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Uduce twa silica dufite imyenge twateguwe hakoreshejwe uburyo bwa sol-gel hamwe n'impinduka zimwe na zimwe kugira ngo haboneke utwo duce dufite imyenge miremire. Utwo duce twakuwemo N-phenylmaleimide-methylvinyl isocyanate (PMI) na styrene binyuze muri polymerization ya reverse chain transfer-fragmentation (RAFT) kugira ngo hakorwe polyamides za N-phenylmaleimide. Styrene (PMP) stationary phase. Inkingi z'icyuma kidafunze zifite umurambararo muto (100 × 1.8 mm imbere) zari zipakiyemo slurry packing. Imikorere ya chromatographic y'inkingi ya PMP yapimwe kugira ngo hatandukanye uruvange rwa peptides za sintetike zigizwe na peptides eshanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) na tryptic hydrolyzate ya human serum albumin (HAS). Mu gihe cyo gusesengura neza, umubare w’amasahani afite uruvange rwa peptides wageze kuri 280.000 plates/sq.m. Ugereranije imikorere yo gutandukanya inkingi yakozwe n’inkingi ya Ascentis Express RP-Amide y’ubucuruzi, byagaragaye ko imikorere yo gutandukanya inkingi ya PMP yari iruta inkingi y’ubucuruzi mu bijyanye no gutandukanya no gukemura ikibazo.
Inganda zikora imiti zikomoka ku binyabuzima zabaye isoko rikura ku isi hose, aho isoko ryiyongereye cyane mu myaka ya vuba aha. Kubera ko inganda zikora imiti zikomoka ku binyabuzima1,2,3 zikomeje kwiyongera cyane, hakenewe cyane isesengura rya peptide na poroteyine. Uretse peptide yibandwaho, imyanda itandukanye iraboneka mu gihe cyo gukora peptide, bityo gusukura chromatographic birakenewe kugira ngo peptide ibe nziza. Gusesengura no kugaragaza poroteyine mu mazi, mu ngingo, no mu turemangingo ni akazi katoroshye cyane bitewe n'umubare munini w'ubwoko bushobora kugaragara mu gipimo kimwe. Nubwo spectrometry y'ubunini ari igikoresho cyiza cyo gukurikirana peptide na poroteyine, niba ingero nk'izo zinjijwe mu buryo butaziguye muri spectrometer y'ubunini, gutandukanya ibintu ntibizaba bihagije. Iki kibazo gishobora gukemurwa no gukora chromatography y'amazi (LC) mbere yo gusesengura MS, bizagabanya umubare w'ibisesengura byinjira muri spectrometer y'ubunini mu gihe runaka4,5,6. Byongeye kandi, ibisesengura bishobora kwibanda mu gace gato mu gihe cyo gutandukanya ibice by'amazi, bityo bigashyira hamwe ibi bisesengura kandi byongera ubushobozi bwo kubona MS. Ubushakashatsi bwa "liquid chromatography" (LC) bwateye imbere cyane mu myaka icumi ishize kandi bwabaye uburyo bukoreshwa cyane mu gusesengura proteomic7,8,9,10.
Kromatografia y'amazi ihinduka (RP-LC) ikoreshwa cyane mu gusukura no gutandukanya uruvange rwa peptides hakoreshejwe octadecyl-modified silica (ODS) nk'urwego ruhoraho11,12,13. Ariko, bitewe n'imiterere yazo ikomeye n'imiterere ya amphoteric, 14,15 RP stationary phases ntishobora gutanga itandukanyirizo rishimishije rya peptides na proteins. Kubwibyo, isesengura rya peptides na proteins zifite ibice bya polar na non-polar risaba imiterere yihariye ya stationary phases kugira ngo ihuze kandi igumane izi analytes16. Kromatografia ivanze, itanga imikoranire myinshi, ishobora kuba ubundi buryo bwa RP-LC bwo gutandukanya peptides, proteins, n'izindi mvange zikomeye. Hateguwe imiterere myinshi ivanze kandi inkingi zuzuye izi mpande zihoraho zakoreshejwe mu gutandukanya peptides na proteins17,18,19,20,21. Bitewe n’uko hari amatsinda y’ibice by’inyuma n’ibice bitari ibice by’inyuma, ibice by’inyuma by’uburyo buvanze (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) birakwiriye mu gutandukanya peptide na poroteyine22,23,24,25,26,27,28. , ibice by’inyuma by’inyuma bifite amatsinda y’ibice by’inyuma bifite ihuriro ry’ibice by’inyuma bigaragaza ubushobozi bwiza bwo gutandukanya no guhitamo byihariye ku bice by’inyuma n’ibice by’inyuma kuko gutandukanya biterwa n’imikoranire iri hagati y’ibice by’inyuma n’igice cy’inyuma Ibiganiro byinshi 29,30,31,32. Vuba aha, Zhang et al. 30 babonye ibice by’inyuma bya polyamine bya behenyl kandi bigatandukanya neza hydrocarbons, imiti igabanya ubukana, flavonoids, nucleosides, estrogens, n’ibindi bisesengura. Ibikoresho by’inyuma bifite amatsinda y’ibice by’inyuma n’ibice by’inyuma, bityo bishobora gukoreshwa mu gutandukanya peptides na poroteyine mu bice by’inyuma n’ibice by’inyuma. Inkingi zo mu gice cy'imbere cy'umubumbe (urugero, inkingi za C18 zifite umurongo wa amide) ziboneka ku izina ry'ubucuruzi rya Ascentis Express RP-Amide, ariko izi nkingi zakoreshejwe gusa mu gusesengura amine 33.
