (MALDI-TOF-MS) he tikanga maha mō te tautuhi i ngā hanganga warowaihā. Nō nā tata nei, ko te Collision-Induced Dissociation (CID) tandem MS o ngā tāpiringa ion konutai kua whakamahia whānuitia hei tautuhi i ngā tapumati e pā ana ki ngā tūranga piri oligosaccharide, ngā whirihoranga anomeric, ngā raupapatanga, me ngā tūranga peka 20, 21.
He taputapu pai rawa te tātaritanga glycan mō te tautuhi hōhonu i ngā here warowaihā22. Ka whakamahia e tēnei tikanga ngā paturopi monoclonal (mAbs) e arohia ana ki te glycan pakitara pūtau tipu hei whakamātautau hei mārama ki ngā hononga warowaihā matatini. Neke atu i te 250 mAbs e wātea ana puta noa i te ao, i hangaia hei ārai i ngā momo oligosaccharides raina me ngā momo peka mā te whakamahi i ngā momo saccharides24. He maha ngā mAbs kua whakamahia whānuitia hei whakaahua i te hanganga, te hanganga, me ngā whakarerekētanga o te pakitara pūtau tipu, nā te mea he rerekētanga nui i runga i te momo pūtau tipu, te okana, te tau, te wāhanga whanaketanga, me te taiao tipu25,26. Nō nā tata nei, kua whakamahia tēnei tikanga hei mārama ki ngā taupori vesicle i roto i ngā pūnaha tipu me te kararehe me ō rātou tūranga i roto i te kawe glycan e whakatauhia ana e ngā tohu pūtau-iti, ngā wāhanga whanaketanga, ngā whakaohooho taiao rānei, me te whakatau i te mahi whākōkī. Ko ētahi o ngā hanganga rerekē o ngā glycans me ngā xylans kua tautuhia ko te pectin (P), te xylan (X), te mannan (M), ngā xyloglucans (XylG), ngā glucans hononga whakauru (MLG), te arabinoxylan (ArbX), te galactomannan (GalG), te waikawa glucuronic-arabinoxylan (GArbX) me te arabino-galactan (ArbG)29.
Heoi, ahakoa ēnei mahi rangahau katoa, he torutoru noa ngā rangahau kua arotahi ki te āhua o te kohikohinga oligosaccharide i te wā o te waikura nui o te totoka (HSL), tae atu ki te tukunga oligosaccharide, ngā huringa roa o te mekameka oligomeric i te wā o te waikura, ngā momo polymer DP iti, me ō rātou pihi. ngā tohatoha 30,31,32. I taua wā, ahakoa kua whakamātauhia te tātari glycan hei taputapu whai hua mō te tātari whānui o te hanganga glycan, he uaua ki te aromatawai i ngā oligosaccharides DP iti wairewa-wai mā te whakamahi i ngā tikanga paturopi. Ko ngā oligosaccharides DP iti ake me te taumaha ngota iti iho i te 5-10 kDa kāore e herea ki ngā pereti ELISA 33, 34, ā, ka horoia atu i mua i te tāpiritanga o te paturopi.
I konei, mō te wā tuatahi, ka whakaatuhia e mātou he whakamātautau ELISA i runga i ngā pereti kua pania ki te avidin mā te whakamahi i ngā paturopi monoclonal, e whakakotahi ana i tētahi tukanga biotinylation kotahi-taahiraa mō ngā oligosaccharides wairewa me te tātari glycome. I whakamanahia tā mātou huarahi ki te tātari glycome mā te tātari MALDI-TOF-MS me te GC-MS o ngā hononga oligosaccharide tautoko mā te whakamahi i te trimethylsilyl (TMS) derivatization o ngā hanganga huka hydrolyzed. Ka taea te whakawhanake i tēnei huarahi auaha hei tikanga whakaputa-teitei a meake nei, ā, ka kitea he tono whānui ake i roto i te rangahau hauora35.
Ko ngā whakarerekētanga i muri i te whakamaoritanga o ngā whākōkī me ngā paturopi, pērā i te glycosylation,36 ka pā ki tā rātou mahi koiora. Hei tauira, he mea nui te mahi a ngā huringa o te glycosylation o ngā pūmua toto ki te mate rumatiki mumura, ā, ka whakamahia ngā huringa o te glycosylation hei tohu tātaritanga37. Kua pūrongohia i roto i ngā tuhinga he maha ngā glycans e puta wawe ana i roto i ngā momo mate, tae atu ki ngā mate mumura mau tonu o te ara kai me te ate, ngā mate huaketo, ngā mate pukupuku ovarian, te ū, me te prostate38,39,40. Mā te mārama ki te hanganga o ngā glycans mā te whakamahi i ngā tikanga glycan ELISA e hangai ana ki te paturopi ka nui ake te maia ki te tātaritanga mate me te kore e whakamahi i ngā tikanga MS uaua.
