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ʻO nā ala hou immunological a me nā ʻano spectrometric nui no ka nānā ʻana paʻakikī o nā oligosaccharides mau loa i loko o ka stover kulina i mālama mua ʻia me AFEX.ʻO ka biomass Lignocellulosic kahi mea hoʻomau i nā wahie mōʻalihaku a hoʻohana nui ʻia e hoʻomohala i nā biotechnologies no ka hana ʻana i nā huahana e like me ka meaʻai, ka meaʻai, nā wahie a me nā kemika.ʻO ke kī o kēia mau ʻenehana ʻo ia ka hoʻomohala ʻana i nā kaʻina hana hoʻokūkū no ka hoʻololi ʻana i nā ʻakika paʻakikī i loaʻa i loko o nā paia o nā mea kanu i nā kō maʻalahi e like me ka glucose, xylose a me ka arabinose.Ma muli o ka paʻakikī loa o ka biomass lignoselulosic, pono e hoʻokō ʻia i nā lāʻau thermochemical (e laʻa, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic liquids (IL)) a me nā lāʻau lapaʻau (eg, enzymatic hydrolysis and microbial fermentation) i hui pū ʻia e loaʻa ai ka huahana i makemake ʻia..Eia nō naʻe, ke hoʻohana ʻia nā enzyme fungal pāʻoihana i ke kaʻina hydrolysis, ʻo 75-85% wale nō o nā sugars soluble i hana ʻia he monosaccharides, a ʻo ke koena 15-25% he soluble, intractable oligosaccharides, ʻaʻole i loaʻa mau i nā microorganisms.Ma mua, ua hoʻokaʻawale maikaʻi mākou a hoʻomaʻemaʻe i nā oligosaccharides paʻakikī me ka hoʻohana ʻana i ke kalapona a me ka diatomaceous honua kaʻawale a me ka nui hoʻokaʻawale chromatography, a ua noiʻi pū i kā lākou enzyme inhibitory waiwai.Ua ʻike mākou he ʻoi aku ka paʻakikī o ka hoʻoponopono ʻana i nā oligosaccharides i loaʻa i ke kiʻekiʻe kiʻekiʻe o ka polymerization (DP) methylated uronic acid substitutions me nā hui enzyme kalepa ma mua o ka DP haʻahaʻa a me nā oligosaccharides kūʻokoʻa.Ma ʻaneʻi, hōʻike mākou i ka hoʻohana ʻana i kekahi mau ʻano hana hou, e like me ka glycan profiling me ka hoʻohana ʻana i nā antibodies monoclonal (mAbs) kikoʻī e kanu i nā glycans biomass e hōʻike i nā paʻa glycan i loko o nā pā hale mea kanu a me nā hydrolysates enzymatic, matrix-assisted laser desorption ionization, time-of-flight mass-specttrometry..MALDI-TOF-MS) hoʻohana i ka structure-informative diagnostic peaks i loaʻa ma ka spectroscopy ma hope o ka pohō lua o nā ion maikaʻi ʻole, gas chromatography a me mass spectrometry (GC-MS) e hoʻohālikelike i nā paʻa oligosaccharide me ka ʻole o ka derivatization.Ma muli o ka liʻiliʻi o nā oligosaccharides (DP 4-20), paʻakikī kēia mau moleke e hoʻohana no ka mAb hoʻopaʻa a me ke ʻano.No ka lanakila ʻana i kēia pilikia, ua hoʻohana mākou i kahi ala oligosaccharide immobilization e pili ana i ka biotin conjugation hou i hoʻopaʻa inoa i ka hapa nui o nā oligosaccharides soluble DP haʻahaʻa ma ka microplate surface, a laila hoʻohana ʻia i kahi ʻōnaehana mAb throughput kiʻekiʻe no ka loiloi ligation kikoʻī.E maʻalahi kēia ʻano hana hou i ka hoʻomohala ʻana i nā hōʻike glycome high throughput kiʻekiʻe i ka wā e hiki mai ana i hiki ke hoʻohana ʻia e hoʻokaʻawale a ʻike i nā oligosaccharides i loaʻa i nā biomarkers no nā kumu diagnostic.
Lignocellulosic biomass, i haku ʻia me ka mahiʻai, ka ululāʻau, ka mauʻu a me nā mea lāʻau, he meaʻai kūpono no ka hana ʻana i nā huahana bio-based, me ka meaʻai, ka meaʻai, ka wahie a me nā mea mua e hana ai i nā huahana waiwai kiʻekiʻe.ʻO nā kalapona (e like me ka cellulose a me ka hemicellulose) i loaʻa i loko o nā paia o nā mea kanu i hoʻokaʻawale ʻia i monosaccharides e ka hana kemika a me ka biotransformation (e like me ka hydrolysis enzymatic a me ka microbial fermentation).ʻO nā hana maʻamau ma mua o ka hoʻonui ʻia ʻana o ka ammonia fiber (AFEX), dilute acid (DA), ka wai ionic (IL), a me ka pahū mahu (SE), e hoʻohana ana i ka hui pū ʻana o nā kemika a me ka wela e hōʻemi i ka hana lignoscellulose ma ka wehe ʻana i nā pā hale mea kanu3,4.'O ka pa'akikī o ka mea, 5. Ho'ohana 'ia ka hydrolysis enzymatic ma kahi ha'aha'a pa'a ki'eki'e me ka ho'ohana 'ana i nā enzymes i loko o ka carbohydrate active carbohydrate-containing enzymes (CAZymes) a me ka microbial fermentation me ka ho'ohana 'ana i nā hū transgenic a i 'ole bacteria e hana i nā wahie a me nā kinikini 6.
Hoʻokumu ʻia nā CAZymes i nā enzymes pāʻoihana me kahi hui ʻana o nā enzymes e hoʻokaʻawale i nā paʻa paʻa ʻana o ka carbohydrate-sugar paʻakikī e hana i nā monosaccharides2,7.E like me kā mākou i hōʻike mua ai, ʻo ka pūnaewele paʻakikī o nā polymers aromatic o ka lignin me nā kaʻa kaʻa e hiki ke hoʻololi ʻia, e alakaʻi ana i ka hoʻololi ʻana i ke kō, e hōʻiliʻili ana i ka 15-25% o nā oligosaccharides sex i hana ʻole ʻia i ka wā enzymatic hydrolysis o ka biomass pretreated.He pilikia maʻamau kēia me nā ʻano hana pretreatment biomass like ʻole.ʻO kekahi mau kumu o kēia bottleneck ʻo ia ka hoʻopaʻa ʻana i ka enzyme i ka wā hydrolysis, a i ʻole ka haʻahaʻa a i ʻole nā ​​pae haʻahaʻa o nā enzyme koʻikoʻi e pono ai e uhaʻi i nā paʻa kō i ka biomass mea kanu.ʻO ka hoʻomaopopo ʻana i ka haku mele ʻana a me nā hiʻohiʻona o nā kō, e like me nā paʻa kō i loko o ka oligosaccharides, e kōkua iā mākou e hoʻomaikaʻi i ka hoʻololi ʻana i ke kō i ka wā hydrolysis, e hoʻokūkū i nā kaʻina biotechnological me nā huahana i loaʻa i ka aila.