Mu bushakashatsi buriho, hateguwe kandi hasuzumwa uburyo bwo gutandukanya peptide (N-phenylmaleimide, embedding polystyrene) kugira ngo hamenyekane uburyo bwo gutandukanya peptide na tryptic HSA cleavage. Ingamba zikurikira zakoreshejwe mu gutegura uburyo bwo gutandukanya. Uduce twa silica dufite imyenge twateguwe hakurikijwe uburyo bwasobanuwe mu bitabo byacu byabanje, hamwe n'impinduka zimwe na zimwe mu buryo bwo gutegura 31, 34, 35, 36, 37, 38, 39. Ibipimo bya urea, polyethylene glycol (PEG), TMOS na aside acetic byahinduwe kugira ngo haboneke utuntu twa silica dufite imyenge minini. Icya kabiri, hakozwe ligand nshya ya phenylmaleimide-methylvinyl isocyanate maze utuntu twa silica twakuwemo dukoreshwa mu gutegura utwo duce twa polar. Uduce twabonetse twashyizwe mu nkingi y'icyuma kitagira umurambararo (umurambararo w'imbere wa mm 100 × 1.8) hakurikijwe uburyo bwo gufunga bwakozwe neza. Gufunga kw'inkingi bifashwa no guhindagura kwa mekanike kugira ngo harebwe ko urwego rumwe mu nkingi rungana. Inkingi yuzuye yasuzumwe kugira ngo harebwe uburyo bwo gutandukanya uruvange rwa peptides rugizwe na peptides eshanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). na tryptic hydrolysates ya human serum albumin (HSA). Byagaragaye ko uruvange rwa peptides na HSA tryptic digest bitandukanye neza kandi neza. Ubushobozi bwo gutandukanya PMP column bwagereranyijwe n'ubw'inkingi ya Ascentis Express RP-Amide. Byagaragaye ko peptides na poroteyine bifite ubushobozi bwo gutandukanya neza kandi bifite ubushobozi bwo gutandukanya cyane ku nkingi ya PMP, kandi ubushobozi bwo gutandukanya PMP column buri hejuru kuruta ubw'inkingi ya Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, aside asetiki, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) kuri HPLC, methanol, 2-propanol na acetone. Isosiyete ya Sigma-Aldrich (St. Louis, Missouri, muri Leta Zunze Ubumwe za Amerika).
Uruvange rwa urea (8 g), polyethylene glycol (8 g) na ml 8 za aside asetiki ya 0.01 N. byakanzwe mu gihe cy'iminota 10, hanyuma hongerwamo ml 24 za TMOS munsi y'ubukonje bw'urubura. Uruvange rw'ibisubizo rwashyuhijwe kuri 40 ° C mu gihe cy'amasaha 6, hanyuma kuri 120 ° C mu gihe cy'amasaha 8 muri autoclave y'icyuma kidafunze. Amazi yarakuwemo maze ibisigazwa byumye kuri 70 ° C mu gihe cy'amasaha 12. Uduce tw'amazi twumye twaseye neza hanyuma dushyirwa mu ziko kuri 550 ° C mu gihe cy'amasaha 12. Hateguwe uduce dutatu kandi twagaragajwe kugira ngo harebwe niba ingano y'uduce, ingano y'imyenge n'ubuso bw'aho duherereye bishobora kongera kugaragara.
Itsinda rya polar n'igice gihoraho cy'iminyururu ya polystyrene. Uburyo bwo kuyitegura burasobanurwa hano hepfo.
N-phenylmaleimide (200 mg) na methyl vinyl isocyanate (100 mg) byashongeshejwe muri toluene idakora, hanyuma 0.1 ml ya 2,2′-azoisobutyronitrile (AIBN) yongerwa mu icupa rya reaction kugira ngo haboneke copolymer ya phenylmaleimide na methyl vinyl isocyanate (PMCP). ) Uruvange rwashyushywe kuri 60°C mu gihe cy'amasaha 3, ruyungururwa kandi rwumishwa mu ziko kuri 40°C mu gihe cy'amasaha 3.
Uduce twa silica twumye (2 g) twajugunywe muri toluene yumye (100 ml), tuvangwa hanyuma dushyirwa mu cyuma mu gihe cy'iminota 10 mu icupa ryo hasi rya mililitiro 500. PMCP (10 mg) yashongeshejwe muri toluene hanyuma yongerwamo agace gato ku icupa rya reaction binyuze mu muyoboro wongeramo. Uruvange rwasubiwemo kuri 100°C mu gihe cy'amasaha 8, ruyungururwa, rwozwa na acetone hanyuma rwumishwa kuri 60°C mu gihe cy'amasaha 3. Hanyuma, utwo duce twa silica dufitanye isano na PMCP (100 g) twashongeshejwe muri toluene (200 ml), hanyuma hongerwamo 4-hydroxy-TEMPO (2 ml) imbere ya 100 μl ya dibutyltin dilaurate nk'umusemburo. Uruvange rwashongeshejwe kuri 50°C mu gihe cy'amasaha 8, ruyungururwa kandi rwumishwa kuri 50°C mu gihe cy'amasaha 3.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) n'uduce twa silica twafatanye na TEMPO-PMCP (1.5 g) byashyizwe muri toluene hanyuma bisukurwa na azote. Polymerazing ya styrene yakozwe kuri 100°C mu gihe cy'amasaha 12. Umusaruro wavuyemo wogejwe na methanoli hanyuma wumishwa ijoro ryose kuri 60°C. Gahunda rusange y'uburyo bwo gusubiza ibintu igaragara ku ishusho ya mbere.
