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ʻO nā ʻano hana hou no ka pale ʻana i ka maʻi a me ka nui o ka spectrometric no ka loiloi paʻakikī o nā oligosaccharides mau loa i loko o ka mea kanu palaoa i mālama mua ʻia me AFEX. ʻO ka biomass lignocellulosic kahi koho hoʻomau i nā wahie fossil a hoʻohana nui ʻia e hoʻomohala i nā biotechnologies no ka hana ʻana i nā huahana e like me ka meaʻai, ka hānai, nā wahie a me nā kemika. ʻO ke kī i kēia mau ʻenehana ka hoʻomohala ʻana i nā kaʻina hana hoʻokūkū kumukūʻai no ka hoʻololi ʻana i nā kalapona paʻakikī i loko o nā paia o nā mea kanu i nā kō maʻalahi e like me ka glucose, xylose a me ka arabinose. No ka mea he paʻakikī loa ka biomass lignocellulosic, pono e hoʻokō ʻia i nā hana thermochemical (e laʻa, ka exfoliation fiber ammonia (AFEX), nā waikawa dilute (DA), nā wai ionic (IL)) a me nā hana biological (e laʻa, ka hydrolysis enzymatic a me ka fermentation microbial) i ka hui pū ʻana e loaʻa ai ka huahana i makemake ʻia. . Eia nō naʻe, i ka wā e hoʻohana ʻia ai nā enzyme fungal kalepa i ke kaʻina hana hydrolysis, ʻo 75-85% wale nō o nā kō hiki ke hoʻoheheʻe ʻia i hana ʻia he monosaccharides, a ʻo ke koena 15-25% he hiki ke hoʻoheheʻe ʻia, nā oligosaccharides paʻakikī, ʻaʻole i loaʻa mau i nā microorganism. Ma mua, ua hoʻokaʻawale a hoʻomaʻemaʻe mākou i nā oligosaccharides paʻakikī hiki ke hoʻoheheʻe ʻia me ka hoʻohana ʻana i ka hui pū ʻana o ke kalapona a me ka diatomaceous earth separation a me ka size exclusion chromatography, a ua noiʻi pū i ko lākou mau waiwai enzyme inhibitory. Ua ʻike mākou he ʻoi aku ka paʻakikī o ka hana ʻana i nā oligosaccharides e loaʻa ana ke kiʻekiʻe o ka polymerization (DP) methylated uronic acid substitutions me nā hui enzyme kalepa ma mua o ka DP haʻahaʻa a me nā oligosaccharides neutral. Maanei mākou e hōʻike nei i ka hoʻohana ʻana i kekahi mau ʻano hana hou aʻe, me ka glycan profiling e hoʻohana ana i nā monoclonal antibodies (mAbs) kikoʻī i nā glycans biomass mea kanu e wehewehe i nā pilina glycan i loko o nā paia cell mea kanu a me nā hydrolysates enzymatic, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. . Hoʻohana ʻo MALDI-TOF-MS) i nā piko diagnostic structure-informative i loaʻa e ka spectroscopy ma hope o ka palaho lua o nā ions maikaʻi ʻole, gas chromatography a me ka mass spectrometry (GC-MS) e wehewehe i nā pilina oligosaccharide me a me ka ʻole o ka derivatization. Ma muli o ka liʻiliʻi o nā oligosaccharides (DP 4–20), he paʻakikī ke hoʻohana ʻana i kēia mau molekala no ka hoʻopaʻa ʻana a me ke ʻano o ka mAb. No ka lanakila ʻana i kēia pilikia, ua hoʻopili mākou i kahi ʻano hana hoʻopaʻa oligosaccharide biotin hou i hoʻokumu ʻia i ka biotin conjugation i hoʻopaʻa pono i ka hapa nui o nā oligosaccharides soluble DP haʻahaʻa ma ka ʻili microplate, a laila hoʻohana ʻia i loko o kahi ʻōnaehana mAb throughput kiʻekiʻe no ka nānā ʻana i ka ligation kikoʻī. E kōkua kēia ʻano hana hou i ka hoʻomohala ʻana o nā assays glycome throughput kiʻekiʻe i ka wā e hiki mai ana e hiki ke hoʻohana ʻia e hoʻokaʻawale a wehewehe i nā oligosaccharides i loaʻa i nā biomarkers no nā kumu diagnostic.
ʻO ka biomass lignocellulosic, i haku ʻia me nā mea mahiʻai, nā ululāʻau, nā mauʻu a me nā mea lāʻau, he mea hānai kūpono ia no ka hana ʻana i nā huahana bio-based, me ka meaʻai, ka hānai, ka wahie a me nā mea mua o ke kemika e hana i nā huahana waiwai kiʻekiʻe1. ʻO nā kalapona (e like me ka cellulose a me ka hemicellulose) i loko o nā paia o nā mea kanu ua hoʻokaʻawale ʻia i loko o nā monosaccharides ma o ka hana kemika a me ka biotransformation (e like me ka hydrolysis enzymatic a me ka fermentation microbial). ʻO nā hana mua maʻamau e pili ana i ka hoʻonui ʻana o ka fiber ammonia (AFEX), dilute acid (DA), ionic liquid (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 lignocellulose ma ka wehe ʻana i nā paia o nā mea kanu3,4. paʻakikī o ka mea, 5. Hana ʻia ka hydrolysis enzymatic ma kahi ukana solids kiʻekiʻe me ka hoʻohana ʻana i nā enzymes carbohydrate-loading commercial active (CAZymes) a me ka fermentation microbial me ka hoʻohana ʻana i nā yeasts transgenic a i ʻole bacteria e hana i nā wahie a me nā kemika bio-based 6.