He mea paʻakikī ka hoʻoholo ʻana i ke ʻano o nā ʻakika a pono e hui pū ʻia nā ʻano e like me ka chromatography wai (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 a me mass spectrometry (MS)17.,ʻumikumāwalu.ʻO nā ʻano MS e like me ka time-of-flight mass spectrometry me ka laser desorption a me ka ionization me ka hoʻohana ʻana i kahi matrix (MALDI-TOF-MS) kahi ala maʻalahi no ka ʻike ʻana i nā hale kaʻa.I kēia mau lā, ua hoʻohana nui ʻia ʻo Collision-Induced Dissociation (CID) tandem MS o ka sodium ion addducts e ʻike i nā manamana lima e pili ana i nā kūlana hoʻopili oligosaccharide, nā hoʻonohonoho anomeric, nā kaʻina, a me nā kūlana lālā 20, 21.
He mea paahana maikaʻi loa ka ʻikepili ʻo Glycan no ka ʻike hohonu ʻana i nā mea paʻa kaʻapona22.Ke hoʻohana nei kēia ʻano i nā antibodies monoclonal (mAbs) i kuhikuhi ʻia e kanu i ka glycan wall cell ma ke ʻano he ʻimi no ka hoʻomaopopo ʻana i nā pilina waipika paʻakikī.Loaʻa ma mua o 250 mAbs ma ka honua holoʻokoʻa, i hoʻolālā ʻia e kūʻē i nā oligosaccharides linear a me nā lālā me ka hoʻohana ʻana i nā saccharides24.Ua hoʻohana nui ʻia kekahi mau mAbs e ʻike i ke ʻano, ka haku ʻana, a me nā hoʻololi ʻana o ka pā hale mea kanu, no ka mea, aia nā ʻokoʻa nui ma muli o ke ʻano o nā mea kanu, okana, makahiki, pae ulu, a me ka ulu ʻana25,26.I kēia mau lā, ua hoʻohana ʻia kēia ʻano hana no ka hoʻomaopopo ʻana i nā heluna vesicle i loko o nā ʻōnaehana mea kanu a me nā holoholona a me kā lākou mau kuleana i ka lawe ʻana i ka glycan e like me ka mea i hoʻoholo ʻia e nā māka subcellular, nā pae hoʻomohala, a i ʻole nā ​​mea hoʻoulu kaiapuni, a no ka hoʻoholo ʻana i ka hana enzymatic.ʻO kekahi o nā ʻano hana like ʻole o nā glycans a me nā xylans i ʻike ʻia ʻo ia ka pectin (P), xylan (X), mannan (M), xyloglucans (XylG), glucans hui pū ʻia (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan (GArbXgaan (GArbX) a me ka arabinoxylan (GArbX) a me ka arabinoxylan.
Eia nō naʻe, ʻoiai kēia mau hana noiʻi a pau, he liʻiliʻi wale nō nā noiʻi i kālele i ke ʻano o ka hōʻiliʻili ʻana o ka oligosaccharide i ka wā o ka hydrolysis high solids load (HSL), me ka hoʻokuʻu ʻana o ka oligosaccharide, hoʻololi ka lōʻihi o ke kaulahao oligomeric i ka wā o ka hydrolysis, nā polymers haʻahaʻa haʻahaʻa haʻahaʻa, a me ko lākou mau pihi.puunaue 30,31,32.Eia nō naʻe, ʻoiai ua hōʻike ʻia ka loiloi glycan he mea pono no ka nānā ʻana i ka ʻōnaehana glycan, paʻakikī ke loiloi i ka wai-soluble DP oligosaccharides me ka hoʻohana ʻana i nā ʻano antibody.ʻO nā oligosaccharides DP liʻiliʻi me ke kaumaha mole ma lalo o 5-10 kDa ʻaʻole e hoʻopaʻa i nā papa ELISA 33, 34 a holoi ʻia ma mua o ka hoʻohui ʻana i ka antibody.
Maʻaneʻi, no ka manawa mua, hōʻike mākou i kahi hōʻike ELISA ma nā papa avidin-coated me ka hoʻohana ʻana i nā antibodies monoclonal, e hui pū ana i kahi kaʻina biotinylation hoʻokahi no ka soluble refractory oligosaccharides me ka nānā ʻana i ka glycome.ʻO kā mākou hoʻokokoke ʻana i ka nānā ʻana i ka glycome ua hōʻoia ʻia e MALDI-TOF-MS a me GC-MS ma muli o ka nānā ʻana o nā loulou oligosaccharide hoʻohui me ka hoʻohana ʻana i ka trimethylsilyl (TMS) derivatization o nā haku mele hydrolyzed.Hiki ke hoʻomohala ʻia kēia ʻano hana hou ma ke ʻano he ʻano hana kiʻekiʻe i ka wā e hiki mai ana a loaʻa ka noi ākea ma ka noiʻi biomedical35.
Hoʻololi ka hoʻololi ʻana ma hope o ka unuhi ʻana o nā enzymes a me nā antibodies, e like me ka glycosylation,36 i kā lākou hana olaola.No ka la'ana, he mea nui ka ho'ololi 'ana i ka glycosylation o nā protein serum i ka arthritis inflammatory, a ho'ohana 'ia nā loli i ka glycosylation ma ke 'ano he diagnostic markers37.Ua hōʻike ʻia nā glycans like ʻole i loko o ka palapala e ʻike koke ʻia i nā maʻi like ʻole, e like me nā maʻi inflammatory mau o ka ʻōpū a me ka ate, nā maʻi viral, ovarian, umauma, a me nā maʻi maʻi prostate38,39,40.ʻO ka hoʻomaopopo ʻana i ke ʻano o nā glycans me ka hoʻohana ʻana i nā ʻano glycan ELISA e pili ana i ka antibody e hāʻawi i ka hilinaʻi hou i ka ʻike maʻi me ka ʻole o ka hoʻohana ʻana i nā ʻano MS paʻakikī.