I whakaatuhia e tā mātou rangahau o mua kāore i te waikura ngā oligosaccharides uaua i muri i te maimoatanga o mua me te waikura whākōkī (Pikitia 1). I roto i tā mātou mahi i whakaputaina i mua, i whakawhanakehia e mātou he tikanga tango waro totoka-wāhanga hei wehe i ngā oligosaccharides mai i te AFEX-pretreated corn stover hydrolyzate (ACSH)8. Whai muri i te tangohanga tuatahi me te wehenga, i wehea anō ngā oligosaccharides mā te chromatography exclusion size (SEC) ā, i kohia kia rite ki te taumaha ngota. I tātarihia ngā monomer huka me ngā oligomer i tukuna mai i ngā maimoatanga o mua mā te tātari hanganga huka. I te whakataurite i te ihirangi o ngā oligomer huka i whiwhihia e ngā tikanga maimoatanga o mua, ko te noho o ngā oligosaccharides uaua he raruraru noa i roto i te huringa o te koiora ki ngā monosaccharides, ā, ka taea te whakaiti i te hua huka o te 10-15% neke atu rānei, tae atu ki te 18%. US. Ka whakamahia tēnei tikanga mō te whakaputa nui ake o ngā wāhanga oligosaccharide. Ko te ACH i puta mai me ōna wāhanga i muri mai me ngā taumaha ngota rerekē i whakamahia hei rauemi whakamātautau mō te whakaahuatanga o ngā oligosaccharides i roto i tēnei mahi.
I muri i te maimoatanga o mua me te waikura whākōkī, kāore i waikura ngā oligosaccharides pumau. Anei (A) he tikanga wehewehe oligosaccharides e wehea ai ngā oligosaccharides mai i te waikura kānga kua tukatukahia i mua e te AFEX (ACSH) mā te whakamahi i tētahi moenga kua whakakīia o te waro whakahohe me te oneone diatomaceous; (B) Tikanga mō te wehenga o ngā oligosaccharides. I wehea anō ngā oligosaccharides mā te chromatography whakakore rahi (SEC); (C) Ngā monomer me ngā oligomer o te saccharide i tukuna mai i ngā maimoatanga o mua (waikawa kua whakaranua: DA, wai ionic: IL me te AFEX). Ngā āhuatanga waikura whākōkī: te uta totoka teitei o te 25% (w/w) (tata ki te 8% te uta glucan), te waikura 96 ​​hāora, te uta whākōkī arumoni 20 mg/g (ōwehenga Ctec2:Htec2:MP-2:1:1) me te (D) Ngā monomer huka me ngā oligomer o te glucose, te xylose me te arabinose i tukuna mai i te waikura kānga kua tukatukahia i mua e te AFEX (ACS).
Kua whakamātauhia te tātaritanga glycan hei taputapu whai hua mō te tātaritanga hanganga whānui o ngā glycan i roto i ngā tangohanga i wehea mai i ngā toenga koiora totoka. Heoi, kāore i te tino kitea ngā saccharides wairewa-wai mā te whakamahi i tēnei tikanga tuku iho41 nā te mea he uaua ki te whakaū i ngā oligosaccharides taumaha ngota iti ki runga i ngā pereti ELISA, ā, ka horoia atu i mua i te tāpiritanga o te paturopi. Nō reira, mō te here me te tautuhi i te paturopi, i whakamahia he tikanga biotinylation kotahi-taahiraa hei pani i ngā oligosaccharides wairewa, kāore e tutuki ana i runga i ngā pereti ELISA kua pania ki te avidin. I whakamatautauria tēnei tikanga mā te whakamahi i tā mātou ACSH i hangaia i mua me te wāhanga i runga i tōna taumaha ngota (te tohu o te polymerization rānei, DP). I whakamahia te biotinylation kotahi-taahiraa hei whakanui ake i te hononga here oligosaccharide mā te tāpiri i te biotin-LC-hydrazide ki te pito whakaiti o te warowaihā (Pikitia 2). I roto i te otinga, ka tauhohe te rōpū hemiacetal i te pito whakaiti ki te rōpū hydrazide o te biotin-LC-hydrazide hei hanga i te here hydrazone. I te aroaro o te matū whakaiti NaCNBH3, ka whakaitihia te here hydrazone ki tētahi hua mutunga biotinylated pumau. Nā te whakarerekētanga o te pito whakaiti huka, ka taea te here o ngā oligosaccharides DP iti ki ngā pereti ELISA, ā, i roto i tā mātou rangahau i mahia tēnei ki runga i ngā pereti kua pania ki te avidin mā te whakamahi i ngā mAbs kua arotahi ki te glycan.
Te tirotiro i ngā paturopi monoclonal i runga i te ELISA mō ngā oligosaccharides biotinylated. Anei (A) te whakakotahitanga o te biotinylation o ngā oligosaccharides me te tirotiro ELISA i muri mai me ngā mAbs e arohia ana ki te glycan i runga i ngā pereti kua pania ki te NeutrAvidin me (B) e whakaatu ana i tētahi tukanga kotahi-taahiraa mō te biotinylation o ngā hua tauhohenga.