ʻO nā CAZymes i loko o nā enzymes kalepa ua haku ʻia me kahi hui paʻakikī o nā enzymes e hoʻokaʻawale synergistically i nā pilina carbohydrate-sugar paʻakikī e hana i nā monosaccharides2,7. E like me kā mākou i hōʻike ai ma mua, ʻo ka pūnaewele paʻakikī o nā polymers aromatic o ka lignin me nā carbohydrates e hoʻolilo iā lākou i mea paʻakikī loa, kahi e alakaʻi ai i ka hoʻololi kō piha ʻole, e hōʻiliʻili ana i 15-25% o nā oligosaccharides sex i hana ʻole ʻia i ka wā hydrolysis enzymatic o ka biomass i mālama mua ʻia. He pilikia maʻamau kēia me nā ʻano hana pretreatment biomass like ʻole. ʻO kekahi mau kumu no kēia bottleneck e pili ana i ka inhibition enzyme i ka wā hydrolysis, a i ʻole ka nele a i ʻole nā ​​​​pae haʻahaʻa o nā enzymes koʻikoʻi e pono ai e uhaki i nā pilina kō i loko o ka biomass mea kanu. ʻO ka hoʻomaopopo ʻana i ka haku mele a me nā ʻano hoʻonohonoho o nā kō, e like me nā pilina kō i loko o nā oligosaccharides, e kōkua iā mākou e hoʻomaikaʻi i ka hoʻololi kō i ka wā hydrolysis, e hana ana i nā kaʻina hana biotechnological i hoʻokūkū kumukūʻai me nā huahana i loaʻa mai ka petroleum.
He mea paʻakikī ka hoʻoholo ʻana i ke ʻano o nā kalapona a koi ʻia ka hui pū ʻana o nā ʻano hana e like me ka chromatography wai (LC)11,12, spectroscopy resonance magnetic nuclear (NMR)13, electrophoresis capillary (CE)14,15,16 a me ka spectrometry mass (MS)17. ,ʻumikumamāwalu. ʻO nā ʻano MS e like me ka spectrometry mass time-of-flight me ka laser desorption a me ka ionization me ka hoʻohana ʻana i kahi matrix (MALDI-TOF-MS) he ʻano hana maʻalahi no ka ʻike ʻana i nā ʻano kalapona. I kēia manawa, ua hoʻohana nui ʻia ka Collision-Induced Dissociation (CID) tandem MS o nā sodium ion adducts e ʻike i nā manamana lima e pili ana i nā kūlana hoʻopili oligosaccharide, nā hoʻonohonoho anomeric, nā moʻo, a me nā kūlana lālā 20, 21.
He mea hana maikaʻi loa ka loiloi Glycan no ka ʻike hohonu ʻana i nā pilina carbohydrate22. Hoʻohana kēia ʻano hana i nā antibodies monoclonal (mAbs) i kuhikuhi ʻia i ka glycan paia cell mea kanu ma ke ʻano he probes e hoʻomaopopo i nā pilina carbohydrate paʻakikī. Loaʻa ma mua o 250 mAbs ma ka honua holoʻokoʻa, i hoʻolālā ʻia e kūʻē i nā oligosaccharides linear a me nā lālā like ʻole me ka hoʻohana ʻana i nā saccharides like ʻole24. Ua hoʻohana nui ʻia kekahi mau mAbs e wehewehe i ke ʻano, ka haku mele ʻana, a me nā hoʻololi ʻana o ka paia cell mea kanu, no ka mea, he mau ʻokoʻa koʻikoʻi ma muli o ke ʻano cell mea kanu, ka ʻōpū, ka makahiki, ke kahua hoʻomohala, a me ke kaiapuni ulu25,26. ʻOi aku nei, ua hoʻohana ʻia kēia ʻano hana e hoʻomaopopo i nā heluna vesicle i nā ʻōnaehana mea kanu a me nā holoholona a me kā lākou kuleana i ka lawe ʻana o ka glycan e like me ka mea i hoʻoholo ʻia e nā māka subcellular, nā pae hoʻomohala, a i ʻole nā ​​​​​​mea hoʻonāukiuki kaiapuni, a e hoʻoholo i ka hana enzymatic. ʻO kekahi o nā ʻano like ʻole o nā glycans a me nā xylans i ʻike ʻia ʻo ia ka pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) a me arabino-galactan (ArbG)29.
Eia nō naʻe, me kēia mau hana noiʻi āpau, he kakaikahi wale nō nā haʻawina i kālele i ke ʻano o ka hōʻiliʻili ʻana oligosaccharide i ka wā o ka hydrolysis ukana paʻa kiʻekiʻe (HSL), me ka hoʻokuʻu ʻana oligosaccharide, nā loli lōʻihi oligomeric chain i ka wā o ka hydrolysis, nā polymers DP haʻahaʻa like ʻole, a me kā lākou mau piʻo. nā hoʻolaha 30,31,32. I kēia manawa, ʻoiai ua hōʻoia ʻia ka loiloi glycan he mea pono no ka loiloi piha ʻana o ka hoʻonohonoho glycan, he paʻakikī ke loiloi i nā oligosaccharides DP haʻahaʻa hiki ke hoʻoheheʻe ʻia i ka wai me ka hoʻohana ʻana i nā ʻano antibody. ʻO nā oligosaccharides DP liʻiliʻi me ke kaumaha molekala ma lalo o 5-10 kDa ʻaʻole e hoʻopaʻa i nā papa ELISA 33, 34 a holoi ʻia ma mua o ka hoʻohui ʻana o ka antibody.
Maanei, no ka manawa mua, ke hōʻike nei mākou i kahi hoʻāʻo ELISA ma nā papa i uhi ʻia e avidin me ka hoʻohana ʻana i nā antibodies monoclonal, e hoʻohui ana i kahi kaʻina hana biotinylation hoʻokahi-ʻanuʻu no nā oligosaccharides refractory soluble me ka nānā ʻana o glycome. Ua hōʻoia ʻia kā mākou ʻano hana i ka nānā ʻana o glycome e ka MALDI-TOF-MS a me ka nānā ʻana o GC-MS o nā pilina oligosaccharide hoʻopihapiha me ka hoʻohana ʻana i ka trimethylsilyl (TMS) derivatization o nā haku mele kō 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 i ka noiʻi biomedical35.
ʻO nā hoʻololi ma hope o ka unuhi ʻana o nā enzymes a me nā antibodies, e like me ka glycosylation,36 e hoʻopilikia i kā lākou hana olaola. No ka laʻana, he kuleana koʻikoʻi nā loli i ka glycosylation o nā protein serum i ka arthritis inflammatory, a hoʻohana ʻia nā loli i ka glycosylation ma ke ʻano he mau māka diagnostic37. Ua hōʻike ʻia nā glycans like ʻole i loko o ka palapala e ʻike koke ʻia i nā maʻi like ʻole, me nā maʻi inflammatory mau loa o ka ʻōpū gastrointestinal a me ke ake, nā maʻi viral, nā ovarian, ka umauma, a me nā maʻi kanesa prostate38,39,40. ʻO ka hoʻomaopopo ʻana i ke ʻano o nā glycans me ka hoʻohana ʻana i nā ʻano glycan ELISA i hoʻokumu ʻia i ka antibody e hāʻawi i ka hilinaʻi hou aʻe i ka ʻike ʻana i ka maʻi me ka hoʻohana ʻole ʻana i nā ʻano MS paʻakikī.
