Ukulungiswa Kwezigaba Ezimisiwe Zemodi Exubile Zokuhlukaniswa Kwama-Peptides Namaprotheni Nge-High Performance Liquid Chromatography

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Izinhlayiya ze-silica ezinama-porous zalungiselelwa indlela ye-sol-gel enezinguquko ezithile ukuze zithole izinhlayiya ezinkulu.Lezi zinhlayiya zithathwe yi-reversible addition fragmentation chain transfer (RAFT) polymerization ne-N-phenylmaleimide-methylvinylisocyanate (PMI) kanye ne-styrene ukuze kulungiswe i-N-phenylmaleide yensimbi ye-polyborene ye-polymaleimide. ama-s (i-id engu-100 × 1.8 mm) ayepakishwe ngokupakishwa okunodaka.Kuhlolwe ukuhlukaniswa kwekholomu ye-PMP yengxube ye-peptide ehlanganisa ama-peptide amahlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arnkeg, i-albhamu ye-leucineps ye-leucineps ye-leucineps ye-leucineps ye-leucineps ye-human performance). ).Ngaphansi kwezimo ezingcono kakhulu ze-elution, isibalo sepuleti lethiyori lengxube ye-peptide liphezulu njengokungu-280,000 amapuleti/m². Uma kuqhathaniswa nokusebenza kokuhlukaniswa kwekholomu ethuthukisiwe nekholomu yezentengiso ye-Ascentis Express RP-Amide, kubonwe ukuthi ukusebenza kokuhlukaniswa kwekholomu ye-PMP bekuphakeme kunekholomu yezentengiso ngokusebenza kahle nokuhlukaniswa.
Eminyakeni yamuva nje, imboni ye-biopharmaceutical isiphenduke imakethe ekhulayo yomhlaba wonke ngokukhula okukhulu kwesabelo semakethe. Ngokukhula okushubile kwemboni ye-biopharmaceutical1,2,3, ukuhlaziywa kwama-peptides namaprotheni kuyadingeka kakhulu. Ngaphezu kwe-peptide eqondiwe, ukungcola okuningana kukhiqizwa ngesikhathi sokuhlanganiswa kwe-peptide, ngaleyo ndlela kudinga ukuhlanzwa kwe-protein ukuze kutholwe isifiso sohlamvu. uketshezi lomzimba, izicubu namaseli kuwumsebenzi oyinselele ngokwedlulele ngenxa yenani elikhulu lezinhlobo ezingase zibonakale kusampula eyodwa.Nakuba i-mass spectrometry iyithuluzi eliphumelelayo lokulandelana kwe-peptide ne-protein, uma amasampula anjalo ejovwa ku-spectrometer enkulu ngephasi eyodwa, ukuhlukana ngeke kube okuhle.Le nkinga ingancishiswa ngokuqalisa ukuhlaziya inombolo ye-MS (ukuhlukanisa inombolo ye-MS), okuzonciphisa uhlaziyo lwe-MS uketshezi lwe-LC, oluzonciphisa uhlaziyo lwe-MS i-spectrometer ngesikhathi esinikeziwe4,5,6.Ngaphezu kwalokho, ngesikhathi sokuhlukaniswa kwesigaba soketshezi, abahlaziyi bangagxila ezifundeni ezincane, ngaleyo ndlela bagxilise lezi zibalo futhi bathuthukise ukuzwela kokutholwa kwe-MS.I-Liquid chromatography (LC) ithuthuke kakhulu kule minyaka eyishumi edlule futhi isiphenduke indlela ethandwayo ekuhlaziyweni kwe-proteomic7,8,9,10.