Ingero zakuwemo umwuka kuri 393 K mu gihe cy'isaha imwe kugeza igihe habonetse igitutu cyasigaye kiri munsi ya 10-3 Torr. Ingano ya N2 yashyizwe ku gitutu P/P0 = 0.99 yakoreshejwe kugira ngo hamenyekane ingano y'imyenge yose. Imiterere y'uduce twa silica twuzuye n'utwometse kuri ligand yasuzumwe hakoreshejwe mikorosikopi ya elegitoroniki yo gupima (Hitachi High Technologies, Tokyo, Ubuyapani). Ingero zumutse (uduce twa silica twuzuye n'utwometse kuri ligand) zashyizwe ku nkoni za aluminiyumu hakoreshejwe karuboni. Zahabu yashyizwe ku ntambwe hakoreshejwe igikoresho cya Q150T cyo gusukamo amazi, maze urwego rwa Au rw'ubugari bwa 5 nm rushyirwa ku ntambwe. Ibi byongera imikorere myiza y'uburyo bwo hasi bw'amashanyarazi kandi bigatanga uburyo bwo gutera amazi akonje. Isesengura ry'ibintu ryakozwe hakoreshejwe Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Isesengura ry'ingano y'uduce twa Malvern (Worcestershire, UK) Mastersizer 2000 ryakoreshejwe kugira ngo haboneke ikwirakwizwa ry'ingano y'uduce. Uduce twa silica tudapfutse n'uduce twa silica dufunganye na ligand (5 mg buri kimwe) twashyizwe muri 5 ml ya isopropanol, dushyirwa mu cyuma mu gihe cy'iminota 10, dushyirwa mu cyuma mu gihe cy'iminota 5, hanyuma dushyirwa ku ntebe y'ikoranabuhanga ya Mastersizer. Isesengura rya Thermogravimetric rikorwa ku gipimo cya 5 °C ku munota mu bushyuhe buri hagati ya 30 na 800 °C.
Inkingi z'icyuma gifunganye zometseho fibre y'ikirahure zifite ingano (ID 100 × 1.8 mm) zapakiwe hakoreshejwe uburyo bwo kuzuza icyuma gifunganye hakurikijwe uburyo nk'uko bigaragara mu nyandiko ya 31. Inkingi y'icyuma gifunganye (ifite ikirahure, ID 100 × 1 .8 mm) n'aho gusohokera birimo 1 µm byahujwe n'imashini ipakira icyuma gifunganye (Alltech Deerfield, IL, USA). Tegura icyuma gifunganye cyometseho icyuma gifunganye 150 mg y'icyuma gifunganye muri 1.2 ml ya methanol hanyuma uyishyire mu nkingi y'ikigega. Methanol yakoreshejwe nk'igikoresho gifunganye kandi gigenzura icyuma. Paka inkingi ukoresheje umurongo w'igitutu cya 100 MP mu minota 10, 80 MP mu minota 15, na 60 MP mu minota 30. Uburyo bwo gupakira bwakoresheje vibrators ebyiri za gaze chromatography column (Alltech, Deerfield, IL, USA) kugira ngo hamenyekane ko inkingi imwe ipakiye. Funga icyuma gifunganye hanyuma urekure umuvuduko buhoro buhoro kugira ngo wirinde kwangirika kw'umugozi. Inkingi yakuwe ku munwa w'amazi maze ikindi gikoresho gishyirwa ku mwobo w'amazi gihuzwa na sisitemu ya LC kugira ngo hagenzurwe imikorere yayo.
Hakozwe MLC yihariye hakoreshejwe pompe ya LC (10AD Shimadzu, mu Buyapani), icyitegererezo gifite umuyoboro wa 50 nL (Valco (USA) C14 W.05), icyuma gikuraho uturemangingo (Shimadzu DGU-14A), n'idirishya rya UV-VIS capillary. Igikoresho cyo gupima (UV-2075) hamwe n'inkingi ntoya. Koresha imiyoboro mito cyane kandi migufi ihuza kugira ngo ugabanye ingaruka zo kwaguka kw'inyongera kw'inkingi. Nyuma yo kuzuza inkingi, shyiramo capillary (50 µm id 365) aho 1/16″ reduction junction isohokera hanyuma ushyiremo capillary (50 µm) y'agace gacukura. Gukusanya amakuru no gutunganya chromatogram bikorwa hakoreshejwe porogaramu ya Multichro 2000. Kuri 254 nm, UV absorbance y'abasesengura ingingo yagenzuwe kuri 0. Amakuru ya chromatographic yasesenguwe hakoreshejwe OriginPro8 (Northampton, MA).
Albumine yo mu maraso y'umuntu, ifu ya lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg ivanze na trypsin (1.5 mg), 4.0 M urea (1 ml) na 0.2 M ammonium bicarbonate (1 ml). Umuti warabyutswe mu minota 10 maze ubibika mu bwogero bw'amazi kuri 37°C mu gihe cy'amasaha 6, hanyuma ukazimya na 1 ml ya 0.1% TFA. Hindura umuti hanyuma ubibike munsi ya 4°C.