Ua hōʻike kā mākou noiʻi mua ʻana ua noho mau nā oligosaccharides paʻakikī ʻaʻole i hoʻoheheʻe ʻia ma hope o ka mālama mua ʻana a me ka hydrolysis enzymatic (Kiʻi 1). I kā mākou hana i paʻi mua ʻia, ua hoʻomohala mākou i kahi ʻano hana hoʻokaʻawale paʻa lanahu i hoʻāla ʻia 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 size exclusion chromatography (SEC) a hōʻiliʻili ʻia ma ke ʻano o ke kaumaha molekala. Ua kālailai ʻia nā monomers kō a me nā oligomers i hoʻokuʻu ʻia mai nā mālama mua like ʻole e ka loiloi haku mele kō. I ka hoʻohālikelike ʻana i ka ʻike o nā oligomers kō i loaʻa e nā ʻano hana mālama mua like ʻole, ʻo ke alo 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 ka hua kō ma ka liʻiliʻi he 10-15% a hiki i ka 18%. US. Hoʻohana ʻia kēia ʻano hana no ka hana nui hou ʻana o nā ʻāpana oligosaccharide. ʻO ka hopena ACH a me kāna mau ʻāpana ma hope me nā kaumaha molekala like ʻole i hoʻohana ʻia ma ke ʻano he mea hoʻokolohua no ke ʻano o nā oligosaccharides ma kēia hana.
Ma hope o ka mālama mua ʻana a me ka hydrolysis enzymatic, ua noho mau nā oligosaccharides paʻa ʻaʻole i hydrolyzed. Eia (A) kahi ʻano hoʻokaʻawale oligosaccharide kahi e hoʻokaʻawale ʻia ai nā oligosaccharides mai ka AFEX-pretreated corn stover hydrolysate (ACSH) me ka hoʻohana ʻana i kahi moena i hoʻopaʻa ʻia o ke kalapona i hoʻāla ʻia a me ka honua diatomaceous; (B) Ke ʻano no ka hoʻokaʻawale ʻana o nā oligosaccharides. Ua hoʻokaʻawale hou ʻia nā oligosaccharides e ka size exclusion chromatography (SEC); (C) Nā monomers Saccharide a me nā oligomers i hoʻokuʻu ʻia mai nā ʻano mālama mua like ʻole (diluted acid: DA, ionic liquid: IL a me AFEX). Nā kūlana hydrolysis enzymatic: ka hoʻouka ʻana o nā solids kiʻekiʻe o 25% (w/w) (ma kahi o 8% glucan loading), 96 hola hydrolysis, 20 mg/g kalepa enzyme hoʻouka (Ctec2:Htec2:MP-2:1:1 ratio) a me (D) Nā monomers kō a me nā oligomers o ka glucose, xylose a me ka arabinose i hoʻokuʻu ʻia mai ka AFEX pre-treated corn stover (ACS).
Ua hōʻoia ʻia ka loiloi Glycan he mea hana pono no ka loiloi hoʻonohonoho piha o nā glycans i loko o nā extracts i hoʻokaʻawale ʻia mai nā koena biomass paʻa. Eia nō naʻe, ʻaʻole i hōʻike nui ʻia nā saccharides wai-soluble me ka hoʻohana ʻana i kēia ʻano kuʻuna41 no ka mea he paʻakikī ke hoʻopaʻa i nā oligosaccharides kaumaha molekala haʻahaʻa ma nā papa ELISA a holoi ʻia ma mua o ka hoʻohui ʻana o ka antibody. No laila, no ka hoʻopaʻa ʻana a me ke ʻano o ka antibody, ua hoʻohana ʻia kahi ʻano biotinylation hoʻokahi-ʻanuʻu e uhi i nā oligosaccharides hiki ke hoʻoheheʻe ʻia, ʻaʻole i kūlike ma nā papa ELISA i uhi ʻia e avidin. Ua hoʻāʻo ʻia kēia ʻano me ka hoʻohana ʻana i kā mākou ACSH i hana mua ʻia a me kahi ʻāpana e pili ana i kona kaumaha molekala (a i ʻole ke kekelē o ka polymerization, DP). Ua hoʻohana ʻia ka biotinylation hoʻokahi-ʻanuʻu e hoʻonui i ka pilina hoʻopaʻa oligosaccharide ma ka hoʻohui ʻana i ka biotin-LC-hydrazide i ka hopena hoʻemi o ka carbohydrate (Kiʻi 2). I loko o ka hopena, hana ka hui hemiacetal ma ka hopena hoʻemi me ka hui hydrazide o biotin-LC-hydrazide e hana i kahi pilina hydrazone. I ke alo o ka mea hoʻemi NaCNBH3, ua hoʻemi ʻia ka pilina hydrazone i kahi huahana hope biotinylated paʻa. Me ka hoʻololi ʻana o ka hopena hoʻemi kō, ua hiki ke hoʻopaʻa ʻia o nā oligosaccharides DP haʻahaʻa i nā papa ELISA, a ma kā mākou noiʻi ua hana ʻia kēia ma nā papa i uhi ʻia e avidin me ka hoʻohana ʻana i nā mAbs i hoʻopili ʻia i ka glycan.
Nānā ʻana o nā antibodies monoclonal e pili ana i ka ELISA no nā oligosaccharides biotinylated. Maanei (A) ka biotinylation i hui pū ʻia o nā oligosaccharides a me ka nānā ʻana o ELISA ma hope me nā mAbs i hoʻopaʻa ʻia me ka glycan ma nā papa i uhi ʻia me NeutrAvidin a me (B) e hōʻike ana i kahi kaʻina hana hoʻokahi no ka biotinylation o nā huahana hopena.
