Waad ku mahadsan tahay booqashada Nature.com.Nooca browserka aad isticmaalayso waxa uu leeyahay taageerada CSS oo xadidan.Waayo-aragnimada ugu fiican, waxaan kugula talineynaa inaad isticmaasho browser-ka la cusboonaysiiyay (ama aad damiso Habka Laqabsiga ee Internet Explorer).Dhanka kale, si loo xaqiijiyo taageerada joogtada ah, waxaan ka dhigi doonaa goobta iyada oo aan lahayn qaabab iyo JavaScript.
Hababka spectrometric cusub ee difaaca jirka iyo tirada badan ee falanqaynta adag ee oligosaccharides joogtada ah ee kariyaha galleyda oo hore loogu sii daayay AFEX.Lignocellulosic biomass waa beddelka joogtada ah ee shidaalka fosil waxaana si weyn loo adeegsadaa horumarinta cilmiga bayoolajiga ee wax soo saarka sida cuntada, quudinta, shidaalka iyo kiimikooyinka.Furaha tiknoolajiyadani waa horumarinta hababka tartanka qiimaha leh ee loogu beddelayo karbohaydraytyada adag ee ku jira gidaarada unugyada dhirta oo loo beddelo sonkor fudud sida glucose, xylose iyo arabinose.Sababtoo ah lignocellulosic biomass waa mid aad u madax adag, waa in lagu sameeyaa daawaynta thermochemical (tusaale, ammonia fiber exfoliation (AFEX), acids dilute acid (DA), dareeraha ionic (IL)) iyo daaweynta noolaha (tusaale, enzymatic hydrolysis iyo halsano microbial) oo isku dhafan si loo helo badeecada la rabo..Si kastaba ha noqotee, marka enzymes fangas ganacsi loo isticmaalo geeddi-socodka hydrolysis, kaliya 75-85% sonkorta milmay ee la sameeyay waa monosaccharides, iyo 15-25% soo haray waa oligosaccharides milmay, oo aan la dabooli karin, kuwaas oo aan had iyo jeer heli karin microorganisms.Markii hore, waxaan si guul leh u go'doominay oo aan sifeynay oligosaccharides madax adag iyadoo la adeegsanayo isku-darka kaarboon iyo diatomaceous kala soocida dhulka iyo cabbirka ka saarida chromatography, sidoo kale waxaan baarnay hantidooda xakameynta enzyme.Waxaan ogaanay in oligosaccharides ay ku jiraan heerka sare ee polymerization (DP) beddelka methylated uronic acid ay aad u adag tahay in lagu farsameeyo isku-dhafka enzyme-ga ganacsiga marka loo eego DP hoose iyo oligosaccharides dhexdhexaad ah.Halkan waxaan ku soo wargelineynaa isticmaalka dhowr habab oo dheeraad ah, oo ay ku jiraan glycan profiling iyadoo la isticmaalayo monoclonal antibodies (mAbs) gaar ah si ay u dhirta biomass glycans si ay u gartaan curaarta glycan ee derbiyada unugyada dhirta iyo enzymatic hydrolysates, matrix-caawiyey ionization laser desorption, wakhtiga-of-flight mass-spectrometry..MALDI-TOF-MS) waxay isticmaashaa meelaha ugu sarreeya ee ogaanshaha qaab-dhismeed-warbixineed oo lagu helo spectroscopy ka dib burburka labaad ee ion taban, chromatography gaaska iyo spectrometry mass (GC-MS) si loo garto curaarta oligosaccharides leh iyo la'aanteed.Sababtoo ah cabbirka yar ee oligosaccharides (DP 4-20), molecules-yadani way adagtahay in loo isticmaalo isku-xidhka mAB iyo sifeynta.Si looga gudbo dhibaatadan, waxaanu isticmaalnay hab cusub oo isku xidhka biotin-ku-salaysan oligosaccharides immobilization kaas oo si guul leh u calaamadiyay inta badan oligosaccharides milmay DP hoose ee dusha sare ee microplate, kaas oo markaa loo adeegsaday nidaamka mAb-ga wax soo saarka sare ee falanqaynta ligation gaar ah.Habkan cusubi wuxuu fududayn doonaa horumarinta glycome-ka-soo-saarka sare ee mustaqbalka kaas oo loo isticmaali karo in lagu go'doomiyo oo lagu garto oligosaccharides ee ku jira biomarkers ujeedooyin ogaanshaha.
Lignocellulosic biomass, oo ka kooban beeralayda, kaymaha, cawska iyo agabka alwaaxyada ah, waa quudin suurtagal ah oo lagu soo saari karo wax soo saarka noolaha, oo ay ku jiraan cunto, quudin, shidaal iyo horudhac kiimikaad si loo soo saaro alaab qiimo sare leh.Kaarbohaydraytyada (sida cellulose iyo hemicellulose) ee ku jira gidaarada unugyada dhirta waxaa lagu kala saaraa monosaccharides iyada oo la adeegsanayo farsamaynta kiimikada iyo biotransformation (sida enzymatic hydrolysis iyo halsano microbial).Daaweynta hore ee caadiga ah waxaa ka mid ah ballaarinta ammonia fiber (AFEX), dilute acid (DA), dareeraha ionic (IL), iyo qarxinta uumiga (SE), kuwaas oo isticmaala isku-darka kiimikooyinka iyo kulaylka si loo yareeyo wax soo saarka lignocellulose iyadoo la furayo derbiyada unugyada dhirta3,4.madax adayga walxaha, 5. Hydrolysis Enzymatic waxaa lagu fuliyaa at load adag sare iyadoo la isticmaalayo ganacsiga firfircoon ee karbohaydrayt-ku jira enzymes (CAZymes) iyo halsano microbial isticmaalaya khamiir transgenic ama bakteeriyada si ay u soo saaraan bio-ku salaysan shidaalka iyo kiimikooyinka 6.