Kātahi ka tāpirihia ngā pereti kua pania ki te avidin me ngā paturopi kua honoa ki te oligosaccharide ki ngā paturopi tuatahi me te tuarua, ā, ka horoia ki roto i tētahi reo e aro ana ki te mārama me te wā. Kia oti te herea o te paturopi, tāpirihia te papa TMB hei whakatō i te pereti. I mutu te tauhohenga ki te waikawa whanariki. I tātarihia ngā pereti kua whakatōkia mā te whakamahi i tētahi kaipānui ELISA hei whakatau i te kaha here o ia paturopi hei kite i te hononga whakawhiti motuhake ki te paturopi. Mō ngā taipitopito me ngā tawhā o te whakamātautau, tirohia te wāhanga e pā ana "Ngā Rauemi me ngā Tikanga".
Ka whakaatuhia e mātou te whai huatanga o tēnei tikanga hou kua whakawhanakehia mō ngā tono motuhake mā te whakaahua i ngā oligosaccharides wairewa kei roto i te ACSH me ngā wāhanga oligosaccharide mata me te parakore i wehea mai i ngā hydrolysates lignocellulosic. E ai ki te Whakaahua 3, ko ngā xylans kua whakakapia ki te epitope e kitea ana i roto i te ACSH mā te whakamahi i ngā tikanga whakamātautau glycome bioacylated ko te uronic (U) methyluronic (MeU) me te pectic arabinogalactans. Ko te nuinga o ēnei i kitea anō i roto i tā mātou rangahau o mua mō te tātaritanga o ngā glycans o ngā totoka kore-waikura (UHS)43.
Te kimi i ngā epitope oligosaccharide uaua mā te whakamahi i tētahi paturopi monoclonal e tukuna ana ki te pakitara pūtau glycan. Ko te wāhanga "kūpapa" ko te wāhanga ACN, ā, ko te wāhanga "waikawa" ko te wāhanga FA. Ko ngā whero kanapa ake i runga i te mahere wera e tohu ana i te nui ake o te ihirangi epitope, ā, ko ngā kikorangi kanapa ake e tohu ana i te papamuri kau. Ko ngā uara tae i runga i te tauine e ahu mai ana i ngā uara OD mata mō ngā hanganga N=2. Ko ngā epitope matua e kitea ana e ngā paturopi e whakaaturia ana i te taha matau.
Kāore i taea te haehae i ēnei hanganga kore-cellulose e ngā cellulases me ngā hemicellulases tino noa i roto i te ranunga whākōkī arumoni kua whakamatautauria, kei roto ko ngā whākōkī arumoni e whakamahia whānuitia ana. Nō reira, me whai whākōkī āwhina hou mō tā rātou waikura. Ki te kore ngā whākōkī tāpiri kore-cellulose e tika ana, ka ārai ēnei hononga kore-cellulose i te hurihanga katoa ki ngā monosaccharides, ahakoa ka waikurahia whānuitia ō rātou polymers huka matua ki ngā mongamonga poto ake, ā, ka whakarewahia mā te whakamahi i ngā ranunga whākōkī arumoni.
I whakaatuhia e tētahi atu rangahau mō te tohatoha tohu me tōna kaha here he iti ake ngā epitope here i roto i ngā wāhanga huka DP teitei (A, B, C, DP tae atu ki te 20+) i ngā wāhanga DP iti (D, E, F, DP) i roto i ngā dimer) (Pikitia 1). He nui ake te kitea o ngā kongakonga waikawa i roto i ngā epitope kore-cellulose i ngā kongakonga kūpapa. He rite tonu ēnei āhuatanga ki te tauira i kitea i roto i tā mātou rangahau o mua, i reira he ātete ake ngā wāhanga DP me te waikawa teitei ki te hydrolysis enzymatic. Nō reira, ko te noho mai o ngā epitope glycan kore-cellulose me ngā whakakapinga U me MeU ka nui te āwhina ki te pumau o ngā oligosaccharides. Me mōhio ko te pai o te here me te kimi ka raru pea mō ngā oligosaccharides DP iti, inā koa mēnā he oligosaccharide dimeric, trimeric rānei te epitope. Ka taea te whakamātautau i tēnei mā te whakamahi i ngā oligosaccharides arumoni o ngā roa rerekē, ia epitope kotahi anake te epitope e herea ana ki tētahi mAb motuhake.
Nō reira, i whakaatuhia e te whakamahinga o ngā paturopi motuhake-hanganga ētahi momo here uaua. I runga i te momo paturopi i whakamahia, te tauira here tika, me te kaha o te tohu e whakaputaina ana e ia (te nuinga me te iti rawa), ka taea te tautuhi me te tāpiri i ngā whākōkī hou mā te haurua-rahi ki te ranunga whākōkī kia tino oti ai te glycoconversion. Mā te tātari i ngā oligosaccharides ACSH hei tauira, ka taea e tātou te hanga i tētahi pātengi raraunga o ngā here glycan mō ia rauemi koiora. Me mārama i konei me whai whakaaro ki te rerekētanga o te hononga o ngā paturopi, ā, ki te kore e mōhiotia tō rātou hononga, ka puta he uauatanga i te whakataurite i ngā tohu o ngā paturopi rerekē. Hei tāpiri, ko te whakataurite i ngā here glycan ka pai ake te mahi i waenga i ngā tauira mō te paturopi kotahi. Kātahi ka taea te hono atu i ēnei here uaua ki te pātengi raraunga CAZyme, mai i reira ka taea e tātou te tautuhi i ngā whākōkī, te whiriwhiri i ngā whākōkī kaitono me te whakamātautau mō ngā whākōkī wawahi hononga, te whakawhanake rānei i ngā pūnaha moroiti hei whakaatu i ēnei whākōkī hei whakamahi i roto i ngā whare hanga koiora44.