Ua hōʻike ʻia kā mākou noiʻi mua ʻana o ka oligosaccharides paʻakikī i noho ʻole i ka hydrolyzed ma hope o ka pretreatment a me ka hydrolysis enzymatic (Figure 1).Ma kā mākou hana i paʻi mua ʻia, ua hoʻomohala mākou i kahi ʻano hoʻoheheʻe ʻana i ka lānahu ikaika e hoʻokaʻawale i nā oligosaccharides mai ka AFEX-pretreated corn stover hydrolyzate (ACSH)8.Ma hope o ka unuhi mua ʻana a me ka hoʻokaʻawale ʻana, ua hoʻokaʻawale hou ʻia nā oligosaccharides e ka nui exclusion chromatography (SEC) a hōʻiliʻili ʻia ma ke ʻano o ke kaumaha molekala.Ua kālailai ʻia nā monomer kō a me nā oligomer i hoʻokuʻu ʻia mai nā ʻano hana mua e ka nānā ʻana i ka haku mele.I ka hoʻohālikelike ʻana i ka ʻike o nā oligomers sugar i loaʻa i nā ʻano hana pretreatment, ʻo ka hele ʻana o nā oligosaccharides paʻakikī he pilikia maʻamau i ka hoʻololi ʻana o ka biomass i monosaccharides a hiki ke alakaʻi i ka hōʻemi ʻana o ke kō i ka liʻiliʻi o 10-15% a hiki i 18%.US.Hoʻohana ʻia kēia ʻano hana no ka hana nui ʻana o nā hakina oligosaccharide.Ua hoʻohana ʻia ka ACH i loaʻa a me kāna mau hakina hope me nā kaupaona molekala like ʻole ma ke ʻano he mea hoʻokolohua no ke ʻano o ka oligosaccharides i kēia hana.
Ma hope o ka hoʻomaʻamaʻa mua ʻana a me ka hydrolysis enzymatic, hoʻomau mau ʻia ka oligosaccharides me ka wai ʻole.Eia (A) he ʻano hoʻokaʻawale oligosaccharide kahi i hoʻokaʻawale ʻia ai nā oligosaccharides mai ka AFEX-pretreated corn stover hydrolyzate (ACSH) me ka hoʻohana ʻana i kahi moena i hoʻopaʻa ʻia o ka carbon activated a me ka honua diatomaceous;(B) Ke ala no ka hoʻokaʻawale ʻana i nā oligosaccharides.Ua hoʻokaʻawale hou ʻia nā oligosaccharides e ka nui exclusion chromatography (SEC);(C) Hoʻokuʻu ʻia nā monomers saccharide a me nā oligomer mai nā ʻano hana mua (ka wai i hoʻoheheʻe ʻia: DA, wai ionic: IL a me AFEX).Nā kūlana hydrolysis Enzymatic: hoʻouka ʻia nā mea paʻa kiʻekiʻe o 25% (w/w) (ma kahi o 8% hoʻouka glucan), 96 mau hola hydrolysis, 20 mg/g hoʻouka ʻana i nā enzyme kalepa (Ctec2:Htec2:MP-2:1:1 ratio) a me (D) Sugar monomers a me oligomers o ka glucose, xylose a me ka arabinose-hoʻokuʻu ʻia mai AFEX prestated AFEX .
Ua hōʻike ʻia ʻo Glycan analysis he mea hana pono no ka nānā ʻana i nā glycans i nā ʻāpana i hoʻokaʻawale ʻia mai nā koena biomass paʻa.Eia naʻe, ʻaʻole i hōʻike ʻia nā saccharides hiki ke hoʻoheheʻe ʻia i ka wai me ka hoʻohana ʻana i kēia ʻano hana kuʻuna41 no ka mea paʻakikī nā oligosaccharides kaumaha haʻahaʻa haʻahaʻa i ka immobilize ma nā pā ELISA a holoi ʻia ma mua o ka hoʻohui ʻana i ka antibody.No laila, no ka hoʻopaʻa ʻana i ka antibody a me ka hōʻike ʻana, ua hoʻohana ʻia kahi ʻano biotinylation hoʻokahi ʻanuʻu e kāhiko i nā oligosaccharides hiki ke hoʻoheheʻe ʻole ʻia ma nā papa ELISA i uhi ʻia avidin.Ua hoʻāʻo ʻia kēia ʻano me ka hoʻohana ʻana i kā mākou ACSH i hana mua ʻia a me kahi hakina e pili ana i kona kaumaha molekala (a i ʻole degere o ka polymerization, DP).Hoʻohana ʻia ka biotinylation hoʻokahi-step e hoʻonui i ka pilina paʻa oligosaccharide ma ka hoʻohui ʻana i ka biotin-LC-hydrazide i ka hopena hoʻemi o ka carbohydrate (Fig. 2).I ka hoʻonā, ka hui hemiacetal ma ka hopena hoʻohaʻahaʻa e hana me ka hui hydrazide o biotin-LC-hydrazide e hana i kahi paʻa hydrazone.Ma ke alo o ka mea ho'ēmi NaCNBH3, ua ho'ēmiʻia ka paʻa hydrazone i kahi huahana hopena biotinylated paʻa.Me ka hoʻololi ʻana o ke kō e hoʻemi i ka hopena, hiki ke hoʻopaʻa ʻia nā oligosaccharides DP haʻahaʻa i nā papa ELISA, a i kā mākou aʻo ʻana ua hana ʻia kēia ma nā papa avidin-coated me ka hoʻohana ʻana i nā mAbs glycan-targeted.
Ka nānā ʻana o nā antibodies monoclonal e pili ana i ka ELISA no nā oligosaccharides biotinylated.Ma ʻaneʻi (A) hui pū ʻia ka biotinylation o nā oligosaccharides a me ka nānā ʻana ʻo ELISA ma hope me nā mAbs glycan-targeted ma nā papa i uhi ʻia ʻo NeutrAvidin a (B) hōʻike i kahi kaʻina hana hoʻokahi no ka biotinylation o nā huahana hopena.
Ua hoʻohui ʻia nā pāpaʻi i uhi ʻia ʻo Avidin me nā antibodies oligosaccharide-conjugated i nā antibodies mua a me ka lua a holoi ʻia i kahi ʻano māmā a me ka manawa.Ma hope o ka pau ʻana o ka hoʻopaʻa ʻana i ka antibody, e hoʻohui i ka substrate TMB e hoʻoulu i ka pā.Ua pau ka hopena me ka sulfuric acid.Ua kālailai ʻia nā papa incubated e hoʻohana ana i ka mea heluhelu ELISA e hoʻoholo i ka ikaika paʻa o kēlā me kēia antibody e ʻike i ka hoʻopili ʻana i ka antibody-specific cross-linking.No nā kikoʻī a me nā ʻāpana o ka hoʻokolohua, e ʻike i ka ʻāpana pili "Nā Mea a me nā Hana".