CAZymes ee ensaymes-yada ganacsigu waxay ka kooban yihiin isku-dhafan adag oo ensaymes ah kuwaas oo si isku mid ah u kala gooya curaarta karbohaydrayt-sonkorta adag si ay u sameeyaan monosaccharides2,7.Sida aan horayba u soo sheegnay, shabakada adag ee polymers caraf udgoon ee lignin leh karbohaydraytyadu waxay ka dhigtaa kuwo aad u jilicsan, taas oo keenta beddelka sonkorta aan dhamaystirnayn, ururinta 15-25% oligosaccharides jinsiga ah oo aan la soo saarin inta lagu jiro enzymatic hydrolysis ee biomass pretreated.Tani waa dhibaato caadi ah oo leh hababka kala duwan ee daaweynta biomass ka hor.Sababaha qaar ee dhalooyinkaan waxaa ka mid ah xannibaadda ensaymka inta lagu jiro hydrolysis, ama maqnaanshaha ama heerarka hoose ee enzymes muhiimka ah ee lagama maarmaanka ah ee looga baahan yahay inay jebiyaan curaarta sonkorta ee bioomass dhirta.Fahmidda qaabka iyo sifooyinka qaabdhismeedka sonkorta, sida curaarta sonkorta ee oligosaccharides, waxay naga caawin doontaa hagaajinta beddelka sonkorta inta lagu jiro hydrolysis, samaynta hababka bayotechnoolajiyada kharash-tartan la leh alaabada laga soo saaro batroolka.
Go'aaminta qaab dhismeedka karbohaydraytyadu waa caqabad waxayna u baahan tahay habab isku dhafan sida chromatography dareere (LC) 11,12, nukliyeerka magnetic resonance spectroscopy (NMR) 13, electrophoresis capillary (CE) 14,15,16 iyo mass spectrometry (MS)17., siddeed iyo toban.Hababka MS sida spectrometry mass-time-of-flight mass spectrometry leh laser desorption iyo ionization iyadoo la isticmaalayo matrix (MALDI-TOF-MS) waa hab isku dhafan oo lagu aqoonsanayo qaababka karbohaydraytyada.Dhawaan, Isku-dhafka Isku-dhafka ah (CID) tandem MS ee soodhiyamka ion adducts ayaa si ballaaran loo isticmaalay si loo aqoonsado faraha u dhigma boosaska lifaaqa oligosaccharides, qaabeynta anomeric, taxanaha, iyo jagooyinka laanta 20, 21.
Falanqaynta Glycan waa qalab aad u fiican oo si qoto dheer loogu aqoonsanayo curaarta karbohaydrayt22.Habkani waxa uu adeegsadaa unugyada difaaca jirka ee monoclonal (mAbs) ee lagu hagayo in lagu beero gidaarka unugga glycan si ay u baaraan si ay u fahmaan isku xirka karbohaydraytyada adag.In ka badan 250 mAbs ayaa laga heli karaa adduunka oo dhan, oo loogu talagalay inay ka soo horjeedaan oligosaccharides toosan oo kala duwan iyadoo la adeegsanayo saccharide24 kala duwan.Dhowr mAbs ayaa si weyn loo isticmaalay si loo tilmaamo qaab dhismeedka, halabuurka, iyo wax ka beddelka gidaarka unugyada dhirta, maadaama ay jiraan farqi weyn oo ku xiran nooca unugyada dhirta, xubinta, da'da, heerka korriinka, iyo deegaanka koritaanka25,26.Dhawaanahan, habkan ayaa loo isticmaalay si loo fahmo dadyowga vesicle ee nidaamka dhirta iyo xayawaanka iyo doorarkooda ku saabsan gaadiidka glycan sida lagu go'aamiyo calaamadaha subcellular, marxaladaha koritaanka, ama kiciyeyaasha deegaanka, iyo si loo go'aamiyo dhaqdhaqaaqa enzymatic.Qaar ka mid ah qaababka kala duwan ee glycans iyo xylans ee la aqoonsaday waxaa ka mid ah pectin (P), xylan (X), mannan (M), xyloglucans (XylG), glucans bond bond (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan (GarbX)
Si kastaba ha ahaatee, inkastoo dhammaan dadaalladan cilmi-baarista ah, kaliya dhowr daraasadood ayaa diiradda saarey dabeecadda isku-ururinta oligosaccharides inta lagu jiro culeyska culus ee culus (HSL), oo ay ku jiraan sii-deynta oligosaccharides, dhererka silsiladda oligomeric isbeddelka xilliga hydrolysis, noocyo kala duwan oo DP polymers ah, iyo curyaashooda.qaybinta 30,31,32.Dhanka kale, inkasta oo falanqaynta glycan ay caddeeyeen inay tahay qalab waxtar leh oo loogu talagalay falanqaynta dhamaystiran ee qaabka glycan, way adagtahay in la qiimeeyo biyo-miirashada hoose ee DP oligosaccharides iyadoo la adeegsanayo hababka antibody.DP yar oligosaccharides oo leh miisaan kelli ah oo ka yar 5-10 kDa kuma xidhna ELISA taarikada 33, 34 waana la dhaqaa ka hor intaan lagu darin antibody.
Halkan, markii ugu horeysay, waxaan ku muujineynaa baaritaanka ELISA ee taarikada avidin-dahaarka leh iyadoo la adeegsanayo unugyada difaaca jirka ee monoclonal, isku darka hal-tallaabo biotinylation-ka oo loogu talagalay oligosaccharides-ka-soo-baxa dareeraha leh iyo falanqaynta glycome.Habkayaga falanqaynta glycome waxaa ansixiyay MALDI-TOF-MS iyo GC-MS oo ku salaysan falanqaynta isku xidhka oligosaccharides ee dhammaystiran iyadoo la adeegsanayo trimethylsilyl (TMS) soo saarista isku-dhafka sonkorta hydrolyzed.Habkan hal-abuurka leh waxaa loo horumarin karaa hab wax-soo-saar sare leh mustaqbalka waxaana laga heli karaa codsi ballaadhan cilmi-baarista biomedical35.
Beddelka tarjumaada ka dib ee ensaymes iyo unugyada difaaca jirka, sida glycosylation,36 waxay saameeyaan dhaqdhaqaaqooda noole.Tusaale ahaan, isbeddelada glycosylation ee borotiinka serum ayaa door muhiim ah ka ciyaara arthritis-ka bararka, iyo isbeddelada glycosylation waxaa loo isticmaalaa calaamadaha ogaanshaha37.Glycans kala duwan ayaa lagu soo warramey suugaanta si ay si diyaar ah ugu soo baxaan cudurro kala duwan, oo ay ku jiraan cudurrada bararka ee joogtada ah ee mareenka caloosha iyo mindhicirka iyo beerka, caabuqyada fayraska, ugxan-sidaha, naaska, iyo kansarka qanjirka 'prostate'38,39,40.Fahamka qaabka glycans iyadoo la adeegsanayo hababka ELISA ee ku salaysan antibody-ku waxay ku siin doontaa kalsooni dheeraad ah ogaanshaha cudurka iyada oo aan la isticmaalin hababka MS ee adag.