Hei aromatawai i te āhua o te tautoko a ngā tikanga ārai mate i ngā tikanga rerekē mō te tautuhi i ngā oligosaccharides taumaha ngota iti kei roto i ngā hydrolysates lignocellulosic, i whakahaerehia e mātou te MALDI (Pikitia 4, S1-S8) me te tātari i ngā saccharides i ahu mai i te TMS i runga i te GC-MS i runga i te wāhanga oligosaccharide kotahi (Pikitia 5). Ka whakamahia te MALDI hei whakatairite mēnā he rite te tohatoha papatipu o ngā ngota oligosaccharide ki te hanganga e hiahiatia ana. I te pikitia 4 e whakaatu ana i te MS o ngā wāhanga kūpapa ACN-A me te ACN-B. I whakaū te tātari ACN-A i te whānuitanga o ngā huka pentose mai i te DP 4–8 (Pikitia 4) ki te DP 22 (Pikitia S1), ko ōna taumaha e rite ana ki ngā oligosaccharides MeU-xylan. I whakaū te tātari ACN-B i te raupapa pentose me te glucoxylan me te DP 8-15. I roto i ngā rauemi tāpiri pēnei i te Pikitia S3, e whakaatu ana ngā mahere tohatoha papatipu wāhanga waikawa FA-C i te whānuitanga o ngā huka pentose kua whakakapia (Me)U me te DP o te 8-15 e rite ana ki ngā xylan kua whakakapia i kitea i roto i te tirotiro mAb e hangai ana ki te ELISA. He rite tonu ngā epitope.
Ko te whānuitanga MALDI-MS o ngā oligosaccharides wairewa kore-tūturu kei roto i te ACS. I konei, (A) Ngā wāhanga taumaha iti o te ACN-A kei roto ngā oligosaccharides glucuroxylan kua whakakapia ki te waikawa uronic methylated (DP 4-8) me (B) ngā oligosaccharides xylan me te waikawa uronic methylated kua whakakapia ki te glucuroxylan (DP 8-15).
Te tātari i te hanganga o te toenga glycan o ngā oligosaccharides ātete. Anei (A) Te hanganga o te saccharide TMS o ngā momo wāhanga oligosaccharide i whiwhi mā te tātari GC-MS. (B) Ngā hanganga o ngā momo huka i ahu mai i te TMS kei roto i ngā oligosaccharides. ACN – te wāhanga acetonitrile kei roto ngā oligosaccharides kūpapa me te FA – te wāhanga waikawa ferulic kei roto ngā oligosaccharides waikawa.
I puta mai tētahi atu whakatau whakamere i te tātaritanga LC-MS o te wāhanga oligosaccharide, e whakaaturia ana i te Pikitia S9 (ka kitea ngā tikanga i roto i te rauemi tāpiri hiko). I kitea tonutia ngā wāhanga o ngā rōpū hexose me -OAc i te wā e herea ana te wāhanga ACN-B. Ehara i te mea ko tēnei kitenga anake te whakaū i te wehenga i kitea i roto i te tātaritanga glycome me te MALDI-TOF, engari ka whakaratohia hoki he mōhiohio hou mō ngā hua warowaihā pea i roto i te koiora lignocellulosic kua tukatukahia i mua.
I whakahaerehia hoki e mātou he tātaritanga hanganga glycan o ngā wāhanga oligosaccharide mā te whakamahi i te TMS glycan derivatization. Mā te whakamahi i te GC-MS, i whakatauhia e mātou te hanganga o ngā huka io (kore-whakaputa) me ngā huka waikawa (GluA me GalA) i roto i te wāhanga oligosaccharide (Pikitia 5). Kei roto i ngā wāhanga waikawa C me D te waikawa glucuronic, ko te waikawa galacturonic ia kei roto i ngā wāhanga waikawa A me B, he wāhanga DP teitei o ngā huka waikawa ēnei e rua. Ehara i te mea ko ēnei hua anake te whakaū i ā mātou raraunga ELISA me MALDI, engari he rite anō hoki ki ā mātou rangahau o mua mō te kohikohinga oligosaccharide. Nō reira, e whakapono ana mātou he rawaka ngā tikanga ārai mate hou e whakamahi ana i te biotinylation o ngā oligosaccharides me te tirotiro ELISA i muri mai hei kite i ngā oligosaccharides uaua wairewa i roto i ngā tauira koiora.