Gutandukanya uruvange rwa peptides na tryptic digest HSA ku nkingi ya PMP byagenzuwe ukwabyo. Reba tryptic hydrolysis y'uruvange rwa peptides na HSA bitandukanyijwe n'inkingi ya PMP hanyuma ugereranye ibyavuye mu bushakashatsi n'inkingi ya Ascentis Express RP-Amide. Umubare w'amaplate y'ibitekerezo ubarwa hakoreshejwe ikigereranyo gikurikira:
Amashusho ya SEM y'uduce twa silica twonyine n'uduce twa silica twometse kuri ligand agaragara ku Ishusho ya 2. Amashusho ya SEM y'uduce twa silica twonyine (A, B) agaragaza imiterere y'uruziga aho uduce tuba turekure cyangwa dufite uburinganire budasanzwe ugereranije n'ubushakashatsi bwacu bwa mbere. Ubuso bw'uduce twa silica twometse kuri ligand (C, D) bworoshye kurusha ubw'uduce twa silica twonyine, bishobora guterwa n'iminyururu ya polystyrene itwikiriye ubuso bw'uduce twa silica.
Gusoma mikorografi za electron z'uduce twa silica (A, B) n'uduce twa silica dufatanye na ligand (C, D).
Ingano y'uduce twa silica nziza n'uduce twa silica dufatanye na ligand igaragara ku Ishusho ya 2. 3 (A). Imirongo y'ingano y'uduce twa volumetric yagaragaje ko ingano y'uduce twa silica yiyongereye nyuma yo guhindura imiti (Ishusho ya 3A). Amakuru y'ingano y'uduce twa silica twavuye mu bushakashatsi buriho ubu n'ubushakashatsi bwabanje agereranyijwe mu Imbonerahamwe ya 1 (A). Ingano y'uduce twa volumetric d (0.5) ya PMP yari 3.36 µm, ugereranije n'agaciro ka ad (0.5) ka 3.05 µm mu bushakashatsi bwacu bwabanje (uduce twa silica dufatanye na polystyrene)34. Bitewe n'impinduka mu gipimo cya PEG, urea, TMOS na aside asetiki mu ruvange rw'ibisubizo, ikwirakwizwa ry'uduce twayo ryari rito ugereranije n'ubushakashatsi bwacu bwabanje. Ingano y'uduce twa PMP ni nini gato ugereranyije n'iy'uduce twa silica dufatanye na polystyrene twabanje kwiga. Ibi bivuze ko imikorere y'ubuso bw'uduce twa silica hamwe na styrene yashyize gusa urwego rwa polystyrene (0.97 µm) ku buso bwa silica, mu gihe mu cyiciro cya PMP ubunini bw'urwego bwari 1.38 µm.
Ingano y'uduce duto (A) n'ingano y'imyenge (B) y'uduce twa silica twonyine n'uduce twa silica dufatanye na ligand.
Ingano y'imyenge, ingano y'imyenge, n'ubuso bw'uduce twa silica twakoreshejwe muri ubu bushakashatsi bigaragara mu mbonerahamwe ya 1 (B). Imiterere ya PSD y'uduce twa silica twonyine n'uduce twa silica twometse kuri ligand igaragara mu ishusho ya 3 (B). Ibisubizo byabonetse byari bisa n'inyigo yacu yabanje34. Ingano y'imyenge y'uduce twa silica twonyine n'uduce twa silica twometse kuri ligand yari 310 Å na 241 Å, uko bikurikirana, bigaragaza ko nyuma yo guhindura shimi, ingano y'imyenge yagabanutseho 69 Å, nk'uko bigaragara mu mbonerahamwe ya 1 (B), kandi umurongo wo guhindura ugaragara mu ishusho. Ubuso bwihariye bw'uduce twa silica muri ubu bushakashatsi ni 116 m2/g, bungana n'ubushakashatsi bwacu bwabanje (124 m2/g). Nk'uko bigaragara mu mbonerahamwe ya 1 (B), ubuso (m2/g) bw'uduce twa silica nyuma yo guhindura shimi nabwo bwagabanutse kuva kuri 116 m2/g bugera kuri 105 m2/g.
Ibisubizo by'isesengura ry'ibanze ry'icyiciro cy'ubudahinduka bigaragazwa mu mbonerahamwe. 2. Ingano ya karuboni mu gice cy'ubudahinduka ni 6.35%, iri hasi ugereranije n'inyigo yacu yabanje (uduce twa silica dufitanye isano na polystyrene, 7.93%35 na 10.21%, uko bikurikirana) 42. Ingano ya karuboni mu gice cy'ubudahinduka iri hepfo, kubera ko zimwe mu ligande za polar nka phenylmaleimide methyl vinyl isocyanate (PCMP) na 4-hydroxy-TEMPO byakoreshejwe hiyongereyeho styrene mu gutegura SP. Ijanisha ry'uburemere bwa azote mu gice cy'ubudahinduka ni 2.21% ugereranije na 0.1735 na 0.85% mu bushakashatsi bwabanje42. Ibi bivuze ko igice cy'ubudahinduka gifite ijanisha ry'uburemere bwinshi bwa azote bitewe na phenylmaleimide. Mu buryo nk'ubwo, ibicuruzwa (4) na (5) bifite ingano ya karuboni ya 2.7% na 2.9%, mu gihe umusaruro wa nyuma (6) ufite ingano ya karuboni ya 6.35%, nk'uko bigaragara mu mbonerahamwe ya 2. Isesengura rya Thermogravimetric (TGA) ryakoreshejwe mu cyiciro cya PMP gihoraho kugira ngo hamenyekane kugabanya ibiro, kandi umurongo wa TGA ugaragara ku Ishusho ya 4. Umurongo wa TGA ugaragaza kugabanuka kw'ibiro kwa 8.6%, bihuye neza n'umubare wa karuboni (6.35%), kubera ko ligands zitarimo C , ahubwo zirimo N, O na H gusa.