A laila ua hoʻohui ʻia nā papa i uhi ʻia me Avidin me nā antibodies i hoʻohui ʻia me ka oligosaccharide i nā antibodies mua a me nā lua a holoi ʻia i loko o kahi medium māmā a me ka manawa. Ma hope o ka pau ʻana o ka hoʻopaʻa ʻana o ka antibody, e hoʻohui i ka substrate TMB e hoʻomoʻa i ka papa. Ua hoʻōki hope ʻia ka hopena me ka waikawa sulfuric. Ua kālailai ʻia nā papa i hoʻomoʻa ʻia me ka hoʻohana ʻana i kahi mea heluhelu ELISA e hoʻoholo i ka ikaika hoʻopaʻa o kēlā me kēia antibody e ʻike i ka cross-linking kikoʻī antibody. No nā kikoʻī a me nā palena o ka hoʻokolohua, e ʻike i ka ʻāpana pili "Nā Mea Hana a me nā ʻAno".
Hōʻike mākou i ka pono o kēia ʻano hana hou i hoʻomohala ʻia no nā noi kikoʻī ma o ka wehewehe ʻana i nā oligosaccharides soluble i loaʻa i loko o ACSH a me nā ʻāpana oligosaccharide maka a me ka hoʻomaʻemaʻe i hoʻokaʻawale ʻia mai nā hydrolysates lignocellulosic. 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 uronic (U) a i ʻole methyluronic (MeU) a me nā arabinogalactans pectic. Ua loaʻa pū ka hapa nui o lākou i kā mākou noiʻi mua ma ka nānā ʻana i nā glycans o nā solids non-hydrolyzed (UHS)43.
ʻIke ʻia nā epitopes oligosaccharide recalcitrant me ka hoʻohana ʻana i kahi antibody monoclonal i kuhikuhi ʻia i ka glycan paia cell. ʻO ka hapa "neutral" ka hapa ACN a ʻo ka hapa "acidic" ka hapa FA. ʻO nā ʻulaʻula ʻoi aku ka mālamalama ma ka heatmap e hōʻike ana i ka nui o ka epitope, a ʻo nā polū ʻoi aku ka mālamalama e hōʻike ana i kahi hope hakahaka. Hoʻokumu ʻia nā waiwai kala ma ka unahi ma 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 ʻoki ʻia kēia mau ʻano hana ʻole-cellulose e nā cellulases maʻamau a me nā hemicellulases i loko o ka hui enzyme kalepa i hoʻāʻo ʻia, ʻo ia hoʻi nā enzyme kalepa i hoʻohana nui ʻia. No laila, pono nā enzyme kōkua hou no kā lākou hydrolysis. Me ka ʻole o nā enzymes kōkua ʻole-cellulose e pono ai, pale kēia mau pilina ʻole-cellulose i ka hoʻololi piha ʻana i nā monosaccharides, ʻoiai inā ua hydrolyzed nui ʻia kā lākou mau polymers kō makua i nā ʻāpana pōkole a hoʻoheheʻe ʻia me ka hoʻohana ʻana i nā hui enzyme kalepa.
Ua hōʻike ʻia kahi noiʻi hou aʻe o ka hoʻolaha ʻana o ka hōʻailona a me kona ikaika hoʻopaʻa ʻana he haʻahaʻa nā epitopes hoʻopaʻa ʻana i nā ʻāpana kō DP kiʻekiʻe (A, B, C, DP a hiki i ka 20+) ma mua o nā ʻāpana DP haʻahaʻa (D, E, F, DP) i nā dimers) (Kiʻi 1). ʻOi aku ka maʻamau o nā ʻāpana waikawa i nā epitopes non-cellulose ma mua o nā ʻāpana kūlike ʻole. Kūlike kēia mau hanana me ke ʻano i ʻike ʻia ma kā mākou noiʻi mua, kahi i ʻoi aku ka pale ʻana o nā ʻāpana DP kiʻekiʻe a me nā ʻāpana waikawa i ka hydrolysis enzymatic. No laila, ʻo ke alo o nā epitopes glycan non-cellulose a me nā pani U a me MeU hiki ke kōkua nui i ke kūpaʻa o nā oligosaccharides. Pono e hoʻomaopopo ʻia he pilikia paha ka hoʻopaʻa ʻana a me ka ʻike ʻana no nā oligosaccharides DP haʻahaʻa, ʻoiai inā he dimeric a trimeric oligosaccharide ka epitope. Hiki ke hoʻāʻo ʻia kēia me ka hoʻohana ʻana i nā oligosaccharides kalepa o nā lōʻihi like ʻole, ʻo kēlā me kēia me hoʻokahi epitope wale nō e hoʻopaʻa ana i kahi mAb kikoʻī.
No laila, ua hōʻike ka hoʻohana ʻana i nā antibodies kikoʻī i ke ʻano i kekahi mau ʻano o nā pilina recalcitrant. Ma muli o ke ʻano o ka antibody i hoʻohana ʻia, ke ʻano ligation kūpono, a me ka ikaika o ka hōʻailona āna e hana ai (ka nui loa a me ka liʻiliʻi loa), hiki ke ʻike ʻia nā enzymes hou a hoʻohui ʻia i ka semi-quantitatively i ka hui enzyme no ka glycoconversion piha. Ma ka lawe ʻana i ka nānā ʻana o nā oligosaccharides ACSH ma ke ʻano he laʻana, hiki iā mākou ke hana i kahi waihona ʻikepili o nā pilina glycan no kēlā me kēia mea biomass. Pono e hoʻomaopopo ʻia ma ʻaneʻi e pono e noʻonoʻo ʻia ka like ʻole o nā antibodies, a inā ʻaʻole ʻike ʻia ko lākou like ʻana, e hana kēia i kekahi mau pilikia i ka hoʻohālikelike ʻana i nā hōʻailona o nā antibodies like ʻole. Eia kekahi, ʻo ka hoʻohālikelike ʻana o nā pilina glycan e hana maikaʻi loa ma waena o nā laʻana no ka antibody like. A laila hiki ke hoʻopili ʻia kēia mau pilina paʻakikī i ka waihona ʻikepili CAZyme, kahi e hiki ai iā mākou ke ʻike i nā enzymes, koho i nā enzymes moho a hoʻāʻo no nā enzymes e uhaki ana i ka pilina, a i ʻole e hoʻomohala i nā ʻōnaehana microbial e hōʻike i kēia mau enzymes no ka hoʻohana ʻana i nā biorefineries44.