Daraasaddeenii hore waxay muujisay in oligosaccharides madax-adayggu ay ku sii jireen biyo la'aan ka dib daaweynta hore iyo enzymatic hydrolysis (Jaantus 1).Shaqadeenii hore ee la daabacay, waxaan ku soo saarnay qaab dhuxul firfircoon oo weji adag laga soo saarayo si loo go'doomiyo oligosaccharides ee AFEX-pretreated galleyda hadhuudhka hydrolyzate (ACSH)8.Ka dib markii la soosaaray iyo kala-soocidda bilawga ah, oligosaccharides ayaa lagu sii kala qaybiyay cabbirka chromatography (SEC) waxaana loo ururiyay siday u kala horreeyaan miisaanka kelli.Sonkorta monomers iyo oligomers oo laga sii daayay daawooyin kala duwan ayaa lagu falanqeeyay falanqaynta halabuurka sonkorta.Marka la barbardhigo waxyaabaha ku jira oligomers sonkorta ee laga helay habab kala duwan oo daaweyn ah, joogitaanka oligosaccharides madax adag waa dhibaato caadi ah oo ku saabsan beddelka biomass ee monosaccharides waxayna u horseedi kartaa hoos u dhigista soosaarka sonkorta ugu yaraan 10-15% iyo xitaa ilaa 18%.USHabkan waxaa loo isticmaalaa wax soo saarka ballaaran ee jajabyada oligosaccharides.Natiijada ACH iyo jajabkeeda xiga ee leh miisaanyo kala duwan ayaa loo isticmaalay qalab tijaabo ah oo loogu talagalay oligosaccharides ee shaqadan.
Ka dib daawaynta ka hor iyo enzymatic hydrolysis, oligosaccharides joogto ah ayaa ku hadhay biyo la'aan.Halkan (A) waa habka kala-soocidda oligosaccharides kaas oo oligosaccharides ay ka go'an tahay AFEX-pretreated hadhuudhka hadhuudhka hydrolyzate (ACSH) iyada oo la adeegsanayo sariir bacaysan oo kaarboon firfircoon iyo dhul diatomaceous ah;(B) Habka loo kala saaro oligosaccharides.Oligosaccharides-ka waxaa lagu sii kala saaray cabbirka chromatography (SEC);(C) Saccharide monomers iyo oligomers oo laga sii daayay daawooyin kala duwan (asiidh la qasi jiray: DA, dareeraha ionic: IL iyo AFEX).Xaaladaha hydrolysis enzymatic: culeyska sare ee culeyska 25% (w / w) (qiyaastii 8% glucan loading), 96 saacadood hydrolysis, 20 mg / g loading enzym ganacsiga (Ctec2: Htec2: MP-2: 1: 1 ratio) iyo (D) Sonkorta monomers iyo oligomers of glucose, xylose la sii daayay oo ka soo horraysin AFcorn.
Falanqaynta Glycan waxay caddaysay inay tahay qalab waxtar leh oo loogu talagalay falanqaynta qaabdhismeedka dhammaystiran ee glycans ee laga soocay hadhaaga bioomass adag.Si kastaba ha ahaatee, saccharide-ka biyaha ku milma ayaa si hoose loo soo bandhigay iyadoo la isticmaalayo habkan soo jireenka ah41 sababtoo ah oligosaccharides miisaan yar ayaa adag in lagu dhaqdhaqaajiyo taarikada ELISA waxaana la dhaqaa ka hor intaan lagu darin antibody.Sidaa darteed, xidhitaanka iyo sifaynta antibody-ka, habka biotinylation hal-tallaabo ah ayaa loo adeegsaday in lagu dahaadho oligosaccharides milmay, aan waafaqsanayn taarikada ELISA ee avidin-dahaarka leh.Habkan waxaa lagu tijaabiyay iyadoo la adeegsanayo ACSH-kii hore ee aan soo saarnay iyo jajab ku salaysan miisaankeeda kelli (ama heerka polymerization, DP).Hal-tallaabo biotinylation ayaa loo isticmaalay si loo kordhiyo xidhiidhka isku xidhka oligosaccharides iyadoo lagu daray biotin-LC-hydrazide si loo yareeyo dhamaadka karbohaydraytyada (Jaantus. 2).Xalka, kooxda hemiacetal ee dhamaadka dhimista waxay la falgalaan kooxda hydrazide ee biotin-LC-hydrazide si ay u sameeyaan bond hydrazone.Joogitaanka wakiilka yaraynta NaCNBH3, dammaanadda hydrazone waxaa lagu dhimay badeecada dhamaadka biotinylated deggan.Wax ka beddelka sonkorta dhimista dhamaadka, xidhitaanka oligosaccharides DP hoose ee taarikada ELISA waxay noqotay suurtagal, daraasaddeenana tan waxaa lagu sameeyay taarikada avidin-dahaarka leh iyadoo la adeegsanayo mAbs-glycan-la beegsado.
Baaritaanka unugyada difaaca jirka ee monoclonal ee ku saleysan ELISA ee oligosaccharides biotinylated.Halkan (A) isku-darka biotinylation ee oligosaccharides iyo baadhista ELISA ee ku xigta oo leh mAbs-ga glycan-ku wajahan ee NeutrAvidin ee dahaarka leh iyo (B) waxay tusinaysaa habraac hal-tallaabo ah oo loogu talagalay biotinylation-ka alaabta falcelinta.
Taarikada dahaarka leh ee oligosaccharides-ka-hortagga unugyada difaaca jirka ayaa markaa lagu daray unugyada difaaca aasaasiga ah iyo kuwa sare waxaana lagu dhaqay dhexdhexaad iftiin iyo waqti xasaasi ah.Ka dib marka xidhidhiyaha difaaca jirka la dhammeeyo, ku dar substrate TMB si aad saxanka ugu shubto.Falcelinta ayaa ugu dambeyntii lagu joojiyay sulfuric acid.Taarikada la kariyey waxaa lagu falanqeeyay iyada oo la adeegsanayo akhristaha ELISA si loo go'aamiyo xoogga isku-xidhka ka-hortagga kasta si loo ogaado isku-xidhka-isku-tallaabta gaarka ah ee anti-body-ga.Faahfaahinta iyo cabbirrada tijaabada, eeg qaybta u dhiganta "Alaabaha iyo Hababka".