I te mea kua whakamanahia ngā tikanga tirotiro mAb e ahu mai ana i te ELISA e ngā tikanga rerekē, i hiahia mātou ki te tūhura atu i te pūmanawa o tēnei tikanga tauanga hou. E rua ngā oligosaccharides arumoni, ko te xylohexasaccharide oligosaccharide (XHE) me te 23-α-L-arabinofuranosyl-xylotriose (A2XX), i hokona, ā, i whakamatautauria mā te whakamahi i tētahi huarahi mAb hou e aro ana ki te glycan pakitara pūtau. E whakaatu ana te Pikitia 6 i tētahi hononga rārangi i waenga i te tohu here biotinylated me te kukū log o te kukū oligosaccharide, e whakaatu ana i tētahi tauira adsorption Langmuir pea. I roto i ngā mAbs, i hono te CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, me te CCRC-M151 ki te XHE, ā, i hono te CCRC-M108, CCRC-M109, me te LM11 ki te A2XX i runga i te whānuitanga o te 1 nm ki te 100 nano. Nā te iti o te wātea o ngā paturopi i te wā o te whakamātautau, he iti noa ngā whakamātautau i mahia me ia kukū oligosaccharide. Me mārama i konei he tino rerekē te tauhohenga a ētahi paturopi ki te oligosaccharide kotahi hei matū, nā te mea pea ka herea ki ngā epitope rerekē, ā, ka rerekē pea ngā hononga here. Ka tino uaua ake ngā tikanga me ngā pānga o te tautuhi epitope tika ina whakamahia te huarahi mAb hou ki ngā tauira tūturu.
E rua ngā oligosaccharides arumoni i whakamahia hei whakatau i te whānuitanga o te kitenga o ngā momo mAb e aro ana ki te glycan. I konei, ko ngā hononga raina me te kukū log o te kukū oligosaccharide e tohu ana i ngā tauira adsorption Langmuir mō (A) XHE me te mAb me (B) A2XX me te mAb. Ko ngā epitopes e rite ana e tohu ana i ngā hanganga o ngā oligosaccharides arumoni i whakamahia hei substrates i roto i te whakamātautau.
He taputapu kaha te whakamahinga o ngā paturopi monoclonal e arohia ana e te glycan (tātari glycocomic, tātari mAb rānei e hangai ana ki te ELISA) mō te whakaahuatanga hōhonu o te nuinga o ngā glycan pakitara pūtau matua e hanga ana i te koiora tipu. Heoi, ko te tātari glycan matarohia ka whakaahua noa i ngā glycan pakitara pūtau nui ake, nā te mea kāore te nuinga o ngā oligosaccharides e mau tonu ana i runga i ngā pereti ELISA. I roto i tēnei rangahau, i whakakōhuatia te oumu kānga i mua i te maimoatanga o te AFEX i te nui o ngā totoka. I whakamahia te tātari huka hei whakatau i te hanganga o ngā warowaihā pakitara pūtau uaua i roto i te hydrolyzate. Heoi, kāore i te tino arohia te tātari mAb o ngā oligosaccharides iti ake i roto i ngā hydrolysates, ā, me whai taputapu atu hei whakakōhua ​​i ngā oligosaccharides i runga i ngā pereti ELISA.
Ka ripoatahia e mātou i konei tētahi tikanga hou, whai hua hoki mō te whakapūmau i te oligosaccharide mō te tirotiro mAb mā te whakakotahi i te oligosaccharide biotinylation, ā, ka whai muri ko te tirotiro ELISA i runga i ngā pereti kua pania ki te NeutrAvidin™. Ko ngā oligosaccharides biotinylated kua whakapūmautia i whakaatu i te kaha o te hononga ki te paturopi kia taea ai te kimi tere me te whai hua i ngā oligosaccharides uaua. Ko te tātari i te hanganga o ēnei oligosaccharides uaua i runga i te spectrometry papatipu i whakaū i ngā hua o tēnei huarahi hou ki te tirotiro ārai mate. Nō reira, e whakaatu ana ēnei rangahau ka taea te whakamahi i te whakakotahitanga o te oligosaccharide biotinylation me te tirotiro ELISA me ngā paturopi monoclonal kua arotahi ki te glycan hei kimi i ngā hononga whakawhiti i roto i ngā oligosaccharides, ā, ka taea te whakamahi whānuitia i roto i ētahi atu rangahau matū koiora e whakaatu ana i te hanganga o ngā oligosaccharides.
Ko tēnei tikanga tātari glycan e ahu mai ana i te biotin te pūrongo tuatahi e āhei ana ki te tirotiro i ngā here warowaihā uaua o ngā oligosaccharides wairewa i roto i te koiora tipu. Mā tēnei ka āwhina i te mārama he aha i uaua ai ētahi wāhanga o te koiora ina tae mai ki te hanga wahie koiora. Ka whakakīia e tēnei tikanga tētahi āputa nui i roto i ngā tikanga tātari glycome, ā, ka whakawhānui ake i tana tono ki tētahi whānuitanga o ngā matūriki i tua atu i ngā oligosaccharides tipu. I te heke mai, ka whakamahia pea e mātou te karetao mō te biotinylation me te whakamahi i te tikanga kua whakawhanakehia e mātou mō te tātari tere-rahi o ngā tauira mā te whakamahi i te ELISA.