No ka loiloi ʻana pehea e hoʻopiha ai nā ʻano hana immunological i nā ʻano hana ʻē aʻe no ke ʻano o nā oligosaccharides kaumaha molekala haʻahaʻa i loaʻa i nā hydrolysates lignocellulosic, ua hana mākou iā MALDI (Kiʻi 4, S1-S8) a me ka nānā ʻana o nā saccharides i loaʻa mai TMS ma muli o GC-MS ma ka ʻāpana oligosaccharide panel like (Kiʻi 5). Hoʻohana ʻia ʻo MALDI e hoʻohālikelike inā kūlike ka hoʻolaha nui ʻana o nā molekala oligosaccharide me ke ʻano i manaʻo ʻia. Ma ke kiʻi 4 hōʻike ʻo MS o nā ʻāpana kūlike ʻole ACN-A a me ACN-B. Ua hōʻoia ka loiloi ACN-A i kahi ʻano o nā kō pentose mai DP 4-8 (Kiʻi 4) a i DP 22 (Kiʻi S1), nona nā kaumaha e pili ana i nā oligosaccharides MeU-xylan. Ua hōʻoia ka loiloi ACN-B i ka moʻo pentose a me glucoxylan me DP 8-15. Ma nā mea hoʻohui e like me ke Kiʻi S3, hōʻike nā palapala hoʻolaha nui o ka ʻāpana waikawa FA-C i kahi laulā o nā kō pentose i pani ʻia (Me)U me ka DP o 8-15 e kūlike ana me nā xylans i pani ʻia i loaʻa ma ka nānā ʻana o ka mAb ma muli o ELISA. Kūlike nā epitopes.
ʻO ka spectrum MALDI-MS o nā oligosaccharides hiki ʻole ke hoʻoheheʻe ʻia i loaʻa i loko o ACS. Maanei, (A) nā ʻāpana haʻahaʻa o ka ACN-A e loaʻa ana ka methylated uronic acid (DP 4-8) i pani ʻia me 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).
ʻO ka nānā ʻana i ka haku mele ʻana o ke koena glycan o nā oligosaccharides refractory. Maanei (A) ka haku mele ʻana o ka TMS saccharide o nā ʻāpana oligosaccharide like ʻole i loaʻa me ka hoʻohana ʻana i ka nānā ʻana o GC-MS. (B) Nā ʻano o nā kō like ʻole i loaʻa mai ka TMS i loko o nā oligosaccharides. ACN - ʻāpana acetonitrile e loaʻa ana nā oligosaccharides neutral a me FA - ʻāpana waikawa ferulic e loaʻa ana nā oligosaccharides acid.
Ua huki ʻia kekahi hopena hoihoi mai ka loiloi LC-MS o ka ʻāpana oligosaccharide, e like me ka mea i hōʻike ʻia ma ke Kiʻi S9 (hiki ke ʻike ʻia nā ʻano hana ma ka mea hoʻohui uila). Ua ʻike pinepine ʻia nā ʻāpana o nā hui hexose a me -OAc i ka wā o ka ligation o ka ʻāpana ACN-B. ʻAʻole wale kēia ʻike e hōʻoia i ka fragmentation i ʻike ʻia ma ka glycome a me ka loiloi MALDI-TOF, akā hāʻawi pū kekahi i ka ʻike hou e pili ana i nā derivatives carbohydrate hiki i ka biomass lignocellulosic i mālama mua ʻia.
Ua hana pū mākou i ka loiloi haku mele glycan o nā ʻāpana oligosaccharide me ka hoʻohana ʻana i ka TMS glycan derivatization. Me ka hoʻohana ʻana i ka GC-MS, ua hoʻoholo mākou i ka haku mele o nā kō neural (non-derivative) a me nā kō acidic (GluA a me GalA) i loko o ka ʻāpana oligosaccharide (Kiʻi 5). Loaʻa ka waikawa Glucuronic i nā ʻāpana acidic C a me D, ʻoiai loaʻa ka waikawa galacturonic i nā ʻāpana acidic A a me B, ʻo ia mau mea ʻelua he mau ʻāpana DP kiʻekiʻe o nā kō acidic. ʻAʻole wale kēia mau hopena e hōʻoia i kā mākou ʻikepili ELISA a me MALDI, akā kūlike pū kekahi me kā mākou mau haʻawina mua o ka hōʻiliʻili oligosaccharide. 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 o ELISA ma hope e ʻike i nā oligosaccharides recalcitrant soluble i nā ʻano laʻana olaola like ʻole.
ʻOiai ua hōʻoia ʻia nā ʻano hana loiloi mAb i hoʻokumu ʻia e ELISA e kekahi mau ʻano hana like ʻole, makemake mākou e ʻimi hou aku i ka hiki o kēia ʻano hana helu hou. ʻElua mau oligosaccharides kalepa, ʻo 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 paia cell. Hōʻike ka Kiʻi 6 i kahi pilina linear ma waena o ka hōʻailona hoʻopaʻa biotinylated a me ka log concentration o ka oligosaccharide concentration, e hōʻike ana i kahi kumu hoʻohālike adsorption Langmuir. Ma waena o nā mAbs, ua pili ʻo CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, a me CCRC-M151 me XHE, a ua pili ʻo CCRC-M108, CCRC-M109, a me LM11 me A2XX ma luna o kahi laulā o 1 nm a 100 nano. Ma muli o ka palena o ka loaʻa ʻana o nā antibodies i ka wā o ka hoʻokolohua, ua hana ʻia nā hoʻokolohua palena me kēlā me kēia oligosaccharide concentration. Pono e hoʻomaopopo ʻia ma ʻaneʻi he ʻokoʻa loa ka pane ʻana o kekahi mau antibodies i ka oligosaccharide like me kahi substrate, malia paha no ka mea e hoʻopaʻa lākou i nā epitopes ʻokoʻa iki a hiki ke loaʻa nā ʻano hoʻopaʻa like ʻole. E ʻoi aku ka paʻakikī o nā ʻano hana a me nā hopena o ka ʻike pololei ʻana o ka epitope ke hoʻopili ʻia ke ʻano mAb hou i nā laʻana maoli.