Hōʻike mākou i ka pono o kēia ʻano hana hou i kūkulu ʻia no nā noi kikoʻī ma ke ʻano o nā oligosaccharides soluble i loaʻa ma ACSH a me nā hakina oligosaccharide crude a hoʻomaʻemaʻe ʻia i hoʻokaʻawale ʻia mai nā lignoscellulosic hydrolysates.E like me ka mea i hōʻike ʻia ma ke Kiʻi 3, ʻo nā xylans i hoʻololi ʻia epitope maʻamau i ʻike ʻia ma ACSH me ka hoʻohana ʻana i nā ʻano hana assay glycome bioacylated he mau uronic (U) a i ʻole methyluronic (MeU) a me pectic arabinogalactans.Ua ʻike ʻia ka hapa nui o lākou i kā mākou noiʻi mua ʻana i ka nānā ʻana i nā glycans o nā mea paʻa ʻole hydrolyzed (UHS)43.
ʻO ka ʻike ʻana i nā epitopes oligosaccharide recalcitrant me ka hoʻohana ʻana i kahi antibody monoclonal i kuhikuhi ʻia i ka glycan wall cell.ʻO ka hakina "neutral" ka hakina ACN a me ka hakina "acidic" ka hakina FA.Hōʻike nā ʻulaʻula ʻulaʻula ma ka palapala wela i ka ʻike epitope kiʻekiʻe, a ʻo nā uliuli ʻoi aku ka maikaʻi e hōʻike i kahi kāʻei hakahaka.Hoʻokumu ʻia nā waiwai kala ma ka pālākiō i nā waiwai OD maka no nā formulations N=2.Hōʻike ʻia nā epitopes nui i ʻike ʻia e nā antibodies ma ka ʻākau.
ʻAʻole hiki ke hoʻokaʻawale ʻia kēia mau hale cellulose ʻole e nā cellulases maʻamau a me nā hemicellulases i hoʻāʻo ʻia i loko o ka hui ʻana o ka enzyme ʻoihana i hoʻāʻo ʻia, ʻo ia hoʻi nā enzyme ʻoihana i hoʻohana pinepine ʻia.No laila, koi ʻia nā enzyme kōkua hou no kā lākou hydrolysis.Me ka loaʻa ʻole o nā enzymes pono ʻole cellulose, ʻo kēia mau mea paʻa ʻole cellulose e pale i ka hoʻololi piha ʻana i nā monosaccharides, ʻoiai inā i hydrolyzed nui ʻia ko lākou mau mākua kō polymers i nā ʻāpana pōkole a hoʻoheheʻe ʻia me ka hoʻohana ʻana i nā hui enzyme kalepa.
ʻO ka noiʻi hou aʻe o ka hāʻawi ʻana i ka hōʻailona a me kona ikaika paʻa i hōʻike ʻia he haʻahaʻa ka haʻahaʻa o nā epitopes paʻa i nā hakina kōpaʻa DP kiʻekiʻe (A, B, C, DP a hiki i 20+) ma mua o nā haʻahaʻa DP haʻahaʻa (D, E, F, DP) i nā dimer) (Fig. 1).ʻOi aku ka maʻamau o nā ʻāpana ʻakika i nā epitopes non-cellulose ma mua o nā ʻāpana kū ʻole.Kūlike kēia mau ʻano me ke ʻano i ʻike ʻia ma kā mākou noiʻi mua, kahi i ʻoi aku ka kūʻē ʻana o ka DP kiʻekiʻe a me nā moieties acid i ka hydrolysis enzymatic.No laila, hiki i ka loaʻa ʻana o nā epitopes glycan non-cellulose a me nā pani U a me MeU hiki ke kōkua nui i ka paʻa o nā oligosaccharides.Pono e hoʻomaopopo ʻia he pilikia ka paʻa ʻana a me ka ʻike ʻana i nā oligosaccharides haʻahaʻa, ʻoiai inā he dimeric a trimeric oligosaccharide ka epitope.Hiki ke ho'āʻo ʻia kēia me ka hoʻohana ʻana i nā oligosaccharides pāʻoihana o nā lōʻihi like ʻole, aia i kēlā me kēia me hoʻokahi epitope e hoʻopaʻa ʻia i kahi mAb kikoʻī.
No laila, ua hōʻike ʻia ka hoʻohana ʻana i nā antibodies kikoʻī kikoʻī i kekahi mau ʻano o nā paʻa recalcitrant.Ma muli o ke ʻano o ka antibody i hoʻohana ʻia, ke ʻano ligation kūpono, a me ka ikaika o ka hōʻailona e hoʻopuka ai (ka hapa nui a me ka liʻiliʻi loa), hiki ke ʻike ʻia nā enzymes hou a hoʻohui ʻia i semi-quantitatively i ka hui enzyme no ka glycoconversion piha loa.Ke lawe nei i ka loiloi o ACSH oligosaccharides ma ke ʻano he laʻana, hiki iā mākou ke hana i kahi waihona o nā paʻa glycan no kēlā me kēia mea biomass.Pono e hoʻomaopopo ʻia ma ʻaneʻi e noʻonoʻo ʻia ka pili like ʻole o nā antibodies, a inā ʻaʻole ʻike ʻia ko lākou pili, e hana kēia i kekahi mau pilikia i ka hoʻohālikelike ʻana i nā hōʻailona o nā antibodies like ʻole.Eia kekahi, ʻoi aku ka maikaʻi o ka hoʻohālikelike ʻana i nā paʻa glycan ma waena o nā laʻana no ka antibody like.Hiki ke hoʻopili ʻia kēia mau mea paʻa paʻa i ka waihona CAZyme, kahi e hiki ai iā mākou ke ʻike i nā enzymes, koho i nā enzymes moho a hoʻāʻo no nā enzymes wawahi paʻa, a i ʻole hoʻomohala i nā ʻōnaehana microbial e hōʻike i kēia mau enzymes no ka hoʻohana ʻana i nā biorefineries44.