Isocyanate ya phenylmaleimide-methylvinyl isocyanate yatoranyijwe kugira ngo ihindure ubuso bw'uduce twa silica bitewe n'amatsinda yayo ya phenylmaleimide na vinylisocyanate. Amatsinda ya isocyanate ya vinyl ashobora kongera gukorana na styrene binyuze mu gukora polymerization ya radical. Impamvu ya kabiri ni ugushyiramo itsinda rifite imikoranire iri hagati na analyte kandi ridafite imikoranire ikomeye ya electrostatic hagati ya analyte na phase stationary, kubera ko moiety ya phenylmaleimide idafite charge virtual kuri pH isanzwe. Polarity y'igice stationary ishobora kugenzurwa n'ingano nziza ya styrene n'igihe cyo gukora reaction ya polymerization ya free radical. Intambwe ya nyuma y'isesengura (polymerization ya free radical) ni ingenzi kuko ihindura polarity y'igice stationary. Isesengura ry'ibanze ryakozwe kugira ngo harebwe ingano ya karuboni muri izi phase stationary. Byagaragaye ko kongera ingano ya styrene n'igihe cyo gukora reaction byongera ingano ya karuboni mu gice stationary na vice. SPs zikozwe zifite ingano zitandukanye za styrene zifite loads zitandukanye za karuboni. Mu buryo nk'ubwo, izi ntambwe zitarahinduka zashyizwe ku nkingi z'icyuma kidasagwa kandi imiterere yazo ya chromatographic (guhitamo, ubushobozi bwo kugaragaza, agaciro ka N, nibindi) yaragenzuwe. Hashingiwe kuri ubu bushakashatsi, hatoranyijwe imiterere myiza yo gutegura intambwe zitarahinduka ya PMP kugira ngo habeho polarity igenzurwa kandi ibungabungwe neza ry'umusesenguzi.
Inkingi ya PMP yanasuzumwe kugira ngo hasesengurwe uruvange rutanu rwa peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) hakoreshejwe ubushobozi bwa phase mobile. 60/40 (v/v) ACN/amazi (0.1% TFA) ku gipimo cy’amazi cya 80 µl/min. Mu gihe cyo gupima neza (200.000 plates/m), umubare wa plates theory (N) kuri buri nkingi (100 × 1.8 mm) ni 20.000 ± 100. Agaciro ka N kuri stencils eshatu za PMP kagaragara muri Table 3 naho chromatograms zigaragara muri Figure 5A. Isesengura ryihuse ku muvuduko mwinshi w’amazi (700 µl/min) ku nkingi ya PMP, peptide eshanu zavuye mu munota umwe, agaciro ka N gahebuje ka 13.500 ± 330 kuri buri nkingi (umurambararo wa mm 100 x 1.8), bingana na plates 135.000/m (Ishusho ya 5B). Inkingi eshatu zifite ingano imwe (umurambararo w’imbere 100 x 1.8 mm) zuzuyemo ibice bitatu bitandukanye bya PMP stationary phase kugira ngo hamenyekane uburyo bwo kongera kubyara. Isesengura ryakozwe kuri buri nkingi ritandukanya uruvange rumwe rw’igerageza kuri buri nkingi hakoreshejwe uburyo bwiza bwo gukuraho, umubare w’plates za teorique N, n’igihe cyo kubika. Amakuru yo kongera kubyara ku nkingi za PMP agaragara muri Imbonerahamwe ya 4. Uburyo bwo kongera kubyara ku nkingi ya PMP bwahuye neza n’agaciro ka %RSD gake cyane nkuko bigaragara muri Imbonerahamwe ya 3.
Gutandukanya uruvange rwa peptide ku nkingi ya PMP (B) n'inkingi ya Ascentis Express RP-Amide (A), igice cyo kugenda cya 60/40 ACN/H2O (TFA 0.1%), ingano z'inkingi ya PMP (100 x 1.8 mm id), isesengura Urutonde rw'ibinyabutabire: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) na 5 (leucic aside enkephalin).
Inkingi ya PMP (umurambararo w'imbere wa mm 100 x 1.8) yapimwe kugira ngo hamenyekane uburyo hydrolyzate ya tryptic ya albumin yo mu maraso y'umuntu itandukanyijwe na HPLC. Chromatogram iri ku Ishusho ya 6 igaragaza ko ingero zitandukanijwe neza kandi zifite ubushobozi bwiza cyane. Ibisubizo bya HSA byasesenguwe hakoreshejwe umuvuduko w'amazi wa 100 μl/min, igice cyo kugenda cya 70/30 acetonitrile/amazi na 0.1% TFA. Gutandukanya HSA byagabanijwemo imisozi 17, nk'uko bigaragara kuri chromatogram (Ishusho ya 6), bihuye na peptide 17. Ingufu zo gutandukanya imisozi imwe n'imwe na hydrolyzate ya HSA zarabazwe kandi agaciro kabyo kagaragazwa mu Imbonerahamwe ya 5.