I-reversed-phase liquid chromatography (RP-LC) isetshenziswa kakhulu ekuhlanzeni nasekuhlukaniseni izingxube ze-peptide kusetshenziswa i-octadecyl-modified silica (ODS) njengesigaba esimile11,12,13.Kodwa-ke, izigaba ezimile ze-RP azihlinzeki ngokuhlukana okwanelisayo kwama-peptide namaprotheni ngenxa yokwakheka kwawo okukhethekile15 kwemvelo. okudingekayo ukuze kuhlaziywe ama-peptide nama-protein ane-polar ne-non-polar moieties ukuze kuhlanganyele futhi kugcinwe lezi analytes16.I-Mixed-mode chromatography, ehlinzeka ngokusebenzisana kwe-multimodal, ingaba enye indlela ye-RP-LC yokuhlukaniswa kwama-peptide, amaprotheni, nezinye izingxube eziyinkimbinkimbi.Iningana yemodi exubile yemodi exubile1 ilungiselelwe, lezi zigaba ezimisiwe1 ze-protein1 zilungiselelwe, lezi zigaba ezimisiwe kanye nezigaba1 ziye zalungiswa, lezi zigaba ze-stationary1 ziye zalungiswa, lezi zigaba ezimisiwe kanye nezigaba1 ziye zalungiswa, lezi zigaba ezimisiwe kanye nezigaba1 zisetshenzisiwe. 8,19,20,21.Izigaba ezimile zemodi exubile (i-WAX/RPLC, i-HILIC/RPLC, i-polar intercalation/RPLC) ifanele ukuhlukaniswa kwe-peptide kanye namaprotheni ngenxa yokuba khona kokubili kwamaqembu e-polar kanye namaqembu angewona ama-polar22,23,24,25,26,27,28 .Ngokufanayo nesigaba samandla esiyingqayizivele se-polar se-polar sibonisa i-polar enhle ye-polar kanye nesiteshi se-polar esiyingqayizivele. ukukhethwa kwama-analyte e-polar kanye ne-non-polar, njengoba ukuhlukana kuncike ekusebenzisaneni phakathi kwe-analyte nesigaba esimile.Ukusebenzisana kwe-Multimodal 29, 30, 31, 32.Muva nje, u-Zhang et al.I-30 ilungise isigaba sokumiswa kwe-dodecyl-terinated polyamine futhi yahlukanisa ngempumelelo ama-hydrocarbon, ama-antidepressants, ama-flavonoid, ama-nucleosides, ama-estrogens, nezinye izihlaziyo ezimbalwa.I-polar intercalator inamaqembu e-polar kanye ne-non-polar, ngakho-ke ingasetshenziswa ukuhlukanisa ama-peptide nama-protein anako kokubili i-hydrophobic kanye ne-hydrophilic-ayidrophilicembed, i-polar-modiumembed columns) ayatholakala ngokwentengiso ngaphansi kwegama lokuhweba elithi Ascentis Express RP-Amide columns, kodwa lawa makholomu asetshenziselwa ukuhlaziywa kwe-amine 33 kuphela.
Ocwaningweni lwamanje, i-polar-embedded stationary phase (N-phenylmaleimide-embedded polystyrene) yalungiswa futhi yahlolwa ukuze kuhlukaniswe ama-peptides kanye ne-trypsin digests ye-HSA.Isigaba esimile salungiswa kusetshenziswa leli qhinga elilandelayo.Izinhlayiya ze-silica ezinama-porous zalungiswa ngokuvumelana nenqubo enikezwe ekushicilelweni kwethu kwangaphambilini ngokunye ukuguqulwa. I-cetic acid yalungiswa ukuze kulungiswe izinhlayiya ze-silica ezinosayizi omkhulu wembotshana.Okwesibili, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yahlanganiswa futhi yasetshenziselwa ukukhipha izinhlayiya ze-silica ukuze kulungiswe isigaba esimile esishunyekiwe se-polar. Isigaba esimile esaphumela kusetshenziswa ikholomu yokupakishwa kwensimbi engagqwali (100 mm isikimu sokupakishwa kwensimbi engagqwali). ukudlidliza komshini ukuze kuqinisekiswe ukuthi umbhede wenziwe ngendlela efanayo phakathi kwekholomu.Hlola ukuhlukaniswa kwekholomu epakishiwe yezingxube ze-peptide ezihlanganisa ama-peptide amahlanu;(Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) kanye ne-Trypsin digest ye-human serum albumin (HAS).Ingxube ye-peptide ne-trypsin digest ye-HSA yaqashelwa ukuze ihlukaniswe ngokulungiswa okuhle nokusebenza kahle kwekholomu ye-AMP ye-Express AMP yayiqhathaniswa nekholomu yokuhlukaniswa kwe-AMP. ama-peptide namaprotheni abonwe exazululwe kahle futhi esebenza kahle kukholamu ye-PMP, eyayisebenza kahle kakhulu kunekholomu ye-Ascentis Express RP-Amide.
I-PEG (i-Polyethylene Glycol), i-Urea, i-Acetic Acid, i-Trimethoxy Orthosilicate (TMOS), i-Trimethyl Chlorosilane (TMCS), i-Trypsin, i-Human Serum Albumin (HSA), i-Ammonium Chloride, i-Urea, i-Hexane Methyldisilazane (HMDS), i-Methacryloyl Chloride, i-Byyldroksidi (MC) I-HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, ne-Acetone Ithengwe kwa-Sigma-Aldrich (St. Louis, MO, USA).