No ka loiloi pehea e hoʻokō ai nā ʻano immunological i nā ʻano hana ʻē aʻe no ka hoʻohālikelike ʻana i nā oligosaccharides kaumaha haʻahaʻa i loaʻa i loko o ka lignoscellulosic hydrolysates, ua hana mākou MALDI (Fig. 4, S1-S8) a me ka nānā ʻana o nā saccharides i loaʻa mai i ka TMS ma muli o GC-MS ma ka papa like (Fig. 5) oligosaccharide hapa.Hoʻohana ʻia ʻo MALDI e hoʻohālikelike inā pili ka hoʻohele nui o nā molekala oligosaccharide i ke ʻano i manaʻo ʻia.Ma ka fig.Hōʻike ka 4 i ka MC o nā ʻāpana kū ʻole ACN-A a me ACN-B.Ua hōʻoia ʻia ka loiloi ACN-A i kahi ʻano o ka pentose sugars mai DP 4-8 (Fig. 4) a i DP 22 (Fig. S1), nona nā paona e like me MeU-xylan oligosaccharides.Ua hōʻoia ʻia ka loiloi ACN-B i ka pūʻulu pentose a me glucoxylan me DP 8-15.Ma nā mea hoʻohui e like me ke Kiʻi S3, hōʻike ʻia nā palapala ʻāina hoʻohele nui o FA-C acidic moiety mass distribution i kahi ʻano o (Me)U i pani ʻia nā pentose sugars me kahi DP o 8-15 i kūlike me nā xylans i hoʻololi ʻia i loaʻa ma ELISA-based mAb screening.Kūlike nā epitopes.
MALDI-MS spectrum of soluble non-compliant oligosaccharides i loaʻa ma ACS.Maʻaneʻi, (A) ACN-A nā hakina haʻahaʻa i loaʻa i ka methylated uronic acid (DP 4-8) i pani i ka glucuroxylan oligosaccharides a me (B) ACN-B xylan a me ka methylated uronic acid oligosaccharides i pani ʻia me ka glucuroxylan (DP 8-15).
Ka nānā ʻana o ka haku mele ʻana o ke koena glycan o nā oligosaccharides refractory.Ma ʻaneʻi (A) TMS saccharide haku mele o nā hakina oligosaccharide like ʻole i loaʻa me ka hoʻohana ʻana i ka loiloi GC-MS.(B) Nā hoʻolālā o nā kō i loaʻa mai i ka TMS i loaʻa i nā oligosaccharides.ACN – ʻāpana acetonitrile i loaʻa nā oligosaccharides kūʻokoʻa a me FA - ʻāpana ʻakika ferulic i loaʻa nā oligosaccharides waikawa.
ʻO kekahi hopena hoihoi i huki ʻia mai ka loiloi LC-MS o ka hakina oligosaccharide, e like me ka mea i hōʻike ʻia ma ke Kiʻi S9 (hiki ke ʻike ʻia nā ʻano ma ka mea hoʻohui uila).Ua nānā mau ʻia nā ʻāpana o nā hui hexose a me -OAc i ka wā ligation o ka hakina ACN-B.ʻAʻole wale kēia ʻike e hōʻoia i ka ʻāpana i ʻike ʻia ma ka nānā ʻana o ka glycome a me MALDI-TOF, akā hāʻawi pū kekahi i ka ʻike hou e pili ana i nā derivatives carbohydrate i loko o ka biomass lignoscellulosic pretreated.
Ua kālailai pū mākou i ke kō o ka hakina oligosaccharide me ka hoʻohana ʻana i ke kōpaʻa TMS derivatization.Ke hoʻohana nei i ka GC-MS, ua hoʻoholo mākou i ka hoʻohui ʻana o nā neural (non-derivative) a me nā sugars acidic (GluA a me GalA) i ka hakina oligosaccharide (Fig. 5).Loaʻa ka ʻakika Glucuronic i loko o nā mea ʻakika C a me D, ʻoiai ʻo ka galacturonic acid i loaʻa i loko o nā ʻāpana acidic A a me B, ʻo ia mau mea ʻelua he mau mea DP kiʻekiʻe o nā kō waikawa.ʻAʻole kēia mau hopena e hōʻoia wale i kā mākou ELISA a me MALDI data, akā ua kūlike pū me kā mākou mau noiʻi mua o ka oligosaccharide accumulation.No laila, ke manaʻoʻiʻo nei mākou ua lawa nā ʻano immunological hou e hoʻohana ana i ka biotinylation o oligosaccharides a me ka nānā ʻana i ka ELISA e ʻike i nā oligosaccharides recalcitrant soluble i nā ʻano laʻana olaola.
Ma muli o ka hōʻoia ʻia ʻana o nā ʻano hōʻike mAb e pili ana i ka ELISA e nā ʻano hana like ʻole, makemake mākou e ʻimi hou i ka hiki o kēia ʻano quantitative hou.ʻElua mau oligosaccharides pāʻoihana, xylohexasaccharide oligosaccharide (XHE) a me 23-α-L-arabinofuranosyl-xylotriose (A2XX), i kūʻai ʻia a hoʻāʻo ʻia me ka hoʻohana ʻana i kahi ala mAb hou e kuhikuhi ana i ka glycan wall cell.Hōʻike ka Figure 6 i ka pilina laina ma waena o ka hōʻailona hoʻopaʻa biotinylated a me ka hoʻopaʻa ʻana o ka log o ka oligosaccharide, e hōʻike ana i kahi kumu hoʻohālike Langmuir adsorption.Ma waena o nā mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, a me CCRC-M151 i hoʻopili ʻia me XHE, a me CCRC-M108, CCRC-M109, a me LM11 i hoʻopili ʻia me A2XX ma luna o ka laulā o 1 nm a 100 nano.Ma muli o ka loaʻa ʻana o nā antibodies i ka wā o ka hoʻokolohua, ua hana ʻia nā hoʻokolohua liʻiliʻi me kēlā me kēia ʻano oligosaccharide.Pono e hoʻomaopopo ʻia ma ʻaneʻi he ʻokoʻa loa ka hana ʻana o kekahi mau antibodies i ka oligosaccharide like me kahi substrate, no ka mea ua nakinaki lākou i nā epitopes ʻokoʻa a hiki ke loaʻa nā pilina paʻa like ʻole.E ʻoi aku ka paʻakikī o nā hana a me nā hopena o ka ʻike epitope pololei ke hoʻohana ʻia ke ala mAb hou i nā laʻana maoli.
Ua hoʻohana ʻia ʻelua oligosaccharides pāʻoihana no ka hoʻoholo ʻana i ka laulā ʻike o nā mAbs-targeting like ʻole.Maʻaneʻi, hōʻike nā laina laina me ka hoʻopaʻa ʻana o ka oligosaccharide i nā ʻano hoʻohālike adsorption Langmuir no (A) XHE me mAb a me (B) A2XX me mAb.Hōʻike nā epitopes pili i nā hale o nā oligosaccharides pāʻoihana i hoʻohana ʻia ma ke ʻano he substrates i ka assay.