Hydrolysates za HSA tryptic zatandukanyijwe ku nkingi ya PMP (umurambararo w'imbere wa mm 100 x 1.8), umuvuduko w'amazi (100 μl/min), igice cyo kugenda cya acetonitrile 60/40/amazi, na TFA 0.1%.
aho L ari uburebure bw'inkingi, η ni ubunini bw'igice cyo kugenda, ΔP ni igitutu cy'inyuma cy'inkingi, naho u ikaba umuvuduko ugororotse w'igice cyo kugenda. Ingufu z'inkingi ya PMP zari 2.5 × 10–14 m2, umuvuduko w'amazi wari 25 µl/min, 60/40 v/v ni byo byakoreshejwe. ACN/amazi. Ingufu z'inkingi ya PMP (ID 100 × 1.8 mm) zari zisa n'izo twakoze mbere ya Ref.34. Ingufu z'inkingi yuzuyemo uduce dufite imyenge mike ni 1.7 × 10 .6 µm, 2.5 × 10-14 m2 kuri uduce twa 5 µm43. Kubwibyo, ingufu z'igice cya PMP zisa n'ingufu z'uduce tw'ibanze dufite ingano ya 5 μm.
aho Wx ari uburemere bw'inkingi yuzuyemo chloroform, Wy ni uburemere bw'inkingi yuzuyemo methanol, naho ρ ikaba ubucucike bw'umusemburo. Ubucucike bwa methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Ubucucike bwose bw'inkingi ya silica-C18 (100 × 1.8 mm ID)34 hamwe n'inkingi yacu ya C18-urea31 yizweho mbere bwari 0.63 na 0.55, uko bikurikirana. Ibi bivuze ko kuba hariho ligande za urea bigabanya ubucucike bw'igice kitahagaze. Ku rundi ruhande, ubucucike bwose bw'inkingi ya PMP (umurambararo w'imbere 100 × 1.8 mm) ni 0.60. Inkingi za PMP ntizishobora kwinjira cyane ugereranyije n'inkingi zipakiye uduce twa silica dufatanye na C18 kuko mu byiciro bitambitse byo mu bwoko bwa C18 ligande za C18 zifatanye n'uduce twa silica mu minyururu igororotse, mu gihe mu byiciro bitambitse byo mu bwoko bwa polystyrene hakorwa polymer nini cyane izengurutse uduce. urwego rwa A. Mu igerageza risanzwe, porosity y'inkingi ibarwa ku buryo bukurikira:
Ku ishusho ya 7A, B yerekana ibishushanyo bya Van Deemter by'inkingi ya PMP (id 100 x 1.8 mm) n'inkingi ya Ascentis Express RP-Amide (id 100 x 1.8 mm) munsi y'imiterere imwe yo kugabanya, 60/40 ACN/H2O na 0 .1% TFA 20 µl/min kugeza 800 µl/min ku nkingi zombi. Agaciro ka HETP ntarengwa ku gipimo cyiza cy'umuvuduko (80 µl/min) kari 2.6 µm na 3.9 µm ku nkingi ya PMP n'inkingi ya Ascentis Express RP-Amide, uko bikurikirana. Agaciro ka HETP kerekana ko ubushobozi bwo gutandukanya inkingi ya PMP (100 x 1.8 mm id) buri hejuru cyane ugereranije n'inkingi ya Ascentis Express RP-Amide (100 x 1.8 mm id). Imbonerahamwe ya van Deemter iri ku Ishusho ya 7 (A) igaragaza ko kugabanuka k'agaciro ka N kutari hejuru cyane iyo amazi agenda yiyongera ugereranije n'ubushakashatsi bwacu bwabanje. Ubushobozi bwo gutandukanya inkingi ya PMP (id 100 × 1.8 mm) ugereranije n'inkingi ya Ascentis Express RP-Amide bushingiye ku miterere n'ingano byazamuwe hamwe n'uburyo bwo gupakira inkingi bugezweho bukoreshwa mu kazi ka none34.
(A) Igishushanyo cya Van Deemter (umuvuduko wa HETP ugereranyije n'umuvuduko wa elegitoroniki) cyabonetse ku nkingi ya PMP (id 100 x 1.8 mm) muri 60/40 ACN/H2O hamwe na 0.1% TFA. (B) Igishushanyo cya Van Deemter (umuvuduko wa HETP ugereranyije n'umuvuduko wa elegitoroniki) cyabonetse ku nkingi ya Ascentis Express RP-Amide (id 100 x 1.8 mm) muri 60/40 ACN/H2O hamwe na 0.1% TFA.