Ingxube ye-urea (8 g), i-polyethylene glycol (8 g), nama-mL angu-8 ka-0.01 N acetic acid anyakaziswa imizuzu engu-10, kwase kwengezwa u-24 mL we-TMOS lapho ngaphansi kwezimo ezibandayo eqhweni. I-70 ° C amahora angu-12. Isisindo esomile esithambile sasiwumhlabathi obushelelezi kuhhavini futhi sibalwa ku-550 ° C amahora angu-12. Amaqoqo amathathu alungiselelwe futhi abonakaliswe ukuhlola ukuzala kabusha ngobukhulu bezinhlayiyana, ubukhulu be-pore kanye nendawo ephezulu.
Ngokuguqulwa kwendawo yezinhlayiya ze-silica nge-ligand yangaphambili ye-phenylmaleimide-methylvinylisocyanate (PCMP) elandelwa i-polymerization ye-radial nge-styrene, i-polar group-containing compound yalungiswa.Isigaba esimile samaketanga ahlanganisiwe namaketanga e-polystyrene.Inqubo yokulungiselela ichazwe ngezansi.
I-N-phenylmaleimide (200 mg) ne-methyl vinyl isocyanate (100 mg) zincibilikisiwe ku-toluene eyomile, futhi u-0.1 mL we-2,2′-azoisobutyronitrile (AIBN) yengezwe ku-reaction flask ukuze kulungiswe i-phenylmaleimide-methyl vinyl isocyanate amahora angu-3 C, i-CP ° yomisiwe ngamahora angu-30PM i-heart 30PM. kuhhavini ku-40 ° C amahora amathathu.
Izinhlayiya ze-silica ezomile (2 g) zahlakazwa ku-toluene eyomile (100 mL), zinyakaziswa futhi zafakwa ku-sonicated ku-500 mL eyindilinga yeflask ephansi ye-10 min.PMCP (10 mg) yachithwa ku-toluene futhi yengezwa i-dropwise ku-flask yokusabela nge-funnel yokudonsa. amahora.Khona-ke, izinhlayiya ze-silica eziboshiwe ze-PMCP (100 g) zahlakazwa ku-toluene (200 ml) kanye ne-4-hydroxy-TEMPO (2 mL) yengezwe phambi kwe-100 µL ye-dibutyltin dilaurate njenge-catalyst.Ingxube yashukunyiswa ku-50 ° C futhi yomiswa ngamahora angu-3 °, ihlungiwe amahora angu-3.
I-Styrene (1 mL), i-benzoyl peroxide BPO (0.5 mL), kanye nezinhlayiya ze-silica ezinamathiselwe i-TEMPO-PMCP (1.5 g) zihlakazwe ku-toluene futhi zahlanzwa nge-nitrogen. I-polymerization ye-styrene yenziwa ku-100 ° C amahora angu-12. Umkhiqizo owenziwe wawashwa nge-methanol 1 ebusuku ku-reactivity ye-Fire ° C jikelele yomiswa.
Amasampula ahlanjululwe ku-393 K ngehora le-1 ukuze athole ingcindezi esele engaphansi kwe-10-3 Torr.Inani le-N2 elikhangisiwe ngengcindezi ehlobene ye-P / P0 = 0.99 yasetshenziselwa ukucacisa ivolumu ye-pore ephelele.I-morphology yezinhlayiya ze-silica ezingenalutho kanye ne-ligand-bonded, isampula ye-electron ye-tachiesD ephezulu ihlolwe nge-microscopy yaseJapan i-Hiscopy e-Japan scanning. (i-silica engenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand) zibekwe kukholomu ye-aluminium kusetshenziswa i-adhesive carbon tape.Igolide yafakwa kumasampula kusetshenziswa i-sputter coater ye-Q150T, futhi ungqimba lwe-5 nm Au lwafakwa kumasampula.Lokhu kuthuthukisa ukusebenza kahle kwenqubo ngokusebenzisa ama-voltage aphansi futhi kunikeza okusanhlamvu okuhle, i-Flash sputtering ebandayo, i-EA2 i-Eleron ebandayo yase-USA1 Isici se-EA2 i-Flash1ron. isetshenziselwa ukuhlaziya okuyisisekelo.I-Malvern (Worcestershire, UK) I-Mastersizer engu-2000 yokuhlaziya usayizi wezinhlayiyana yasetshenziswa ukuze kutholwe ukusatshalaliswa kosayizi wezinhlayiyana.Izinhlayiya ze-silica ezinqunu kanye nezinhlayiya ze-silica eziboshwe nge-ligand (5 mg ngayinye) zihlakazwe ku-5 mL we-isopropanol, i-sonicated for 10 min, vortexed, and the Master of the wasmotric analysis of the 5 mg. kwenziwa ngesilinganiso esingu-5 °C ngomzuzu phezu kwezinga lokushisa elingu-30 kuya ku-800 °C.