Ua hoʻohana ʻia ʻelua mau oligosaccharides kalepa e hoʻoholo ai i ka laulā ʻike o nā ʻano mAbs glycan-targeting like ʻole. Maanei, hōʻike nā pilina linear me ka log concentration o ka oligosaccharide concentration i nā ʻano adsorption Langmuir no (A) XHE me mAb a me (B) A2XX me mAb. Hōʻike nā epitopes pili i nā ʻano o nā oligosaccharides kalepa i hoʻohana ʻia ma ke ʻano he substrates i ka assay.
ʻO ka hoʻohana ʻana i nā antibodies monoclonal i hoʻopaʻa ʻia i ka glycan (ka nānā ʻana o ka glycocomic a i ʻole ka nānā ʻana o ka mAb ma muli o ka ELISA) he mea hana ikaika ia no ka wehewehe hohonu ʻana i ka hapa nui o nā glycans paia cell nui e hana ana i ka biomass mea kanu. Eia nō naʻe, ʻo ka loiloi glycan maʻamau e wehewehe wale i nā glycans paia cell nui, ʻoiai ʻaʻole i hoʻopaʻa pono ʻia ka hapa nui o nā oligosaccharides ma nā papa ELISA. Ma kēia haʻawina, ua hydrolyzed enzymatically ʻia ka AFEX-pretreated corn stover ma kahi kiʻekiʻe o nā solids. Ua hoʻohana ʻia ka loiloi kō e hoʻoholo i ka haku mele ʻana o nā carbohydrates paia cell recalcitrant i loko o ka hydrolyzate. Eia nō naʻe, ua hoʻohaʻahaʻa ʻia ka loiloi mAb o nā oligosaccharides liʻiliʻi i loko o nā hydrolysates, a pono nā mea hana hou e hoʻopaʻa pono i nā oligosaccharides ma nā papa ELISA.
Ke hōʻike nei mākou ma ʻaneʻi i kahi ʻano hana hoʻopaʻa oligosaccharide hou a maikaʻi hoʻi no ka nānā ʻana o mAb ma ka hoʻohui ʻana i ka biotinylation oligosaccharide i ukali ʻia e ka nānā ʻana o ELISA ma nā papa i uhi ʻia ʻo NeutrAvidin™. Ua hōʻike nā oligosaccharides biotinylated i hoʻopaʻa ʻia i ka lawa ʻana o ka pilina no ka antibody e hiki ai ke ʻike wikiwiki a maikaʻi i nā oligosaccharides recalcitrant. Ua hōʻoia ka nānā ʻana i ka haku mele ʻana o kēia mau oligosaccharides paʻakikī e pili ana i ka spectrometry mass 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 nānā ʻana o ELISA me nā antibodies monoclonal i hoʻopaʻa ʻia e glycan e ʻike i nā crosslinks i loko o nā oligosaccharides a hiki ke hoʻohana nui ʻia i nā haʻawina biochemical ʻē aʻe e hōʻike ana i ke ʻano o nā oligosaccharides.
ʻO kēia ʻano hana hoʻopili glycan i hoʻokumu ʻia ma ka biotin ka hōʻike mua i hiki ke noiʻi i nā pilina carbohydrate recalcitrant o nā oligosaccharides soluble i loko o ka biomass mea kanu. Kōkua kēia i ka hoʻomaopopo ʻana i ke kumu he paʻakikī loa kekahi mau ʻāpana o ka biomass i ka wā e pili ana i ka hana biofuel. Hoʻopiha kēia ʻano hana i kahi hakahaka koʻikoʻi i nā ʻano hana loiloi glycome a hoʻonui i kāna noi i kahi ākea o nā substrates ma mua o nā oligosaccharides mea kanu. I ka wā e hiki mai ana, hiki iā mākou ke hoʻohana i ka robotics no ka biotinylation a hoʻohana i ke ʻano a mākou i hoʻomohala ai no ka loiloi throughput kiʻekiʻe o nā laʻana me ka hoʻohana ʻana iā ELISA.
Ua ʻohi ʻia ka ʻōpala kulina (CS) i ulu ʻia mai nā hua hybrid Pioneer 33A14 i ka makahiki 2010 mai Kramer Farms ma Ray, Colorado. Me ka ʻae ʻia e ka mea nona ka ʻāina, hiki ke hoʻohana ʻia kēia biomass no ka noiʻi. Ua mālama ʻia nā hāpana maloʻo < 6% makū i loko o nā ʻeke zip-lock i ka mahana o ka lumi. Ua mālama ʻia nā hāpana maloʻo < 6% makū i loko o nā ʻeke zip-lock i ka mahana o ka lumi. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Ua mālama ʻia nā laʻana maloʻo ma ka haʻahaʻa <6% i loko o nā ʻeke i hoʻopaʻa ʻia me ka zipper ma ka mahana o ka lumi.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%. Mālama ʻia nā laʻana i loko o nā ʻeke zipper ma ka mahana o ka lumi me ka haʻahaʻa <6%.Ua kūlike ke aʻo ʻana me nā alakaʻi kūloko a me ka ʻāina. Ua hana ʻia ka loiloi haku mele me ka hoʻohana ʻana i ke kaʻina hana NREL. Ua ʻike ʻia aia i loko o 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 ka cellulase, β-glucosidase a me Cellic® HTec2 (157 mg protein/ml, lot VHN00001) mai Novozymes (Franklinton, NC, USA)). Ua hāʻawi ʻia ʻo Multifect Pectinase® (72 mg protein/mL), he hui paʻakikī o nā enzymes pectin degradation, e DuPont Industrial Biosciences (Palo Alto, CA, USA). Ua hoʻoholo ʻia nā ʻano protein enzyme ma ka helu ʻana i ka nui o ka protein (a me ka unuhi ʻana i ka hāʻawi ʻana o ka nitrogen non-protein) me ka hoʻohana ʻana i ka loiloi nitrogen Kjeldahl (ʻano AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Ua kūʻai ʻia ka honua Diatomaceous 545 mai EMD Millipore (Billerica, MA). Ua kūʻai ʻia ke kalapona i hoʻāla ʻia (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, a me nā kemika ʻē aʻe a pau mai Sigma-Aldrich (St. Louis, MO).