ʻO ka hoʻohana ʻana i nā antibodies monoclonal glycan-targeted (glycocomic analysis or ELISA-based mAb screening) he mea hana ikaika no ka hōʻike hohonu ʻana o ka hapa nui o nā glycans pā hale nui i hana i ka biomass mea kanu.Eia nō naʻe, ʻike wale ʻia ka loiloi glycan maʻamau i nā glycans pā hale nui, no ka mea, ʻaʻole i hoʻokuʻu ʻia ka hapa nui o nā oligosaccharides ma nā pā ELISA.I loko o kēia noiʻi ʻana, ua hoʻoheheʻe ʻia ʻo AFEX-pretretreated corn stover i ka hydrolyzed ma kahi ʻano paʻa kiʻekiʻe.Ua hoʻohana ʻia ka nānā ʻana i ke kō no ka hoʻoholo ʻana i ke ʻano o ka recalcitrant cell wall carbohydrates i loko o ka hydrolyzate.Eia naʻe, hoʻohaʻahaʻa ʻia ka loiloi mAb o nā oligosaccharides liʻiliʻi i loko o nā hydrolysates, a pono nā mea hana hou e hoʻoneʻe pono i nā oligosaccharides ma nā papa ELISA.
Ke hōʻike nei mākou ma aneʻi i kahi ʻano hoʻomaʻamaʻa oligosaccharide immobilization no ka nānā ʻana i ka mAb ma o ka hoʻohui ʻana i ka biotinylation oligosaccharide a ukali ʻia e ka nānā ʻana ELISA ma nā papa i uhi ʻia ʻo NeutrAvidin™.Ua hōʻike ʻia nā oligosaccharides biotinylated immobilized i ka pili pono no ka antibody e hiki ai i ka ʻike wikiwiki a me ka maikaʻi o nā oligosaccharides recalcitrant.ʻO ka nānā ʻana o ka haku ʻana o kēia mau oligosaccharides paʻakikī e pili ana i ka spectrometry nui i hōʻoia i nā hopena o kēia ala hou i ka immunoscreening.No laila, hōʻike kēia mau haʻawina e hiki ke hoʻohana ʻia ka hui ʻana o ka oligosaccharide biotinylation a me ka ELISA screening me glycan-targeted monoclonal antibodies e ʻike i nā crosslinks i nā oligosaccharides a hiki ke hoʻohana nui ʻia i nā noiʻi biochemical ʻē aʻe e hōʻike ana i ke ʻano o ka oligosaccharides.
ʻO kēia kaʻina hana glycan profiling biotin-based ka hōʻike mua hiki ke noiʻi i nā paʻa kaʻa kaʻa recalcitrant o nā oligosaccharides soluble i loko o ka biomass mea kanu.Kōkua kēia i ka hoʻomaopopo ʻana i ke kumu o ka paʻakikī o kekahi mau ʻāpana o ka biomass i ka hana ʻana i ka biofuel.Hoʻopiha kēia ʻano hana i kahi āpau koʻikoʻi i nā ʻano loiloi glycome a hoʻonui i kāna noi i kahi ākea o nā substrates ma mua o nā oligosaccharides kanu.I ka wā e hiki mai ana, hiki iā mākou ke hoʻohana i nā robotics no ka biotinylation a hoʻohana i ke ʻano a mākou i hoʻomohala ai no ka loiloi kiʻekiʻe-throughput o nā laʻana e hoʻohana ana iā ELISA.
Ua hōʻiliʻili ʻia ka mauʻu kulina (CS) i ulu ʻia mai Pioneer 33A14 hua hybrid i ka makahiki 2010 mai Kramer Farms ma Ray, Colorado.Me ka ʻae o ka mea nona ka ʻāina, hiki ke hoʻohana ʻia kēia biomass no ka noiʻi. Ua mālama ʻia nā laʻana i kahi maloʻo <6% o ka wai i loko o nā ʻeke paʻa zip i ka mahana wela. Ua mālama ʻia nā laʻana i kahi maloʻo <6% o ka wai i loko o nā ʻeke paʻa zip i ka mahana wela. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Mālama ʻia nā laʻana ma kahi maloʻo ma lalo o 6% humidity i loko o nā ʻeke zippered i ka lumi wela.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%. Hoʻopaʻa ʻia nā laʻana i loko o nā ʻeke zipper ma ka lumi wela me ka haʻahaʻa <6%.Ua hoʻokō ka haʻawina i nā alakaʻi kūloko a me ka ʻāina.Ua hoʻohana ʻia ka hoʻohana ʻana i ka protocol NREL.Ua ʻike ʻia ka haku mele he 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein a me 13. 4% lehu.
ʻO Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) he hui paʻakikī o cellulase, β-glucosidase a me Cellic® HTec2 (157 mg protein/ml, lot VHN00001) mai Novozymes (Franklinton, NC, USA)).ʻO Multifect Pectinase® (72 mg protein/mL), he hui paʻakikī o nā enzymes degrading pectin, i hāʻawi ʻia e DuPont Industrial Biosciences (Palo Alto, CA, USA).Ua hoʻoholo ʻia ka ʻike ʻana o ka protein enzyme ma ke koho ʻana i ka ʻike protein (a me ka unuhi ʻana i ka hāʻawi ʻana o ka nitrogen non-protein) me ka hoʻohana ʻana i ka Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Ua kūʻai ʻia ka honua Diatomaceous 545 mai EMD Millipore (Billerica, MA).Ua kūʻai ʻia mai ʻo Sigma-Aldrich (St. Louis, MO) nā kalapona i hana ʻia (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, a me nā mea ʻē aʻe a pau.
Hana ʻia ka AFEX pretreatment ma GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Hana ʻia ka hana mua ma 140 ° C. no 15 mau minuke.46 manawa noho ma 1:1 ratio o ka anhydrous ammonia i biomass ma 60% (w/w) e hoʻouka ana i loko o kahi mea hoʻoheheʻe ʻauʻau kuhili ʻole (Parr Instruments Company).He 30 mau minuke.Lawe ʻia ka reactor i 140 ° C a ua hoʻokuʻu koke ʻia ka ammonia, e ʻae i ka biomass e hoʻi koke i ka lumi wela.Ua like ka hana o ka AFEX pre-treated corn stover (ACS) me ka untreated corn stover (UT-CS).