Waxaan muujineynaa isticmaalka habkan cusub ee la sameeyay ee codsiyada gaarka ah anagoo tilmaamayna oligosaccharides-ka milmaya ee ku jira ACSH iyo sidoo kale jajabyada oligosaccharides ee cayriin iyo nadiifsan oo ka soocaya lignocellulosic hydrolysates.Sida ku cad Jaantuska 3, Xylans-ka ugu caansan ee lagu beddelo epitope-ka ee lagu aqoonsaday ACSH iyadoo la adeegsanayo hababka baaritaanka bioacylated glycome assay badanaa waa uronic (U) ama methyluronic (MeU) iyo pectic arabinogalactans.Intooda badan ayaa sidoo kale laga helay daraasaddeennii hore ee falanqaynta glycans ee adkaha aan-hydrolyzed (UHS)43.
Ogaanshaha epitopes oligosaccharides recalcitrant iyadoo la isticmaalayo antibody monoclonal ah oo lagu hagayo glycan gidaarka unugga.Jajabka "dhexdhexaad ah" waa jajabka ACN iyo "asiidhka" jajabku waa jajabka FA.Casaan ifaya oo ku yaal khariidadda kulaylka ayaa muujinaya tusmooyin sare oo buluug ah, buluugyo dhalaalayana waxay muujinayaan asal madhan.Qiimaha midabka ee miisaanka waxay ku salaysan yihiin qiyamka OD cayriin ee qaababka N=2.Xuubabka ugu muhiimsan ee ay aqoonsan yihiin unugyada difaaca jirka ayaa lagu muujiyay dhanka midig.
Qaab-dhismeedyadan aan unugyadu ahayn waxaa lagu kala furfuri kari waayay unugyada ugu caansan ee unugyada iyo hemicellulases ee isku dhafka ganacsiga ee la tijaabiyay, kaas oo ay ku jiraan enzymes-yada ganacsiga ee inta badan la isticmaalo.Sidaa darteed, enzymes caawiye cusub ayaa looga baahan yahay hydrolysiskooda.La'aanteed enzymes-ka aan-cellulose-ka ahayn ee lagama maarmaanka ah, curaartan aan-cellulose-ka ahayn waxay ka hortagayaan beddelaad dhammaystiran oo loogu talagalay monosaccharides, xitaa haddii polymers-ka sonkorta waalidkood si weyn loogu daadiyo jajabyo gaaban oo lagu kala diro iyadoo la adeegsanayo isku-darka ensaymes-ka ganacsiga.
Daraasad dheeraad ah oo ku saabsan qaybinta calaamadaha iyo xoogga ku-xidhka ayaa muujisay in epitopes-ku-xirnaanta ay ka hooseeyaan jajabyada sonkorta DP ee sarreeya (A, B, C, DP ilaa 20+) marka loo eego jajabyada DP ee hooseeya (D, E, F, DP) ee dimers) (Jaantus. 1).Qaybaha aysiidhdu waxay ku badan yihiin xuubabka unugyada aan unugga ahayn marka loo eego jajabyada dhexdhexaadka ah.Ifafaalayaashani waxay la socdaan qaabkii lagu arkay daraasaddeennii hore, halkaas oo DP sare iyo unugyada aashitada ay aad uga adkeysinaayeen hydrolysis enzymatic.Sidaa darteed, joogitaanka epitopes glycan ee aan-cellulose ahayn iyo beddelka U iyo MeU waxay si weyn uga qayb qaadan karaan xasilloonida oligosaccharides.Waa in la ogaadaa in wax ku oolnimada ku-xidhka iyo ogaanshaha ay noqon karto dhibaato DP oligosaccharides hooseeya, gaar ahaan haddii epitope-ku yahay dimeric ama trimeric oligosaccharides.Tan waxaa lagu tijaabin karaa iyadoo la adeegsanayo oligosaccharides ganacsi oo dhererkoodu kala duwan yahay, mid walbana wuxuu ka kooban yahay hal epitope oo keliya oo ku xiran mAb gaar ah.
Sidaa darteed, isticmaalka unugyada difaaca jirka ee qaab-dhismeedku waxay daaha ka qaadeen noocyo ka mid ah curaarta dib-u-celinta.Iyada oo ku xidhan nooca antibody ee loo isticmaalo, qaabka ligation ku habboon, iyo xoogga calaamada ay soo saartaa (inta ugu badan iyo ugu yaraan), enzymes cusub ayaa la aqoonsan karaa oo lagu dari karaa nus-tiro ah isku-darka enzyme si loo helo glycoconversion dhamaystiran.Qaadashada falanqaynta ACSH oligosaccharides tusaale ahaan, waxaan u abuuri karnaa xogta curaarta glycan walxo kasta oo noole ah.Waa in lagu ogaadaa halkan in la tixgeliyo xidhiidhka kala duwan ee unugyada difaaca jirka, iyo haddii xiriirkooda aan la garanayn, tani waxay abuuri doontaa dhibaatooyin gaar ah marka la barbardhigo calaamadaha unugyada difaaca jirka ee kala duwan.Intaa waxaa dheer, isbarbardhigga curaarta glycan ayaa laga yaabaa inay si fiican u shaqeyso inta u dhaxaysa shaybaarada isla antibody-ka.Xirmooyinkan madax-adayggu waxay markaa ku xidhnaan karaan xogta CAZyme, kaas oo aan ka aqoonsan karno enzymes, dooran karno ensaymes musharaxa iyo tijaabinta enzymes-jabinta, ama horumarinta hababka microbial si ay u muujiyaan enzymes-yadan si loogu isticmaalo biorefineries44.