I hauhaketia te kakau witi kānga (CS) i whakatipuria mai i ngā purapura ranu Pioneer 33A14 i te tau 2010 mai i Kramer Farms i Ray, Colorado. Me te whakaaetanga a te kaipupuri whenua, ka taea te whakamahi i tēnei koiora mō ngā rangahau. I rongoatia ngā tauira i roto i ngā putea raka-whiri maroke i te pāmahana rūma, me te kore e neke ake i te 6% te makuku. I rongoatia ngā tauira i roto i ngā putea raka-whiri maroke i te pāmahana rūma, me te kore e neke ake i te 6% te makuku. Образцы хранились сухими при влажности < 6% ki te пакетах с застежкой-молнией при комнатной температуре. I rongoatia ngā tauira kia maroke i te haumākū <6% i roto i ngā pēke kua herea ki te reti i te pāmahana rūma.样品在室温下以干燥< 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%. Ka rongoatia ngā tauira ki roto i ngā putea reti i te pāmahana rūma me te haumākū < 6%.I tutuki te rangahau i ngā aratohu ā-rohe me ā-motu. I whakahaerehia te tātaritanga hanganga mā te whakamahi i te kawa NREL. I kitea kei roto i te hanganga he 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% pūmua me te 13.4% pungarehu.
Ko te Cellic® CTec2 (138 mg pūmua/ml, puranga VCNI 0001) he ranunga matatini o te cellulase, te β-glucosidase me te Cellic® HTec2 (157 mg pūmua/ml, puranga VHN00001) mai i Novozymes (Franklinton, NC, USA)). Ko te Multifect Pectinase® (72 mg pūmua/mL), he ranunga matatini o ngā whākōkī e whakaiti ana i te pectin, i kohaina e DuPont Industrial Biosciences (Palo Alto, CA, USA). I ​​whakatauhia ngā kukū pūmua whākōkī mā te whakatau tata i te ihirangi pūmua (me te tango i te takoha o te hauota kore-pūmua) mā te whakamahi i te tātari hauota Kjeldahl (tikanga AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). I ​​hokona te oneone diatomaceous 545 mai i EMD Millipore (Billerica, MA). I hokona mai i Sigma-Aldrich (St. Louis, MO) te waro whakahohe (DARCO, 100 mesh granules), Avicel (PH-101), te beech xylan, me ngā matū katoa.
I whakahaerehia te maimoatanga-mua o te AFEX i te GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). I ​​whakahaerehia te maimoatanga-mua i te 140° C mō te 15 meneti. 46 meneti te roa o te noho i te ōwehenga 1:1 o te haukini waikore ki te koiora i te 60% (w/w) te uta i roto i te tauhohenga puranga papa-papa kore waikura (Parr Instruments Company). E 30 meneti te roa. I kawea te tauhohenga ki te 140°C, ā, i tukuna wawetia te haukini, kia tere ai te hoki o te koiora ki te pāmahana rūma. He rite te hanganga o te oumu kānga kua tukatukahia-mua o te AFEX (ACS) ki te oumu kānga kāore i tukatukahia (UT-CS).
I whakaritea ngā totoka teitei ACSH 25% (w/w) (tata ki te 8% te utaina o te dextran) hei rauemi tīmatanga mō te hanga oligosaccharides nui. I mahia te hydrolysis whākōkī o te ACS mā te whakamahi i tētahi ranunga whākōkī arumoni tae atu ki te Cellic® Ctec2 10 mg pūmua/g glucan (i roto i te koiora kua tukatukahia), Htec2 (Novozymes, Franklinton, NC), 5 mg pūmua/g glucan, me te Multifect Pectinase (Genencor Inc, USA). ), 5 mg pūmua/g dextran. I mahia te hydrolysis whākōkī i roto i tētahi bioreactor 5-rita me te rōrahi mahi o te 3 rita, pH 4.8, 50°C me te 250 rpm. I muri i te hydrolysis mō te 96 hāora, i kohia te hydrolyzate mā te centrifugation i te 6000 rpm mō te 30 meneti, kātahi ka kohia i te 14000 rpm mō te 30 meneti hei tango i ngā totoka kāore i hydrolyzed. Kātahi ka tātarihia te waikura mā roto i tētahi kapu tātari 0.22 mm. I rongoatia te waikura kua tātarihia ki roto i ngā pounamu waikura i te pāmahana 4° C, kātahi ka whakawehewehea ki te waro.
Te tātari i te hanganga o ngā tauira koiora i ahu mai i te tangohanga e ai ki ngā tikanga tātari taiwhanga NREL: te whakarite tauira mō te tātari hanganga (NREL/TP-510-42620) me te whakatau i ngā warowaihā hanganga me te lignin i roto i te koiora (NREL/TP-510 – 42618)47.
I whakahaerehia te tātaritanga oligosaccharide o te awa hydrolyzate i runga i te tauine 2 ml mā te whakamahi i te tikanga hydrolysis waikawa e ahu mai ana i te autoclave. Whakaranua te tauira hydrolyzate ki te 69.7 µl o te waikawa whanariki 72% i roto i te ngongo ahurea taupoki tīwiri 10 ml ka whaowhia mō te 1 hāora i runga i te papa mahi i te 121 °C, whakamataohia ki runga i te hukapapa ka tātarihia ki roto i te ipu chromatography wai mahi teitei (HPLC). I whakatauhia te kukū o ngā oligosaccharides mā te tango i te kukū o ngā monosaccharides i roto i te tauira kāore i hydrolyzed mai i te kukū huka katoa i roto i te tauira hydrolyzed-waikawa.