Amakholomu ensimbi engenacala anemigqa emincane enobukhulu (i-id engu-100 × 1.8 mm) aye apakishwa kusetshenziswa indlela yokupakisha enodaka, kusetshenziswa inqubo efanayo esetshenziswe ku-Ref.31.Ikholomu yensimbi engagqwali (efakwe ingilazi, i-id engu-100 × 1.8 mm) ene-outlet fitting equkethe i-1 µm frit yayixhunywe ku-slurry packer (Alltech Deerfield, IL, USA). Lungiselela i-slurry yesigaba esimile ngokumisa u-150 mg wesigaba esimisiwe se-mL futhi thumela ikholomu ye-Menotha ku-menotha esetshenziswa ku-menotha 1. i-solvent slurry kanye ne-propelling solvent.Gcwalisa ikholomu ngokulandelana ngokusebenzisa izingcindezi ze-100 MP imizuzu engu-10, i-80 MP imizuzu engu-15, kanye ne-60 MP imizuzu engu-30. Ngesikhathi sokupakisha, ukudlidliza komshini kwasetshenziswa ngama-shaker amabili ekholomu ye-GC (Alltech, Deerfield, IL, IL, IL, IL, I-USA) ukupakisha ukucindezela kwekholomu ukuze kuvinjwe ingcindezi yokukhishwa kancane kancane. ikholomu.Nqamula ikholomu kuyunithi yokupakisha okunodaka bese uxhuma okunye ukufaka endaweni yokungena kanye nohlelo lwe-LC ukuze uhlole ukusebenza kwayo.
Iphampu ye-LC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) ene-50nL injection loop, i-membrane degasser (Shimadzu DGU-14A), iwindi le-capillary le-UV-VIS lakhiwe Isitholi sedivayisi esikhethekile se-µLC (UV-2075) kanye nomphumela omncane oxhumanisa ikholomu encane yengilazi kumashubhu amancane anengilazi ebanzi kakhulu kumashubhu amancane anemigqa ebanzi. Ening.Ngemva kokupakishwa, ama-capillary (50 μm id 365 kanye nokunciphisa ama-union capillaries (50 μm) afakwe endaweni engu-1/16″ yokuncishiswa kwenyunyana. Ukuqoqwa kwedatha nokucutshungulwa kwe-chromatographic kwenziwa kusetshenziswa isofthiwe ye-Multichro 2000. Ukuqapha ku-254 μm were tested UVtes Analyze Analyse i-Chromatographic ye-Chromatographic. Northampton, MA).
I-Albumin ephuma ku-serum yomuntu, impushana e-lyophilized, ≥ 96% (i-agarose gel electrophoresis) 3 mg exutshwe ne-trypsin (1.5 mg), 4.0 M urea (1 mL), kanye ne-0.2 M ammonium bicarbonate (1 mL).Isixazululo sanyakaziswa imizuzu engu-10 futhi sigcinwa kubhavu wamanzi amahora angu-60 % 1 % . Hlunga isixazululo bese ugcine ngaphansi kuka-4 °C.
Ukuhlukaniswa kwezingxube ze-peptide kanye nokugaya kwe-HSA trypsin kwahlaziywa ngokuhlukana kumakholomu e-PMP.Hlola ukuhlukaniswa kwengxube ye-peptide kanye ne-trypsin digest ye-HSA ngekholomu ye-PMP bese uqhathanisa imiphumela nekholomu ye-Ascentis Express RP-Amide.Inombolo yepuleti lethiyori ibalwa kanje:
Izithombe ze-SEM zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-FIG.2 .Sem Izithombe zezinhlayiya ze-Silica ezingenalutho (a, b) zikhombise lokho, ngokungafani nezifundo zethu zangaphambili, lezi zinhlayiyana zibushelelezi noma izihlayikhali ezingajwayelekile.