Si loo qiimeeyo sida hababka difaaca jirka u buuxiyaan habab kale oo lagu garto oligosaccharides miisaanka molecular hooseeya ee ku jira lignocellulosic hydrolysates, waxaan samaynay MALDI (Jaantus. 4, S1-S8) iyo falanqaynta saccharide TMS-ka soo jeeda oo ku salaysan GC-MS oo isku mid ah (Jaantus. 5) qayb oligosaccharides.MALDI waxaa loo isticmaalaa in lagu barbardhigo in qaybinta tirada guud ee molecules oligosaccharides ay la mid tahay qaab dhismeedka loogu talagalay.On berde.4 wuxuu muujinayaa MS qaybaha dhexdhexaadka ah ACN-A iyo ACN-B.Falanqaynta ACN-A waxay xaqiijisay noocyo kala duwan oo sonkor pentose ah oo u dhexeeya DP 4-8 (Jaantus. 4) ilaa DP 22 (Jaantus. S1), kuwaas oo miisaankoodu u dhigmayo MeU-xylan oligosaccharides.Falanqaynta ACN-B waxay xaqiijisay taxanaha pentose iyo glucoxylan oo leh DP 8-15.Walaxda dheeriga ah sida Jaantuska S3, maabab qaybinta unugyada aashitada FA-C waxay muujinayaan noocyo kala duwan oo ah (Me) U oo lagu beddelay sonkor pentose ah oo leh DP 8-15 kuwaas oo la jaan qaadaya xylansyada la beddelay ee laga helay ELISA-ku salaysan baadhista mAb.Xuubyadu waa joogto.
MALDI-MS spectrum oligosaccharides milma oo aan waafaqsanayn oo ku jira ACS.Halkan, (A) ACN-A jajabyo kala duwan oo miisaan hooseeya oo ay ku jiraan methylated uronic acid (DP 4-8) beddelka glucuroxylan oligosaccharides iyo (B) ACN-B xylan iyo methylated uronic acid oligosaccharides oo lagu beddelay glucuroxylan (DP 8-15).
Falanqaynta halabuurka glycan hadhaaga oligosaccharides refractory.Halkan (A) Isku-dhafka TMS saccharide ee jajabyada oligosaccharides ee kala duwan oo la helay iyadoo la adeegsanayo falanqaynta GC-MS.(B) Qaab-dhismeedka sonkorta kala duwan ee TMS-ka laga keeno ee ku jirta oligosaccharides.ACN - jajabka acetonitrile oo ka kooban oligosaccharides dhexdhexaad ah iyo FA - jajab acid ferulic oo ka kooban oligosaccharides acid.
Gabagabo kale oo xiiso leh ayaa laga soo qaatay falanqaynta LC-MS ee jajabka oligosaccharides, sida ku cad Jaantuska S9 (hababka waxaa lagu arki karaa qalabka dheeriga ah ee elektiroonigga ah).Qaybaha hexose iyo -OAc ayaa si isdaba joog ah loo arkay inta lagu gudajiro jajabka ACN-B.Helitaankani kaliya ma xaqiijinayo kala qaybsanaanta lagu arkay glycome iyo falanqaynta MALDI-TOF, laakiin sidoo kale waxay bixisaa macluumaad cusub oo ku saabsan soo-saareyaasha karbohaydrayt ee suurtagalka ah ee biomass-ka hore loo daweeyay.
Waxaan sidoo kale samaynay falanqaynta halabuurka glycan ee jajabyada oligosaccharides iyadoo la adeegsanayo derivatization TMS glycan.Isticmaalka GC-MS, waxaanu go'aaminnay isku-dhafka neerfaha (aan-ka-soo-saarka) iyo sonkorta acidic (GluA iyo GalA) ee jajabka oligosaccharides (Jaantus. 5).Glucuronic acid waxaa laga helaa qaybaha aashitada C iyo D, halka galacturonic acid laga helo qaybaha aashitada A iyo B, oo labaduba ah qaybaha DP ee sare ee sonkorta acidic.Natiijooyinkani kaliya ma xaqiijinayaan xogtayada ELISA iyo MALDI, laakiin sidoo kale waxay la jaan qaadayaan daraasadaheena hore ee ururinta oligosaccharides.Sidaa darteed, waxaan aaminsanahay in hababka difaaca jirka ee casriga ah iyadoo la adeegsanayo biotinylation of oligosaccharides iyo baaritaanka ELISA ee xiga ay ku filan yihiin in lagu ogaado oligosaccharides recalcitrant-ka-soo-baxa ee shaybaarada noolaha ee kala duwan.
Maadaama hababka baarista mAb ee ku saleysan ELISA lagu ansixiyay dhowr habab oo kala duwan, waxaan rabnay inaan sii sahamno awoodda habkan cusub ee tirada.Laba oligosaccharides ganacsi, xylohexasaccharide oligosaccharides (XHE) iyo 23-α-L-arabinofuranosyl-xylotriose (A2XX), ayaa la iibsaday oo la tijaabiyay iyadoo la adeegsanayo hab cusub oo mAb ah oo lagu beegsanayo glycan gidaarka unugyada.Jaantuska 6 waxa uu muujinayaa xidhiidhka toosan ee u dhexeeya calaamada xidhitaanka biotinylated iyo kaydka loga ee fiirsashada oligosaccharides, taas oo soo jeedinaysa qaabka xayeysiinta Langmuir ee suurtogalka ah.Waxaa ka mid ah mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, iyo CCRC-M151 oo la xidhiidha XHE, iyo CCRC-M108, CCRC-M109, iyo LM11 oo la xidhiidha A2XX oo ka badan 10 namno.Sababtoo ah helitaanka xaddidan ee unugyada difaaca jirka inta lagu jiro tijaabada, tijaabooyin xaddidan ayaa lagu sameeyay uruurinta oligosaccharide kasta.Waa in la ogaadaa in qaar ka mid ah unugyada difaaca jirka ay si aad u kala duwan uga falceliyaan isla oligosaccharides sida substrate, malaha sababtoo ah waxay ku xiran yihiin qaybo yar oo kala duwan waxayna yeelan karaan xiriiro isku xiran oo aad u kala duwan.Hababka iyo saamaynta aqoonsiga epitope-ka saxda ah ayaa aad uga sii adkaan doona marka habka cusub ee mAb lagu dabaqo muunado dhab ah.
Laba oligosaccharides ganacsi ayaa loo isticmaalay si loo go'aamiyo kala duwanaanshaha ogaanshaha ee mAbs-ka-bartilmaameedka kala duwan.Halkan, isku xidhka toosan ee kaydinta oligosaccharides waxay tilmaamaysaa qaababka xayeysiinta Langmuir ee (A) XHE oo leh mAb iyo (B) A2XX oo leh mAb.Qaybaha u dhigma waxay muujinayaan qaab-dhismeedka oligosaccharides-ganacsiyeedka loo isticmaalo sida substrates ee baaritaanka.