I tātarihia ngā kukū hukahuka, xylose, me te arabinose i roto i te koiora waikawa kua waikurahia mā te whakamahi i tētahi pūnaha Shimadzu HPLC kua rite ki tētahi autosampler, he whakamahana pou, he papu isocratic, me tētahi pūoko taupū whakaata i runga i tētahi pou Bio-Rad Aminex HPX-87H. I puritia te pou i te 50°C, ā, i whakawāteahia ki te 0.6 ml/min 5 mM H2SO4 i roto i te wai.
I whakawaiwaitia te wai i roto i te wai hydrolyzate, ā, i tātarihia te ihirangi o te monomer me te oligosaccharide. Ko ngā huka monomeric i whiwhihia i muri i te wai enzymatic i tātarihia mā te HPLC me te pou Bio-Rad (Hercules, CA) Aminex HPX-87P me te pou tiaki pungarehu. I puritia te pāmahana o te pou i te 80°C, i whakamahia te wai hei wāhanga neke me te tere rere o te 0.6 ml/min. I whakatauhia ngā oligosaccharides mā te wai hydrolysis i roto i te waikawa wai i te 121°C e ai ki ngā tikanga kua whakaahuatia i roto i ngā tohutoro 41, 48, 49.
I whakahaerehia te tātaritanga saccharide ki runga i ngā toenga koiora mata, kua tukatukahia i mua i te AFEX me ngā toenga koiora katoa kāore i te waikurahia (tae atu ki te whakaputa i ngā tangohanga pakitara pūtau raupapa me tā rātou tirotiro mAb) mā te whakamahi i ngā tikanga kua whakaahuahia i mua 27, 43, 50, 51. Mō te tātaritanga glycome, ka whakaritea ngā toenga kore-waipiro o te rauemi pakitara pūtau tipu mai i ngā toenga koiora, ā, ka tukuna ki te tangohanga raupapa me ngā tauhohenga kaha haere pērā i te ammonium oxalate (50 mM), te konutai kāponāte (50 mM me te 0.5% w/v), CON. (1M me te 4M, e rua me te 1% w/v konutai borohydride) me te waikawa chlorite e ai ki te whakaahuatanga i mua 52,53. Kātahi ka tukuna ngā tangohanga ki te ELISA ki tētahi papa matatini o ngā mAb50 i tukuna ki te glycan pakitara pūtau, ā, ka whakaatuhia ngā tauhohenga here mAb hei mahere wera. I hokona ngā mAb e aro ana ki te glycan pakitara pūtau tipu mai i ngā taonga taiwhanga (CCRC, JIM me te raupapa MAC).
Te whakapūmautanga kotahi-taahiraa o ngā oligosaccharides. I whakahaerehia te hononga o ngā warowaihā me te biotin-LC-hydrazide mā te whakamahi i te tikanga e whai ake nei. I whakarewaina te Biotin-LC-hydrazide (4.6 mg/12 μmol) ki roto i te dimethyl sulfoxide (DMSO, 70 μl) mā te whakaoho kaha me te whakamahana i te 65° C mō te 1 meneti. I tāpirihia te waikawa acetic glacial (30 µl) ā, i ringihia te ranunga ki runga i te konutai cyanoborohydride (6.4 mg/100 µmol) ā, i whakarewaina katoatia i muri i te whakamahana i te 65° C mō te 1 meneti pea. Kātahi ka tāpirihia mai i te 5 ki te 8 μl o te ranunga tauhohenga ki te oligosaccharide maroke (1-100 nmol) kia whiwhi ai i te 10-ngā wā, neke atu rānei te taikaha molar o te tapanga ki runga i te pito whakaiti. I whakahaerehia te tauhohenga i te 65°C mō te 2 hāora, ā, i muri iho ka purea tonutia ngā tauira. Kāore i whakamahia te konutai cyanoborohydride i roto i ngā whakamātautau tapanga me te kore whakaiti, ā, i tauhohehia ngā tauira i te 65° C mō ngā haora 2.5.
Te paninga ELISA me te horoinga o ngā tauira o ngā oligosaccharides biotinylated. E 25 μl o ngā tauira biotinylated (100 μl o ia tauira kukū i whakaranua ki te 5 ml o te 0.1 M Tris buffer solution (TBS)) i tāpirihia ki ia poka o te pereti kua pania ki te avidin. I pania ngā poka whakahaere ki te 50 μl o te biotin i te kukū o te 10 μg/ml i roto i te 0.1 M TBS. I whakamahia te wai tango-ion hei paninga mō ngā inenga kau. I whakatōkia te papa mō ngā hāora e 2 i te pāmahana rūma i roto i te pōuri. Horoia te pereti kia 3 ngā wā ki te miraka ngako 0.1% i roto i te 0.1 M TBS mā te whakamahi i te kaupapa nama 11 mō Grenier flat 3A.