Hateguwe kandi hasuzumwa uburyo bwo gutandukanya uruvange rwa peptides za sintetike na tryptic hydrolyzate ya human serum albumin (HSA) mu gupima amazi mu buryo bwo hejuru. Imikorere ya chromatographic ya PMP columns ku mvange za peptide ni myiza cyane mu bijyanye no gutandukanya no gukemura ibibazo. Uburyo bwiza bwo gutandukanya PMP columns buterwa n'impamvu nyinshi nko ingano y'uduce twa silica n'ingano y'imyenge, guhuza ibice bya stationary, n'ibikoresho bigoye gupakira. Uretse ubushobozi bwo gutandukanya cyane, ikindi cyiza cy'iki cyiciro cya stationary ni urwego rwo hasi rw'inyuma ku muvuduko mwinshi w'amazi. PMP columns zishobora kongera kubyara kandi zishobora gukoreshwa mu gusesengura uruvange rwa peptides no gusya tryptic ya proteins zitandukanye. Turateganya gukoresha iyi column mu gutandukanya ibintu bikomoka ku binyabuzima biva mu bimera karemano, ibikomoka ku bimera bivura n'ibihumyo muri chromatography y'amazi. Mu gihe kizaza, PMP columns nazo zizasuzumwa mu gutandukanya poroteyine na antibodies za monoclonal.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Iperereza ku buryo bwo gutandukanya peptide bwa chromatography yahinduwe igice cya mbere: Iterambere rya protocole yo kugaragaza inkingi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Iperereza ku buryo bwo gutandukanya peptide bwa chromatography yahinduwe igice cya mbere: Iterambere rya protocole yo kugaragaza inkingi.Field, JK, Owerby, MR, Lau, J., Togersen, H., na Petersson, P. Iperereza ku buryo bwo gutandukanya Peptide hakoreshejwe Chromatographie y'icyiciro cya kabiri, Igice cya I: Gutegura Porotokole yo Gushushanya Inkingi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Iperereza ku buryo bwo gutandukanya peptide bwa chromatography yahinduwe igice cya mbere: Iterambere rya protocole y'imiterere y'inkingi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Iperereza ku buryo bwo gutandukanya peptide bwa chromatography yahinduwe igice cya mbere: Iterambere rya protocole y'imiterere y'inkingi.Field, JK, Owerby, MR, Lau, J., Togersen, H., na Petersson, P. Iperereza ku buryo bwo gutandukanya Peptide hakoreshejwe Chromatographie y'icyiciro cya kabiri, Igice cya I: Gutegura Porotokole yo Gushushanya Inkingi.J. 色谱法。 1603，113-129。 https://doi.org/10.1016/j.chroma.2019.05.038 (2019)。
Gomez, B. n'abandi. Uburyo bwo gukora peptides nziza zikora mu kuvura indwara zandura. Biotechnology. Ibyo byagezweho 36(2), 415–429. https://doi.org/10.1016/j.biotechadv.2018.01.004 (2018).
Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Peptide zivura zikozwe mu buryo bwa synthetic: Siyansi n'isoko. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Peptide zivura zikozwe mu buryo bwa synthetic: Siyansi n'isoko.Vliege P, Lisowski V, Martinez J na Chreschatyski M. Peptide zivura zikozwe mu buryo bwa synthetic: siyansi n'isoko.Vliege P, Lisowski V, Martinez J na Khreschatsky M. Peptide zivura zikozwe mu buryo bwa synthetic: siyansi n'isoko. kuvumbura imiti. Uyu munsi 15 (1–2), 40–56. https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Koromatografia y'amazi ya proteomiki iteye imbere. Xie, F., Smith, RD & Shen, Y. Koromatografia y'amazi ya proteomiki iteye imbere.Reba F., Smith RD na Shen Yu. Koromatografia y'amazi ya proteomiki iteye imbere. Xie, F., Smith, RD & Shen, Y. 高级蛋白质组液相色谱。 Xie, F., Smith, RD & Shen, Y. Ibigize poroteyine bigezweho 液相色谱。Reba F., Smith RD na Shen Yu. Koromatografia y'amazi ya proteomiki iteye imbere.J. Kromatography. A 1261, 78–90 (2012).
Liu, W. n'abandi. Isuzumabumenyi ry'ibinyabutabire rigezweho rya chromatography y'amazi-spectrometry y'ubunini rishobora guhuza metabolomike ishingiye ku buryo bwagutse na proteomics. anus. Chim. Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Uruhare rwa UHPLC mu iterambere ry'imiti. Chesnut, SM & Salisbury, JJ Uruhare rwa UHPLC mu iterambere ry'imiti.Chesnut, SM na Salisbury, JJ Uruhare rwa UHPLC mu iterambere ry'imiti.Chesnut, SM na Salisbury, JJ Uruhare rwa UHPLC mu iterambere ry'imiti. J. Sept Science. 30(8), 1183–1190 (2007).
Wu, N. & Clausen, AM Ingingo z'ibanze kandi zifatika za chromatography y'amazi afite umuvuduko mwinshi cyane kugira ngo atandukane vuba. Wu, N. & Clausen, AM Ingingo z'ibanze kandi zifatika za chromatography y'amazi afite umuvuduko mwinshi cyane kugira ngo atandukane vuba.Wu, N. na Clausen, AM Ingingo z'ibanze kandi zifatika za chromatography y'amazi afite umuvuduko mwinshi kugira ngo atandukane vuba. Wu, N. & Clausen, AM 用于快速分离的超高压液相色谱的基础和实践方面。 Wu, N. & Clausen, AM Ingingo z'ibanze n'ingirakamaro za chromatography y'amazi afite umuvuduko mwinshi cyane kugira ngo atandukane vuba.Wu, N. na Clausen, AM Ingingo z'ibanze kandi zifatika za chromatography y'amazi afite umuvuduko mwinshi kugira ngo atandukane vuba.J. Nzeri. Science. 30(8), 1167–1182. https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitchef, P. Gukoresha chromatography y'amazi ikora neza cyane mu iterambere ry'imiti. Wren, SA & Tchelitchef, P. Gukoresha chromatography y'amazi ikora neza cyane mu iterambere ry'imiti.Ren, SA na Chelischeff, P. Ikoreshwa rya chromatography y'amazi ifite imikorere myiza cyane mu iterambere ry'imiti. Wren, SA & Tchelitcheff, P. 超高效液相色谱在药物开发中的应用。 Wren, SA na Tchelitchef, P.Ren, SA na Chelischeff, P. Gukoresha chromatography y'amazi ikora neza cyane mu iterambere ry'imiti.J. Kromatography. 1119(1-2), 140-146. https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. n'abandi. Hydrogel ifite imyenge minini ikomoka kuri emulsion y'amavuta iri mu mazi ifite urwego rwo hejuru rw'imbere mu gusukura neza enterovirus 71. Umushinga w'ibinyabutabire. Ikinyamakuru 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. na Wilkins, JA Uruhare rwa chromatography y'amazi muri poroteyine. Shi, Y., Xiang, R., Horváth, C. na Wilkins, JA Uruhare rwa chromatography y'amazi muri poroteyine.Shi, Y., Xiang, R., Horvath, C. na Wilkins, JA Uruhare rwa chromatography y'amazi muri poroteyine. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA 液相色谱在蛋白质组学中的作用。 Shi, Y., Xiang, R., Horváth, C. & Wilkins, JAShi, Y., Xiang, R., Horvath, C. na Wilkins, JA Uruhare rwa chromatography y'amazi muri poroteyine.J. Kromatography. A 1053 (1-2), 27-36 (2004).