Isticmaalka unugyada difaaca jirka ee glycan-bartilmaameedka monoclonal (falanqaynta glycocomic ama ELISA-based mAb screening) waa qalab awood leh oo si qoto dheer loogu tilmaamayo inta badan glycans gidaarka unugyada waaweyn ee ka kooban bioomass dhirta.Si kastaba ha noqotee, falanqaynta glycan ee qadiimiga ah ayaa kaliya tilmaameysa glycans gidaarka unugyada weyn, maadaama oligosaccharides intooda badan aysan si hufan u dhaqdhaqaaqin taarikada ELISA.Daraasaddan, AFEX-ku-diyaar gareeyay kariyaha hadhuudhka ayaa sisaymaati ahaan hydrolyzed ka kooban yahay adkaha sare.Falanqaynta sonkorta ayaa loo isticmaalay si loo go'aamiyo ka kooban karbohaydraytyada gidaarka unugga ee dib u celinta ee ku jira hydrolyzate.Si kastaba ha ahaatee, falanqaynta mAb ee oligosaccharides yar yar ee hydrolysates waa la dhayalsaday, iyo qalab dheeraad ah ayaa loo baahan yahay si wax ku ool ah u dhaqdhaqaaqa oligosaccharides ee taarikada ELISA.
Waxaan halkaan uga warbixineynaa hab cusub oo wax ku ool ah oligosaccharides immobilization for mAB iyadoo la isku darayo oligosaccharides biotinylation oo ay ku xigto baaritaanka ELISA ee taarikada dahaarka leh ee NeutrAvidin™.Oligosaccharides-ka biotinylated ee aan dhaqdhaqaaqa lahayn ayaa muujiyay xidhiidh ku filan antibody-ka si ay awood ugu yeelato ogaanshaha degdegga ah oo hufan ee oligosaccharides dib-u-celinta.Falanqaynta ka kooban oligosaccharides-adag adag oo ku salaysan spectrometry mass ayaa xaqiijiyay natiijooyinka habkan cusub ee difaaca jirka.Sidaa daraadeed, daraasadahani waxay muujinayaan in isku-darka oligosaccharides biotinylation iyo baaritaanka ELISA ee unugyada unugyada monoclonal ee glycan-bartilmaameedka ah loo isticmaali karo in lagu ogaado isku-xirnaanta oligosaccharides waxaana si ballaaran loogu dabaqi karaa daraasadaha kale ee biochemical ee tilmaamaya qaabka oligosaccharides.
Habkan glycan-ku-saleysan biotin-ku-saleysan waa warbixintii ugu horreysay ee awood u leh in lagu baaro curaarta karbohaydrayt-ka-soo-baxa ee oligosaccharides-ka milmay ee baomass-ka dhirta.Tani waxay kaa caawinaysaa in la fahmo sababta qaybo ka mid ah bioomass ay aad ugu madax adag yihiin marka ay timaado wax soo saarka bayooloji.Habkani wuxuu buuxinayaa farqiga muhiimka ah ee hababka falanqaynta glycome wuxuuna ku fidiyaa codsigeeda ilaa tiro balaadhan oo ah substrates oo ka baxsan oligosaccharides dhirta.Mustaqbalka, waxaa laga yaabaa inaan u isticmaalno robotics biotinylation oo aan u isticmaalno habka aan u soo saarnay falanqaynta sare ee muunadaha iyadoo la adeegsanayo ELISA.
Cawska galleyda (CS) ee ka soo baxay abuuraha isku-dhafan ee Pioneer 33A14 ayaa laga soo goostay 2010kii Beeraha Kramer ee Ray, Colorado.Ogolaansho mulkiilaha, baayoomass-kan waxa loo isticmaali karaa cilmi-baadhis. Tijaabooyinku waxa lagu kaydiyay qalalan <6% qoyaan bacaha sibka-qufulka ee heerkulka qolka. Tijaabooyinku waxa lagu kaydiyay qalalan <6% qoyaan bacaha sibka-qufulka ee heerkulka qolka. Образцы хранились сухими при влажности < 6% в пакетах Tusaalooyinka waxa lagu kaydiyay qalalan <6% huurka bacaha zippered ee heerkulka qolka.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%. Tusaalooyinka waxaa lagu kaydiyaa bacaha jibbaarka heerkulka qolka leh huurka <6%.Daraasadu waxay u hogaansantay xeerarka deegaanka iyo qaranka.Falanqaynta curinta waxaa lagu sameeyay hab-maamuuska NREL.Halabuurka ayaa la ogaaday inuu ka kooban yahay 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% borotiin iyo 13. 4% dambas.
Celic® CTec2 (138 mg protein/ml, lot VCNI 0001) waa isku dhafka kakan ee cellulase, β-glucosidase iyo Cellic® HTec2 (157 mg protein/ml, lot VHN00001) oo ka yimid Novozymes (Franklinton, NC, USA)).Multifect Pectinase® (72 mg protein/mL), isku darka adag ee pectin-ka wax-jabiya enzymes, waxaa ku deeqay DuPont Industrial Biosciences (Palo Alto, CA, USA).Heerarka borotiinka Enzyme waxaa lagu go'aamiyay qiyaasidda nuxurka borotiinka (iyo kala-goynta ka qayb qaadashada nitrogen-ka aan borotiinka ahayn) iyadoo la adeegsanayo falanqaynta nitrogen ee Kjeldahl (habka AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Dhulka Diatomaceous 545 waxaa laga soo iibsaday EMD Millipore (Billerica, MA).Kaarboon firfircoon (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, iyo dhammaan kiimikooyinka kale ayaa laga soo iibsaday Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment waxaa lagu sameeyay GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Daawaynta ka hor waxaa lagu sameeyay 140 ° C. 15 daqiiqo.46 wakhtiga daganaanshaha 1:1 saamiga ammonia anhydrous iyo bayoomass 60% (w/w) ku shubashada dab-dhaliye dufcad bir ah oo aan birta lahayn (Shirkadda Qalabka Qalabka).Waxay qaadatay 30 daqiiqo.Reactor-ka ayaa la keenay 140°C ammonia-na si degdeg ah ayaa loo sii daayay, taas oo u oggolaanaysa bayoolajiga inuu si dhakhso ah ugu soo laabto heerkulka qolka.Halabuurka AFEX shooladda gallayda ee horay loo daaweeyay (ACS) waxay la mid tahay tan kariyaha galleyda aan la daweyn (UT-CS).