Ua hana ʻia ka mālama mua ʻana o AFEX ma GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Ua hana ʻia ka mālama mua ʻana ma 140° C. no 15 mau minuke. 46 manawa noho ma ka lakio 1:1 o ka ammonia anhydrous i ka biomass ma 60% (w/w) hoʻouka ʻana i loko o kahi reactor batch benchtop kila kila (Parr Instruments Company). Ua hala he 30 mau minuke. Ua lawe ʻia ka reactor i 140°C a ua hoʻokuʻu koke ʻia ka ammonia, e ʻae ana i ka biomass e hoʻi koke i ka mahana o ka lumi. Ua like ke ʻano o ka AFEX pre-treated corn stover (ACS) me ka corn stover i mālama ʻole ʻia (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 ʻana o nā oligosaccharides. Ua hana ʻia ka hydrolysis enzymatic o ACS me ka hoʻohana ʻana i kahi hui enzyme kalepa e komo pū ana me Cellic® Ctec2 10 mg protein/g glucan (i loko o ka biomass i mālama mua ʻia), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, a me Multifect Pectinase (Genencor Inc, USA). ), 5 mg protein/g dextran. Ua hana ʻia ka hydrolysis enzymatic i loko o kahi bioreactor 5-lita me ka nui 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 ma o ka centrifugation ma 6000 rpm no 30 mau minuke a laila ma 14000 rpm no 30 mau minuke e wehe i nā mea paʻa i hoʻoheheʻe ʻole ʻia. A laila ua hoʻokomo ʻia ka hydrolyzate i ka kānana sterile ma o kahi kīʻaha kānana 0.22 mm. Ua mālama ʻia ka hydrolyzate i kānana ʻia i loko o nā ʻōmole sterile ma 4° C. a laila ua hoʻokaʻawale ʻia ma luna o ke kalapona.
Ka nānā ʻana i ka haku mele ʻana o nā laʻana biomass i hoʻokumu ʻia i ka extract e like me nā kaʻina hana loiloi keʻena hoʻokolohua NREL: ka hoʻomākaukau ʻana i nā laʻana no ka nānā ʻana i ka haku mele (NREL/TP-510-42620) a me ka hoʻoholo ʻana i nā kalapona kūkulu a me ka lignin i loko o ka biomass (NREL/TP-510 - 42618)47.
Ua hana ʻia ka loiloi Oligosaccharide o ke kahawai hydrolyzate ma kahi unahi 2 ml me ka hoʻohana ʻana i kahi ʻano hana hydrolysis waikawa autoclave. E kāwili i ka hāpana hydrolyzate me 69.7 µl o 72% sulfuric acid i loko o kahi paipu moʻomeheu pāpale wili 10 ml a hoʻomoʻa no 1 hola ma luna o kahi benchtop ma 121 °C, e hoʻomaʻalili ma luna o ka hau a kānana i loko o kahi vial chromatography wai hana kiʻekiʻe (HPLC). Ua hoʻoholo ʻia ka nui o nā oligosaccharides ma ka unuhi ʻana i ka nui 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 nā pae glucose, xylose, a me arabinose i loko o ka biomass hydrolyzed acid me ka hoʻohana ʻana i kahi ʻōnaehana Shimadzu HPLC i lako me kahi autosampler, mea hoʻomehana kolamu, pamu isocratic, a me ka mea ʻike refractive index ma kahi kolamu Bio-Rad Aminex HPX-87H. Ua mālama ʻia ke kolamu ma 50°C a ua eluted me 0.6 ml/min 5 mM H2SO4 i loko o ka wai. kahe.
Ua hoʻoheheʻe ʻia ka supernatant hydrolyzate a ua kālailai ʻia no ka ʻike monomer a me oligosaccharide. Ua kālailai ʻia nā kō 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 mālama ʻia ka mahana o ke kolamu ma 80°C, ua hoʻohana ʻia ka wai ma ke ʻano he pae mobile me ka nui o ke kahe ʻana o 0.6 ml/min. Ua hoʻoholo ʻia nā Oligosaccharides e ka hydrolysis i loko o ka waikawa dilute ma 121°C e like me nā ʻano i wehewehe ʻia ma nā refs. 41, 48, 49.
Ua hana ʻia ka loiloi Saccharide ma nā koena biomass maka, AFEX i mālama mua ʻia a me nā koena biomass āpau i hydrolyzed ʻole (me ka hana ʻana o nā extracts paia cell serial a me kā lākou mAb screening) me ka hoʻohana ʻana i nā kaʻina hana i wehewehe mua ʻia 27, 43, 50, 51. No ka loiloi glycome, ua hoʻomākaukau ʻia nā koena waiʻona-insoluble o ka mea paia cell mea kanu mai nā koena biomass a ua hoʻokomo ʻia i ka serial extraction me nā reagents ikaika 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 acid chlorite e like me ka mea i wehewehe mua ʻia 52,53. A laila ua hoʻokomo ʻia nā extracts iā ELISA e kūʻē i kahi panela paʻakikī o mAb50s i kuhikuhi ʻia i ka glycan paia cell, a ua hōʻike ʻia nā hopena hoʻopaʻa mAb ma ke ʻano he palapala wela. Ua kūʻai ʻia nā mAbs e kuhikuhi ana i ka glycan paia cell mea kanu mai nā waihona hale hana (CCRC, JIM a me MAC series).
ʻO ka biotinylation hoʻokahi-ʻanuʻu o nā oligosaccharides. Ua hana ʻia ka hoʻohuihui ʻana o nā kalapona me ka biotin-LC-hydrazide me ka hoʻohana ʻana i ke kaʻina hana ma lalo nei. Ua hoʻoheheʻe ʻia ʻo Biotin-LC-hydrazide (4.6 mg/12 μmol) i loko o ka dimethyl sulfoxide (DMSO, 70 μl) ma o ka hoʻoulu ikaika ʻana a me ka hoʻomehana ʻana ma 65° C. no 1 min. Ua hoʻohui ʻia ka waikawa acetic glacial (30 µl) a ua ninini ʻia ka hui ʻana ma luna o ka sodium cyanoborohydride (6.4 mg/100 µmol) a ua hoʻoheheʻe loa ʻia ma hope o ka hoʻomehana ʻana ma 65° C. no kahi o 1 minuke. A laila, mai 5 a 8 μl o ka hui hopena i hoʻohui ʻia i ka oligosaccharide maloʻo (1-100 nmol) e loaʻa ai kahi 10-fold a ʻoi aku paha ka nui o ka molar o ka lepili ma luna o ka hopena hōʻemi. Ua hana ʻia ka hopena ma 65°C no 2 h, ma hope o ka hoʻomaʻemaʻe koke ʻia ʻana o nā laʻana. ʻAʻohe sodium cyanoborohydride i hoʻohana ʻia i nā hoʻokolohua lepili me ka ʻole o ka hōʻemi ʻana, a ua hana ʻia nā laʻana ma 65° C. no 2.5 mau hola.
