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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 no ke ʻano o ke ʻano, ka haku ʻana, a me ka hoʻololi ʻana o ka paia o ka mea kanu, no ka mea, aia nā ʻokoʻa nui ma muli o ke ʻano o nā mea kanu, ʻokena, makahiki, pae hoʻomohala, a me ka ulu 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 hanana 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 maʻi 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 i 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 DP 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 hana 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 ʻana o ka 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 i ka hoʻokō ʻana o nā ʻano immunological i nā ʻano hana ʻē aʻe no ka hōʻike ʻana i nā oligosaccharides kaumaha haʻahaʻa i loaʻa i ka lignoscellulosic hydrolysates, ua hana mākou i MALDI (Fig. 4, S1-S8) a me ka nānā ʻana i nā saccharides i loaʻa 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 ʻo 4 iā MS 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 ʻo ACN-B i ka moʻo 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 hana pū mākou i ka nānā ʻana i ka haku mele glycan o nā hakina oligosaccharide me ka hoʻohana ʻana i ka derivatization glycan TMS. 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 kaʻina hana screening 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 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 papa 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 pū 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 paʻa unhydrolyzed. 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 ka 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/iole kekona antibody (diluted 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. Incubate laʻana ma ka lumi wela no 30 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).