Ua hoʻomākaukau ʻia nā mea paʻa kiʻekiʻe ACSH 25% (w/w) (ma kahi o 8% dextran loading) ma ke ʻano he mea hoʻomaka no ka hana nui o nā oligosaccharides.Hana ʻia ka hydrolysis Enzymatic o ACS me ka hoʻohana ʻana i kahi hui enzyme kalepa me Cellic® Ctec2 10 mg protein/g glucan (i ka biomass pretreated), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, a me Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein/g dextran.Hana ʻia ka hydrolysis Enzymatic i kahi bioreactor 5-lita me ka nui o ka hana o 3 lita, pH 4.8, 50 ° C a me 250 rpm.Ma hope o ka hydrolysis no 96 mau hola, ua hōʻiliʻili ʻia ka hydrolyzate e ka centrifugation ma 6000 rpm no 30 mau minuke a laila ma 14000 rpm no 30 mau minuke e wehe i nā mea paʻa ʻole.Hoʻopili ʻia ka hydrolyzate i ka kānana sterile ma o ka beaker kānana 0.22 mm.Mālama ʻia ka hydrolyzate kānana i loko o nā ʻōmole sterile ma 4 ° C. a laila hoʻokaʻawale ʻia ma ke kalapona.
'Ikepili o ka haku mele 'ana o ka extract-ma muli biomass la'ana e like me NREL keena hoʻokolohua kaʻina hana: hoʻomākaukau i nā laʻana no ka haku mele 'ana (NREL/TP-510-42620) a me ka hoʻoholo 'ana i ka structural carbohydrates a me ka lignin i biomass (NREL/TP-510 - 42618)47.
Ua hana ʻia ka loiloi oligosaccharide o ke kahawai hydrolyzate ma kahi pālākiō 2 ml me ka hoʻohana ʻana i kahi ʻano hydrolysis acid-based autoclave.E hoʻohui i ka hāpana hydrolyzate me 69.7 µl o 72% sulfuric acid i loko o ka 10 ml screw cap culture tube a hoʻomoʻa no 1 hola ma luna o kahi pae ma 121 °C, e hoʻomaha i ka hau a kānana i loko o kahi hue wai chromatography (HPLC) kiʻekiʻe.Hoʻoholo ʻia ka ʻike o ka oligosaccharides ma ka unuhi ʻana i ka ʻike o nā monosaccharides i loko o ka hāpana non-hydrolyzed mai ka nui o ke kō i loko o ka hāpana acid-hydrolyzed.
Ua kālailai ʻia ka ʻike ʻana o ka glucose, xylose, a me ka arabinose i loko o ka waiuma hydrolysed biomass me ka hoʻohana ʻana i kahi ʻōnaehana Shimadzu HPLC i hoʻolako ʻia me kahi autosampler, kolamu heater, isocratic pump, a me refractive index detector ma kahi kolamu Bio-Rad Aminex HPX-87H.Ua mālama ʻia ke kolamu ma 50 ° C a hoʻoheheʻe ʻia me 0.6 ml / min 5 mM H2SO4 i ka wai.kahe.
Ua hoʻoheheʻe ʻia ka supernatant hydrolyzate a nānā ʻia no ka monomer a me ka oligosaccharide maʻiʻo.Ua kālailai ʻia nā sugar monomeric i loaʻa ma hope o ka hydrolysis enzymatic e HPLC i lako me kahi kolamu Bio-Rad (Hercules, CA) Aminex HPX-87P a me kahi kolamu kiaʻi lehu.Ua mālamaʻia ka mahana o ke kolamu ma 80 ° C, ua hoʻohanaʻia ka wai e like me ke kaʻina hana me ka holoʻana o 0.6 ml / min.Ua hoʻoholo ʻia nā oligosaccharides e ka hydrolysis i ka dilute acid ma 121 ° C e like me nā ʻano i wehewehe ʻia ma refs.41, 48, 49.
Hana ʻia ka ʻike Saccharide ma ka maka, AFEX pre-treated a me nā koena biomass non-hydrolysed āpau (me ka hana ʻana o nā ʻāpana o ka pā kelepona serial a me kā lākou mAb screening) me ka hoʻohana ʻana i nā kaʻina hana 27, 43, 50, 51.No ka nānā ʻana i ka glycome, ua hoʻomākaukau ʻia nā koena hiki ʻole i ka waiʻona o ka pā hale mea kanu mai nā koena biomass a hoʻopaʻa ʻia i ka unuhi serial me nā mea hoʻoulu hou e like me ka ammonium oxalate (50 mM), sodium carbonate (50 mM a me 0.5% w/v), CON.(1M a me 4M, ʻelua me 1% w/v sodium borohydride) a me ka waikawa chlorite e like me ka mea i wehewehe mua ʻia52,53.A laila ua hoʻokau ʻia nā extracts iā ELISA e kūʻē i kahi papa paʻakikī o mAb50s i kuhikuhi ʻia i ka glycan wall cell, a ua hōʻike ʻia nā hopena paʻa mAb ma ke ʻano he palapala wela.Ua kūʻai ʻia nā mAbs e ʻimi ana i ka glycan pā hale mea kanu mai nā waihona hale hana (CCRC, JIM a me MAC series).
Hoʻokahi-ʻanuʻu biotinylation o oligosaccharides.Ua hana ʻia ka hui ʻana o nā kalapona me ka biotin-LC-hydrazide me ka hoʻohana ʻana i kēia kaʻina hana.Ua hoʻoheheʻeʻia ka Biotin-LC-hydrazide (4.6 mg / 12 μmol) i ka dimethyl sulfoxide (DMSO, 70 μl) ma ka hoʻoulu ikaika a me ka hoʻomehanaʻana ma 65 ° C. no 1 min.Hoʻohui ʻia ka ʻakika acetic glacial (30 µl) a ninini ʻia ka hui ʻana ma luna o ka sodium cyanoborohydride (6.4 mg/100 µmol) a hoʻoheheʻe ʻia ma hope o ka hoʻomehana ʻana ma 65 ° C. no kahi o 1 mau minuke.A laila, mai 5 a 8 μl o ka hui ʻana i hoʻohui ʻia i ka oligosaccharide maloʻo (1-100 nmol) no ka loaʻa ʻana o kahi 10-fold a ʻoi aku ka nui o ka molar o ka lepili ma luna o ka hopena hoʻemi.Ua hoʻokō ʻia ka hopena ma 65 ° C no 2 mau hola, ma hope o ka hoʻomaʻemaʻe koke ʻana i nā laʻana.ʻAʻole i hoʻohana ʻia ka sodium cyanoborohydride i nā hoʻokolohua lepili me ka ʻole o ka hoʻemi ʻana, a ua hana ʻia nā mea hoʻohālike ma 65 ° C. no 2.5 mau hola.