ʻO ka uhi ʻana o ELISA a me ka holoi ʻana i nā laʻana o nā oligosaccharides biotinylated. Ua hoʻohui ʻia he 25 μl o nā laʻana biotinylated (100 μl o kēlā me kēia laʻana i hoʻohuihui ʻia i loko o 5 ml o 0.1 M Tris buffer solution (TBS)) i kēlā me kēia lua o ka pā i uhi ʻia me avidin. Ua uhi ʻia nā lua hoʻomalu me 50 μl o ka biotin ma kahi nui o 10 μg/ml i loko o 0.1 M TBS. Ua hoʻohana ʻia ka wai deionized ma ke ʻano he uhi no nā ana hakahaka. Ua hoʻomoʻa ʻia ka papa no 2 mau hola ma ka mahana o ka lumi i ka pouli. E holoi i ka pā i 3 mau manawa me ka waiū skimmed 0.1% i loko o 0.1 M TBS me ka hoʻohana ʻana i ka papahana helu 11 no Grenier flat 3A.
Hoʻohui a me ka holoi ʻana i nā antibodies mua. E hoʻohui i 40 µl o ka antibody mua i kēlā me kēia lua. E hoʻomoʻa i ka microplate no 1 hola ma ka mahana o ka lumi i ka pouli. A laila ua holoi ʻia nā papa i 3 mau manawa me ka waiū 0.1% i loko o 0.1M TBS me ka hoʻohana ʻana i ka polokalamu holoi #11 no Grenier Flat 3A.
E hoʻohui i ka antibody lua a holoi. E hoʻohui i 50 µl o ka antibody lua o ka ʻiole/ʻiole (hoʻoheheʻe ʻia 1:5000 i ka waiū 0.1% i loko o 0.1 M TBS) i kēlā me kēia lua. E hoʻomoʻa i ka microplate no 1 hola ma ka mahana o ka lumi i ka pouli. A laila ua holoi ʻia nā microplate i 5 mau manawa me ka waiū 0.1% i loko o 0.1 M TBS me ka hoʻohana ʻana i ka papahana holoi pā Grenier Flat 5A #12.
Ke hoʻohui nei i kahi substrate. E hoʻohui i 50 µl o 3,3′,5,5′-tetramethylbenzidine (TMB) i ke 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). E hoʻomoʻa i ka microplate ma ka mahana o ka lumi no 30 mau minuke. I ka pōʻeleʻele.
E hoʻopau i ke kaʻina hana a heluhelu i ka papa. E hoʻohui i 50 µl o 1 N sulfuric acid i kēlā me kēia lua a hoʻopaʻa i ka absorbance mai 450 a 655 nm me ka hoʻohana ʻana i kahi mea heluhelu ELISA.
E hoʻomākaukau i nā hopena 1 mg/ml o kēia mau analytes i loko o 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 (kūlana kūloko). Ua hoʻomākaukau ʻia ʻelua mau kūlana ma ka hoʻohui ʻana i nā hopena kō 1 mg/mL i hōʻike ʻia ma ka Papa 1. Hoʻopaʻahau ʻia nā laʻana a hoʻopaʻa ʻia ma -80° C. a hiki i ka wā e wehe ʻia ai nā wai āpau (ma kahi o 12-18 mau hola).
E hoʻohui i 100–500 µg o ka hāpana i nā paipu poʻi wili ma kahi kaulike analytical. E hoʻopaʻa i ka nui i hoʻohui ʻia. ʻOi aku ka maikaʻi o ka hoʻoheheʻe ʻana i ka hāpana i loko o kahi ʻano kikoʻī o ka solvent a hoʻohui iā ia i ka paipu ma ke ʻano he aliquot wai. E hoʻohana i 20 µl o 1 mg/ml inositol ma ke ʻano he maʻamau kūloko no kēlā me kēia paipu hāpana. Pono ka nui o ke maʻamau kūloko i hoʻohui ʻia i ka hāpana e like me ka nui o ke maʻamau kūloko i hoʻohui ʻia i ka paipu maʻamau.
E hoʻohui i 8 ml o ka methanol anhydrous i loko o kahi ʻōmole poʻi wili. A laila 4 ml o 3 N. methanolic HCl solution, i uhi ʻia a lulu ʻia. ʻAʻole hoʻohana kēia kaʻina hana i ka wai.
E hoʻohui i 500 µl o ka hopena methanol 1 M HCl i nā laʻana oligosaccharide a me nā paipu TMS maʻamau. Ua hoʻomoʻa 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 mahana o ka lumi me ka hoʻohana ʻana i kahi manifold maloʻo. E hoʻohui i 200 µl MeOH a maloʻo hou. Hana hou ʻia kēia kaʻina hana i ʻelua manawa. E hoʻohui i 200 µl o ka methanol, 100 µl o ka pyridine a me 100 µl o ka acetic anhydride i ka laʻana a hui maikaʻi. E hoʻomoʻa i nā laʻana ma ka mahana o ka lumi 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 hoʻomehana i ka ʻōmole i uhi ʻia no 20 mau minuke. 80°C, a laila e hoʻomaʻalili ʻia i ka mahana o ka lumi. E hoʻohana i kahi manifold maloʻo e hoʻomaloʻo hou aku i ka hāpana i kahi nui o 50 µl. He mea nui e hoʻomaopopo ʻaʻole mākou i ʻae i nā hāpana e maloʻo loa.
E hoʻohui i 2 ml o ka hexane a kāwili maikaʻi ma ka vortexing. E hoʻopiha i nā wēlau o nā pipettes Pasteur (5-8 mm) me kahi ʻāpana huluhulu aniani ma ke kau ʻana i ka huluhulu aniani ma luna o kahi pipette 5-3/4 ʻīniha ke anawaena. Ua centrifuged ʻia nā laʻana ma 3000 g no 2 mau minuke. Hoʻopūʻiwa ʻia nā koena hiki ʻole ke hoʻoheheʻe ʻia. E hoʻomaloʻo i ka laʻana i 100-150 µl. Ua hoʻokomo ʻia kahi 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 mau minuke (Papa 2).