Te tāpiri me te horoi i ngā paturopi tuatahi. Tāpirihia he 40 µl o te paturopi tuatahi ki ia poka. Whakatōkia te pereti iti mō te 1 hāora i te pāmahana rūma i roto i te pōuri. Kātahi ka horoia ngā pereti e 3 ngā wā ki te miraka 0.1% i roto i te 0.1M TBS mā te whakamahi i te kaupapa horoi #11 mō Grenier Flat 3A.
Tāpirihia he paturopi tuarua, ka horoia. Tāpirihia he 50 µl o te paturopi tuarua kiore/kiore (kua whakaranua ki te 1:5000 i roto i te miraka 0.1% i roto i te 0.1 M TBS) ki ia poka. Whakatōkia te pereti iti mō te 1 hāora i te pāmahana rūma i roto i te pōuri. Kātahi ka horoia ngā pereti iti e 5 ngā wā ki te miraka 0.1% i roto i te 0.1 M TBS mā te whakamahi i te kaupapa horoi pereti Grenier Flat 5A #12.
Te tāpiri i tētahi papa. Tāpirihia he 50 µl o te 3,3′,5,5′-tetramethylbenzidine (TMB) ki te papa turanga (mā te tāpiri i te 2 pata o te pūwero, te 3 pata o te TMB, te 2 pata o te hauwai waikura ki te 15 ml o te wai whakakore iona). Whakaritea te papa TMB. me te vortex i mua i te whakamahinga). Whakatōkia te pereti iti ki te pāmahana rūma mō te 30 meneti. I te pōuri.
Whakaotia te taahiraa ka pānui i te papa. Tāpirihia he 50 µl o te 1 N waikawa whanariki ki ia poka ka tuhia te mimiti mai i te 450 ki te 655 nm mā te whakamahi i te kaipānui ELISA.
Whakapaia ngā otinga 1 mg/ml o ēnei tātaritanga ki roto i te wai whakakore iona: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine (glcNAc), N-acetylgalactosamine (galNAc), inositol (paerewa ā-roto). E rua ngā paerewa i whakaritea mā te tāpiri i ngā otinga huka 1 mg/mL e whakaaturia ana i te Ripanga 1. Ka tiohia ngā tauira, ka whakamataohia ki te -80° C tae noa ki te tangohia katoatia o te wai (ko te tikanga mō te 12-18 hāora).
Tāpirihia he 100–500 µg o te tauira ki ngā ngongo taupoki tīwiri i runga i te pauna tātari. Tuhia te nui i tāpirihia. He pai ake te whakarewa i te tauira ki tētahi kukū motuhake o te whakarewa ka tāpiri ki te ngongo hei wai whakapūmau. Whakamahia he 20 µl o te 1 mg/ml inositol hei paerewa ā-roto mō ia ngongo tauira. Me rite te nui o te paerewa ā-roto i tāpirihia ki te tauira ki te nui o te paerewa ā-roto i tāpirihia ki te ngongo paerewa.
Tāpirihia he 8 ml o te methanol kore wai ki roto i te ipu taupoki tīwiri. Kātahi ka tāpirihia he 4 ml o te 3 N o te otinga methanolic HCl, taupokina, ka rūrūtia. Kāore tēnei tukanga e whakamahi i te wai.
Tāpirihia he 500 µl o te otinga methanol 1 M HCl ki ngā tauira oligosaccharide me ngā ngongo TMS paerewa. I whaowhia ngā tauira mō te pō (168 hāora) i te 80° C. i roto i te poraka wera. Whakamarokehia te hua methanolysis i te pāmahana rūma mā te whakamahi i te manifold whakamaroke. Tāpirihia he 200 µl MeOH ka whakamaroke anō. E rua ngā wā ka whakahokia tēnei tukanga. Tāpirihia he 200 µl o te methanol, he 100 µl o te pyridine me te 100 µl o te acetic anhydride ki te tauira ka whakaohokia kia pai. Whakawhaowhia ngā tauira i te pāmahana rūma mō te 30 meneti. ka whakamaroke. Tāpirihia he 200 µl o te methanol ka whakamaroke anō.
Tāpirihia he 200 µl o Tri-Sil ka whakamahana i te ngongo kua taupokina mō te 20 meneti. 80°C, kātahi ka whakamatao ki te pāmahana rūma. Whakamahia he mīhini whakamaroke hei whakamaroke anō i te tauira kia tae ki te rahinga tata ki te 50 µl. He mea nui kia mōhio kāore mātou i tuku kia maroke rawatia ngā tauira.
Tāpirihia kia 2 ml o te hexane, ka whakaohokia kia pai mā te vortexing. Whakakīia ngā pito o ngā pipettes Pasteur (5-8 mm) ki tētahi wahi huruhuru karāhe mā te whakauru i te huruhuru karāhe ki runga i tētahi pipettes 5-3/4 inihi te whānui. I whakatōngia ngā tauira ki te 3000 karamu mō te 2 meneti. Ka whakatōngia ngā toenga kore e rewa. Whakamaroketia te tauira ki te 100-150 µl. I werohia he rōrahi tata ki te 1 μl ki roto i te GC-MS i te pāmahana tīmatanga o te 80 °C me te wā tīmatanga o te 2.0 meneti (Ripanga 2).