Adag sare ACSH 25% (w/w) (qiyaastii 8% raritaanka dextran) ayaa loo diyaariyey sidii qalab bilow ah oo loogu talagalay soosaarka baaxadda weyn ee oligosaccharides.Hydrolysis Enzymatic ee ACS waxaa lagu sameeyay iyadoo la adeegsanayo isku dhafka enzyme ganacsiga oo ay ku jiraan Cellic® Ctec2 10 mg borotiinka / g glucan (ku jira biomass pretreated), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, iyo Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein/g dextran.Hydrolysis Enzymatic waxaa lagu sameeyay bioreactor 5-litir ah oo leh mugga shaqada ee 3 litir, pH 4.8, 50 ° C iyo 250 rpm.Hydrolysis ka dib 96 saacadood, hydrolyzate waxaa lagu soo ururiyay centrifugation 6000 rpm 30 daqiiqo ka dibna 14000 rpm 30 daqiiqo si looga saaro adkaha aan biyaha lahayn.Hydrolyzate-ka ayaa markaa lagu sammeeyay sifaynta nadiifka ah iyada oo loo marayo 0.22 mm shaandhaynta shaandhada.Hydrolyzate-ka la sifeeyay waxaa lagu kaydiyay dhalooyin nadiif ah oo ah 4° C. ka dibna la jajabiyey kaarboonka.
Falanqaynta qayb ka mid ah shaybaarada bioomass-ka-soo-baxa ku salaysan iyadoo loo eegayo hababka falanqaynta shaybaarka ee NREL: diyaarinta shaybaarada falanqaynta halabuurka (NREL/TP-510-42620) iyo go'aaminta karbohaydraytyada qaab dhismeedka iyo lignin ee bayomass (NREL/TP-510-42618)47.
Falanqaynta Oligosaccharides ee qulqulka hydrolyzate waxaa lagu sameeyay qiyaasta 2 ml iyadoo la adeegsanayo habka hydrolysis-ku salaysan autoclave.Ku qas muunadda hydrolyzate-ka 69.7 µl of 72% sulfuric acid ee 10 ml tuubada koofiyadda caaga ah oo ku dheji 1 saac korka sare ee 121 ° C, ku qabooji barafka oo shaandhayso fiilada dareeraha ah ee chromatography (HPLC).Isku-duubnaanta oligosaccharides waxaa lagu go'aamiyay in la gooyo uruurinta monosaccharides ee muunada aan biyo la'aanta ahayn ee wadarta qiyaasta sonkorta ee saamiga acid-hydrolyzed.
Glucose, xylose, iyo arabinose-ka ee ku jira biomass-ka acid hydrolysed ayaa lagu falanqeeyay iyadoo la adeegsanayo nidaamka Shimadzu HPLC oo ku qalabaysan autosampler, kuleyliyaha tiirarka, bamka isocratic, iyo index index refractive on a Bio-Rad Aminex HPX-87H column.Tiirka waxaa lagu ilaaliyay 50°C waxaana lagu soo saaray 0.6 ml/min 5 mM H2SO4 oo biyo ah.socodka.
Hydrolyzate supernatant ayaa la qasi jiray oo lagu falanqeeyay maadada monomer iyo oligosaccharides.Sonkorta monomeric ee la helay ka dib hydrolysis enzymatic waxaa falanqeeyay HPLC oo ku qalabaysan tiirka Bio-Rad (Hercules, CA) Aminex HPX-87P iyo tiir ilaaliyaha dambaska.Heerkulka tiirka waxaa lagu ilaaliyay 80 ° C, biyaha waxaa loo isticmaalay sidii marxaladda wareega oo leh heerka socodka 0.6 ml/min.Oligosaccharides waxaa lagu go'aamiyay hydrolysis ee aashitada milmaha leh ee 121 ° C iyadoo loo eegayo hababka lagu qeexay tixraacyada.41, 48, 49.
Falanqaynta saccharide waxaa lagu sameeyay cayriin, AFEX horay loo daaweeyay iyo dhammaan hadhaaga biomass-ka ee aan-hydrolysed (oo ay ku jiraan wax soo saarka derbiyada unugyada serial cell iyo baaritaankooda mAb) iyadoo la adeegsanayo hababka hore ee 27, 43, 50, 51.Falanqaynta glycome, hadhaaga aalkolada aan milmi karin ee walxaha gidaarka unugyada dhirta ayaa laga diyaariyey hadhaaga biomass waxaana lagu sakhraamo soosaar taxane ah oo leh reagents sii kordheysa oo gardarrada ah sida ammonium oxalate (50 mM), carbonate sodium (50 mM iyo 0.5% w / v), CON.(1M iyo 4M, labadaba leh 1% w/v sodium borohydride) iyo chlorite acid sidii hore loo sharaxay52,53.Soosaarayaasha ayaa markaa lagu soo rogay ELISA oo ka dhan ah guddi adag oo mAb50s ah oo lagu hagayo glycan gidaarka unugga, iyo falcelinta isku xidhka mAb ayaa loo soo bandhigay sidii khariidad kuleyl ah.mAbs bartilmaameedsanaysa unugyada gidaarka glycan waxaa laga soo iibsaday kaydka shaybaadhka (CCRC, JIM iyo MAC taxane).
Hal-tallaabo biotinylation of oligosaccharides.Isku xirka karbohaydraytyada leh biotin-LC-hydrazide waxaa lagu sameeyay habkan soo socda.Biotin-LC-hydrazide (4.6 mg/12 μmol) ayaa lagu milmay dimethyl sulfoxide (DMSO, 70 μl) iyada oo la walaaqayo iyo kuleylinta 65 ° C. 1 min.Glacial acetic acid (30 µl) ayaa lagu daray oo isku dar ah ayaa lagu shubay sodium cyanoborohydride (6.4 mg/100 µmol) oo si buuxda u milmay ka dib kuleylka 65 ° C. ilaa 1 daqiiqo.Ka dib, laga bilaabo 5 ilaa 8 μl ee isku dhafka falcelinta ayaa lagu daray oligosaccharide engegan (1-100 nmol) si loo helo 10-laab ama ka badan oo molar ah oo ka badan calaamadda dhamaadka dhimista.Dareen-celinta waxaa lagu sameeyay 65 ° C 2 saacadood, ka dib shaybaarada ayaa isla markiiba la nadiifiyey.Ma jiro sodium cyanoborohydride loo isticmaalay tijaabooyinka calaamadaynta iyada oo aan la dhimin, shaybaaradana waxaa lagu falceliyay 65 ° C. 2.5 saacadood.