Iskena izithombe zesibonakhulu se-electron zezinhlayiya ze-silica ezingenalutho (A, B) kanye nezinhlayiya ze-silica eziboshwe nge-ligand (C, D).
Ukusabalalisa usayizi wezinhlayiyana zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-Figure 3 (A). Amajika okusabalalisa usayizi wezinhlayiyana ezisekelwe kuvolumu abonise ukuthi ubukhulu bezinhlayiya ze-silica banda ngemva kokuguqulwa kwamakhemikhali (Fig. 3A) .Idatha yokusabalalisa usayizi wezinhlayiyana zezinhlayiya ze-silica kusukela ocwaningweni lwamanje kanye nocwaningo lwangaphambilini lwezinhlayiyana ze-P, i-MP iqhathaniswa ne-P. 3.36 μm, uma kuqhathaniswa nocwaningo lwethu lwangaphambilini ngenani lesikhangiso (0.5) lika-3.05 μm (izinhlayiya ze-silica eziboshwe nge-polystyrene) 34.Leli qoqo libe nokusabalalisa usayizi wezinhlayiyana ezincane uma kuqhathaniswa nocwaningo lwethu lwangaphambilini ngenxa yezilinganiso ezihlukene ze-PEG, urea, TMOS, ne-acetic acid engxubeni yokusabela enkulu kunezinhlayiyana ze-polycarene zesigaba sangaphambilini se-silica esifundwe ngaphambilini. .Lokhu kusho ukuthi ukusebenza okungaphezulu kwezinhlayiya ze-silica ezine-styrene kubeka kuphela isendlalelo se-polystyrene (0.97 µm) endaweni ye-silica, kanti esigabeni se-PMP ukujiya kwesendlalelo kwakungu-1.38 µm.
Ukusabalalisa usayizi we-particle (A) nokusabalalisa usayizi we-pore (B) wezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand.
Ubukhulu bembotshana, ivolumu yembotshana kanye nendawo engaphezulu yezinhlayiya ze-silica zocwaningo lwamanje zinikezwe kuThebula 1(B) .Amaphrofayili e-PSD ezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa kuMfanekiso 3(B). Imiphumela iqhathaniswa nesifundo sethu sangaphambilini.Amasayizi wembotshana we-bare kanye nezinhlayiya ze-ligand abonisa ukuthi i-silica eboshwe nge-23, i-23 eboshwe ngokulinganayo, i-silica eboshwe nge-23. usayizi uyancipha ngo-69 ngemva kokuguqulwa kwamakhemikhali, njengoba kuboniswe kuThebula 1 (B), futhi ukuguqulwa kwejika kuboniswe ku-Fig. 3 (B) .Ngokufanayo, umthamo we-pore wezinhlayiya ze-silica wehla ukusuka ku-0.67 kuya ku-0.58 cm3 / g ngemva kokuguqulwa kwamakhemikhali.Indawo ethile ecacile ye-silica efundwayo okwamanje i-11 m / i-2g yezinhlayiya ze-silica ezifundwayo ze-2 / 2g yangaphambilini (2g / 2g) ).Njengoba kuboniswe kuThebula 1(B), indawo engaphezulu (m2/g) yezinhlayiya ze-silica nayo yehla isuka ku-116 m2/g yaya ku-105 m2/g ngemva kokuguqulwa kwamakhemikhali.
Imiphumela yokuhlaziywa okuyisisekelo kwesigaba esimile ikhonjisiwe kuThebula 2.Ukulayishwa kwekhabhoni kwesigaba esimile samanje kungu-6.35%, okungaphansi kokulayishwa kwekhabhoni ocwaningweni lwethu lwangaphambilini (izinhlayiya ze-silica eziboshwe nge-polystyrene, 7.93%35 kanye no-10.21%, ngokulandelana) 42. Ukulayishwa kwekhabhoni kwesigaba esiphansi se-polar, i-ligarene yamanje, ukulungiswa kwe-ligarene yamanje Kusetshenziswe i-phenylmaleimide-methylvinylisocyanate (PCMP) kanye ne-4-hydroxy-TEMPO.Isilinganiso se-nitrogen sesisindo sesigaba samanje esimile singu-2.21%, uma siqhathaniswa ne-0.1735 kanye ne-0.85% ngesisindo se-nitrogen ezifundweni zangaphambilini, ngokulandelana.Lokhu kusho ukuthi i-wt % yemikhiqizo ye-nitrogen ye-carbonnymilar ephezulu, i-carbonnymile lemikhiqizo ye-carbonnymile iphakeme I-4) ne-(5) yayiyi-2.7% kanye ne-2.9%, ngokulandelana, kuyilapho ukulayishwa kwekhabhoni yomkhiqizo wokugcina (6) kwakungu-6.35%, njengoba kuboniswe kuThebula 2. Ukulahlekelwa kwesisindo kuhlolwe ngesigaba sokumisa se-PMP, futhi ijika le-TGA liboniswa ku-Figure 4.Ijika le-TGA libonisa ukulahlekelwa kwesisindo lapho i-carbon ilayisha khona i-8.6% kuphela, kodwa i-carbon equkethe isivumelwano se-8.6% equkethe i-ligas enhle kuphela. futhi N, O, kanye no-H.