ʻO ka uhi ʻana a me ka holoi ʻana o ELISA o nā laʻana o nā oligosaccharides biotinylated.25 μl o nā laʻana biotinylated (100 μl o kēlā me kēia hāpana i hoʻoheheʻe ʻia i 5 ml o 0.1 M Tris buffer solution (TBS)) i hoʻohui ʻia i kēlā me kēia luawai o ka pā avidin-coated.Ua uhi ʻia nā pūnāwai mana me 50 μl o ka biotin ma kahi ʻano o 10 μg / ml i 0.1 M TBS.Ua hoʻohana ʻia ka wai deionized ma ke ʻano he uhi no nā ana hakahaka.Hoʻokomo ʻia ka papa no 2 mau hola ma ka lumi wela i ka pōʻeleʻele.Holoi i ka papa 3 manawa me 0.1% ka waiu waiu i 0.1 M TBS me ka polokalamu no.11 no Grenier flat 3A.
Hoʻohui a holoi i nā antibodies mua.E hoʻohui i 40 µl o ka antibody mua i kēlā me kēia pūnāwai.Hoʻomoʻa i ka microplate no 1 hola ma ka lumi wela i ka pōʻeleʻele.Holoi ʻia nā papa i ʻekolu manawa me ka waiū 0.1% i 0.1M TBS me ka hoʻohana ʻana i ka papahana holoi #11 no Grenier Flat 3A.
E hoʻohui i ka antibody lua a holoi.E hoʻohui i 50 µl o ka ʻiole/ʻiole kekona antibody (hoʻoheheʻe ʻia 1:5000 i 0.1% waiu i 0.1 M TBS) i kēlā me kēia luawai.Hoʻomoʻa i ka microplate no 1 hola ma ka lumi wela i ka pōʻeleʻele.Holoi ʻia nā microplates i 5 manawa me ka waiū 0.1% i 0.1 M TBS me ka hoʻohana ʻana i ka papahana holoi pāpaʻi Grenier Flat 5A #12.
Hoʻohui i kahi substrate.E hoʻohui i 50 µl o 3,3′,5,5′-tetramethylbenzidine (TMB) i ka substrate kumu (ma ka hoʻohui ʻana i 2 kulu o ka buffer, 3 kulu o TMB, 2 kulu o ka hydrogen peroxide i 15 ml o ka wai deionized).E hoʻomākaukau i ka substrate TMB.a me ka vortex ma mua o ka hoʻohana ʻana).Hoʻomoʻa i ka microplate ma ka lumi wela no 30 mau minuke.I ka pōʻeleʻele.
E hoʻopau i ka ʻanuʻu a heluhelu i ka papa.E hoʻohui i 50 µl o 1 N sulfuric acid i kēlā me kēia pūnāwai a hoʻopaʻa i ka hoʻopaʻa ʻana mai 450 a 655 nm me ka hoʻohana ʻana i ka mea heluhelu ELISA.
E hoʻomākaukau i 1 mg/ml solutions o kēia mau analytes i ka wai deionized: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine.(glcNAc), N-acetylgalactosamine (galNAc), inositol (maʻamau o loko).Ua hoʻomākaukau ʻia ʻelua mau mea maʻamau ma ka hoʻohui ʻana i ka 1 mg/mL kōpaʻa wai i hōʻike ʻia ma ka Papa 1. Hoʻopili ʻia nā laʻana i ka hau a lyophilized ma -80 ° C. a hiki i ka hemo ʻana o ka wai (ma kahi o 12-18 mau hola).
E hoʻohui i 100–500 µg o ka laʻana e wili i nā paipu poʻi ma kahi kaulike analytical.E hoʻopaʻa i ka nui i hoʻohui ʻia.ʻOi aku ka maikaʻi o ka hoʻoheheʻe ʻana i ka hāpana ma kahi kikoʻī o ka solvent a hoʻohui iā ia i ka paipu e like me ka aliquot wai.E hoʻohana i 20 µl o 1 mg/ml inositol ma ke ʻano he maʻamau kūloko no kēlā me kēia paipu hāpana.ʻO ka nui o ka maʻamau kūloko i hoʻohui ʻia i ka hāpana e like me ka nui o ka maʻamau kūloko i hoʻohui ʻia i ka paipu maʻamau.
E hoʻohui i 8 ml o ka methanol anhydrous i loko o kahi hue pahu pahu.A laila 4 ml o 3 N. methanolic HCl solution, uhi ʻia a hoʻoluliluli ʻia.ʻAʻole hoʻohana kēia kaʻina i ka wai.
E hoʻohui i 500 µl o 1 M HCl methanol solution i nā laʻana oligosaccharide a me nā paipu TMS maʻamau.Hoʻokomo ʻia nā laʻana i ka pō (168 hola) ma 80 ° C. i loko o kahi poloka wela.E hoʻomaloʻo i ka huahana methanolysis ma ka lumi wela me ka hoʻohana ʻana i ka manifold maloʻo.E hoʻonui i 200 µl MeOH a maloʻo hou.Hana ʻia kēia kaʻina hana ʻelua.E hoʻohui i 200 µl o ka methanol, 100 µl o pyridine a me 100 µl o ka acetic anhydride i ka hāpana a hui maikaʻi.Hoʻomoʻa ʻia nā laʻana ma ka lumi wela no 30 mau minuke.a maloʻo.E hoʻohui i 200 µl o ka methanol a maloʻo hou.
E hoʻohui i 200 µl o Tri-Sil a me ka paipu wela no 20 mau minuke.80 ° C, a laila hoʻoluʻu i ka lumi wela.E hoʻohana i kahi mea hoʻomaloʻo e hoʻomaloʻo hou i ka hāpana a hiki i ka 50 µl.He mea nui e hoʻomaopopo ʻaʻole mākou i ʻae i nā laʻana e maloʻo loa.
E hoʻohui i 2 ml o ka hexane a hui maikaʻi me ka vortexing.E hoʻopiha i ka piko o nā paipu Pasteur (5-8 mm) me kahi ʻāpana huluhulu aniani ma ka hoʻokomo ʻana i ka huluhulu aniani ma luna o kahi pipette 5-3/4 iniha ke anawaena.Ua centrifuged nā laʻana ma 3000 g no 2 mau minuke.Hoʻopili ʻia nā koena insoluble.E hoʻomaloʻo i ka hāpana i 100-150 µl.Ua hoʻokomoʻia ka nui o kahi 1 μl i loko o ka GC-MS ma kahi mahana mua o 80 ° C a me kahi manawa mua o 2.0 minuke (Table 2).