Fekete, S., Vutey, J.-L. & Guillarme, D. Imiterere mishya mu gutandukanya peptide na poroteyine by’amazi mu buryo bwa "reverse-phase": Inyigisho n'ikoreshwa. & Guillarme, D. Imiterere mishya mu gutandukanya peptide na poroteyine by’amazi mu buryo bwa "reverse-phase": Inyigisho n'ikoreshwa. & Guillarme, D. Н. & Guillarme, D. Imiterere mishya mu gutandukanya peptides na poroteyine zivura hakoreshejwe chromatography y'amazi ihinduka: inyigisho n'ikoreshwa. & Guillarme, D. 治疗性肽和蛋白质的反相液相色谱分离的新趋势：理论和应用。 na Guillarme, D.na Guillarmé, D. Imiterere mishya mu gutandukanya peptides na poroteyine zivura hakoreshejwe chromatography y'amazi ihinduka: inyigisho n'ikoreshwa.J. Pharm. Ubumenyi bw'Ubuvuzi. ikibuno. 69, 9–27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Gutandukanya peptide mu buryo bw'ibice bibiri hakoreshejwe sisitemu ya RP-RP-HPLC ifite pH itandukanye mu bipimo bya mbere n'ibya kabiri byo gutandukanya. Gilar, M., Olivova, P., Daly, AE & Gebler, JC Gutandukanya peptide mu buryo bw'ibice bibiri hakoreshejwe sisitemu ya RP-RP-HPLC ifite pH itandukanye mu bipimo bya mbere n'ibya kabiri byo gutandukanya.Gilar M., Olivova P., Dali AE na Gebler JK Gutandukanya peptide mu buryo bw'ibice bibiri hakoreshejwe sisitemu ya RP-RP-HPLC ifite pH itandukanye mu bipimo bya mbere n'ibya kabiri byo gutandukanya.Gilar M., Olivova P., Dali AE na Gebler JK Gutandukanya peptide mu buryo bubiri hakoreshejwe pH zitandukanye mu bipimo bya mbere n'ibya kabiri byo gutandukanya hakoreshejwe sisitemu ya RP-RP-HPLC. J. Sept Science. 28 (14), 1694–1703 (2005).
Fellitti, S. n'abandi. Iperereza ku ihererekanya ry'uburemere n'imiterere ya kinetiki y'inkingi za chromatography zikora neza zipakiye uduce twa C18 dufite imyenge yuzuye kandi dufite imyenge mikeya ntoya kurusha 2 µm. J. Sept Science. 43 (9–10), 1737–1745 (2020).
Piovesana, S. n'abandi. Impinduka ziherutse n'imbogamizi mu isesengura mu kwitandukanya, kumenya no kwemeza peptides zikora ku bimera. anus. Creature anal. Chemical. 410(15), 3425-3444. https://doi.org/10.1007/s00216-018-0852-x (2018).
Muller, JB n'abandi. Imiterere y'ubwami bw'ubuzima mu buryo bwa Proteomic. Nature 582 (7813), 592–596. https://doi.org/10.1038/s41586-020-2402-x (2020).
De Luca, K. n'abandi. Nyuma yo kuvura peptides zivura hakoreshejwe chromatography y'amazi itegura. Molecules (Basel, Ubusuwisi) 26 (15), 4688 (2021).
Yang, Y. & Geng, X. Koromatografia y'uburyo butandukanye n'ikoreshwa ryayo kuri biopolymers. Yang, Y. & Geng, X. Koromatografia y'uburyo butandukanye n'ikoreshwa ryayo kuri biopolymers.Yang, Yu. na Geng, X. Koromatografia y'uburyo butandukanye n'ikoreshwa ryayo kuri biopolymers. Yang, Y. & Geng, X. 混合模式色谱及其在生物聚合物中的应用。 Yang, Y. & Geng, X. Koromatografia y'uburyo butandukanye n'ikoreshwa ryayo muri biopolymers.Yang, Yu. na Gene, X. Koromatografia y'uburyo butandukanye n'ikoreshwa ryayo kuri biopolymers.J. Kromatography. A 1218(49), 8813–8825 (2011).