Dahaarka ELISA iyo dhaqidda shaybaarada oligosaccharides biotinylated.25 μl oo muunado biotinylated ah (100 μl ee muunad kasta oo la ururiyey oo lagu qasi jiray 5 ml of 0.1 M Tris buffer solution (TBS)) ayaa lagu daray ceel kasta oo saxan avidin-dahaarka ah.Ceelasha xakamaynta waxaa lagu dahaadhay 50 μl oo biotin ah oo lagu uruurinayo 10 μg/ml ee 0.1 M TBS.Biyaha deionized ayaa loo isticmaalay dahaar ahaan cabbirrada bannaan.Kiniinka waxaa lagu duray 2 saacadood heerkulka qolka mugdiga dhexdiisa.Ku dhaq saxanka 3 jeer 0.1% caano la shubay oo ku jira 0.1 M TBS adoo isticmaalaya barnaamijka Lr.11 ee Grenier flat 3A.
Ku daridda iyo dhaqidda unugyada difaaca aasaasiga ah.Ku dar 40 µl oo ah antibody aasaasiga ah ceel kasta.Ku dheji microplate ilaa 1 saac heerkulka qolka mugdiga.Taarikada waxaa lagu dhaqay 3 jeer iyadoo 0.1% caano ah 0.1M TBS iyadoo la isticmaalayo barnaamijka dhaqida #11 ee Grenier Flat 3A.
Ku dar antibody secondary oo dhaq.Ku dar 50 µl oo ah jiirka/jiirka difaaca jirka sare (la qasay 1:5000 0.1% caano ah 0.1 M TBS) ceel kasta.Ku dheji microplate ilaa 1 saac heerkulka qolka mugdiga.Microplates-yada ayaa markaa lagu maydhay 5 jeer 0.1% caano ah 0.1 M TBS iyadoo la isticmaalayo barnaamijka saxanka ee Grenier Flat 5A #12.
Ku darida substrateKu dar 50 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate-ka saldhigga ah (adigoo ku daraya 2 dhibcood oo bakhaar ah, 3 dhibcood oo TMB ah, 2 dhibcood oo hydrogen peroxide ilaa 15 ml oo biyo ah).Diyaari substrate-ka TMB.iyo vortex ka hor inta aan la isticmaalin).Ku dheji microplate heerkulka qolka 30 daqiiqo.Madawga.
Dhammaystir tillaabada oo akhri kiniinka.Ku dar 50 µl oo ah 1 N sulfuric acid ceel kasta oo duub nuugista 450 ilaa 655 nm adoo isticmaalaya akhristaha ELISA.
U diyaari 1 mg / ml xalalka falanqaynta kuwaas oo ku jira biyaha 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 (heer gudaha).Labo heerar ayaa la diyaariyey iyadoo lagu daray 1 mg / mL sonkorta sonkorta lagu muujiyey shaxda 1. Tijaabooyinku waa la qaboojiyey oo lyophilized at -80 ° C. ilaa dhammaan biyaha laga saaro (sida caadiga ah saacadaha 12-18).
Ku dar 100-500 µg oo muunad ah si aad u maroojiso tuubooyinka koofiyadda ee dheelitirka gorfaynta.Qor inta lagu darayWay fiicantahay in lagu milo muunada meel gaar ah oo dareere ah oo lagu daro tuubada sida aliquot dareere ah.Isticmaal 20 µl oo ah 1 mg/ml inositol sida heerka gudaha ee tuubo kasta.Qadarka heerka gudaha ee lagu daray muunadku waa inuu la mid yahay inta heerka gudaha ee lagu daray tuubada caadiga ah.
Ku dar 8 ml oo methanol anhydrous ah fiilada fur furka ah.Dabadeed 4 ml oo ah 3 N. methanol HCl xal, la daboolay oo la ruxay.Habkani ma isticmaalo biyo.
Ku dar 500 µl oo 1 M HCl methanol shaybaarada oligosaccharides iyo tuubooyinka caadiga ah ee TMS.Muunado ayaa lagu duray habeenkii (168 saacadood) at 80° C.Ku qalaji badeecada methanolysis heerkulka qolka adoo isticmaalaya kala duwanaansho qallajin.Ku dar 200µl MeOH oo mar kale qalaji.Habkan waxa lagu celiyaa laba jeer.Ku dar 200 µl oo methanol ah, 100 µl oo pyridine ah iyo 100 µl oo acetic anhydride ah muunada oo si fiican isku walaaq.Sambalyada ku rid heerkulka qolka 30 daqiiqo.oo la qalajiyey.Ku dar 200 µl oo methanol ah oo mar kale qalaji.
Ku dar 200 µl oo Tri-Sil ah iyo kuleyl dabool daboolan 20 daqiiqo.80 ° C, ka dibna la qaboojiyey heerkulka qolka.Isticmaal manifoldka qalajinta si aad u sii engejiso muunadda qiyaas ahaan 50 µl.Waxaa muhiim ah in la ogaado in aynaan u oggolaan in shaybaarrada ay si buuxda u engegaan.
Ku dar 2 ml oo hexane ah oo si fiican isku walaaq adiga oo gariiraya.Ku buuxi talooyinka pipettes Pasteur (5-8 mm) gabal dhogor galaas ah adigoo gelinaya dhogorta dhalada ah ee dusha sare ee pipette dhexroor 5-3/4 inch ah.Tusaalooyinka waxaa lagu daray 3000 g 2 daqiiqo.Hadhaaga aan la milmi karin ayaa soo degdegay.Ku qalaji saamiga ilaa 100-150 µl.Mugga qiyaastii 1 μl ayaa lagu duray GC-MS heerkulkii ugu horreeyay ee 80 °C iyo wakhtiga bilowga ah ee 2.0 daqiiqo (Shaxda 2).