I-phenylmaleimide-methylvinylisocyanate ligand ikhethelwe ukuguqulwa kwendawo yezinhlayiya ze-silica ngoba inamaqembu e-polar phenylmaleimide kanye namaqembu e-vinylisocyanate.Amaqembu e-vinylisocyanate angakwazi ukuqhubeka asabele nge-styrene ngokuphila kwe-polymerization e-radical.Isizathu sesibili ukufaka iqembu elinokuxhumana okumaphakathi nesiteshi se-analyte ne-analyte electrony kanye nokuxhumana okuqinile kwe-analyte ne-analyte ye-analyte. I-mide moiety ayinakho ukushaja okubonakalayo ku-pH evamile.I-polarity yesigaba esimile ingalawulwa ngenani eliphelele le-styrene kanye nesikhathi sokusabela se-polymerization ye-radical yamahhala.Isinyathelo sokugcina sokusabela (i-polymerization yamahhala) sibalulekile futhi singashintsha i-polarity yesigaba esimile.Ukuhlaziywa kwe-Elemental kwenziwa ukuze kuhlolwe ukulayishwa kwekhabhoni yalezi zigaba ezikhulayo kanye nenani le-carbon ekhulayo lesigaba esikhulayo kanye nenani elikhulayo lesigaba sekhabhoni. okuphambene nalokho.Ama-SP alungiselelwe ngokugxila okuhlukene kwe-styrene anokulayisha okuhlukile kwekhabhoni.Futhi, layisha lezi zigaba ezimile kumakholomu ensimbi engagqwali futhi uhlole ukusebenza kwazo kwe-chromatographic (ukukhethwa, ukulungiswa, inani le-N, njll.).Ngokusekelwe kulezi zivivinyo, ukwakheka okuthuthukisiwe kwakhethwa ukuze kulungiswe isigaba sokumisa se-PMP ukuze kuqinisekiswe ukubuyiswa kwe-polarity okulawulwayo kanye nokuhlaziya okuhle.
Izingxube ze-peptide ezinhlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nayo yahlolwa kusetshenziswa ikholomu ye-PMP kusetshenziswa isigaba seselula;60/40 (v/v) i-acetonitrile/amanzi (0.1% TFA) ngenani lokugeleza elingu-80 μL/min. Ngaphansi kwezimo ezingcono kakhulu zokucacisa, inombolo yepuleti lethiyori (N) ikholomu ngayinye (i-id engu-100 × 1.8 mm) ingu-20,000 ± 100 (amapuleti angu-200, amakholomu amathathu angu-P² anikeza ikholomu engu-000). ama-chromatogram akhonjiswe kuMfanekiso 5A.Ukuhlaziywa okusheshayo kukholomu ye-PMP ngenani lokugeleza okuphezulu (700 μL/min), ama-peptide amahlanu akhishwa kungakapheli umzuzu owodwa, amanani e-N ayemahle kakhulu, 13,500 ± 330 ikholomu ngayinye (100 × 1.8 mm id), Ihambisana no-00Thdensm ikholomu i-0. (I-id engu-100 × 1.8 mm) ibigcwele izigaba ezintathu ezihlukene zesigaba sokuma se-PMP ukuze kuhlolwe ukukhiqizwa kabusha.Ukugxiliswa kwe-analyte kwekholomu ngayinye kwarekhodwa kusetshenziswa izimo ezingcono kakhulu ze-elution kanye nenani lamapuleti etiyetha N kanye nesikhathi sokugcinwa ukuze kuhlukaniswe ingxube efanayo yokuhlola kukholamu ngayinye.Idatha yokukhiqiza kabusha yamakholomu e-PMP anekholomu ye-PMP ephansi kakhulu yokuphindaphinda kwekholomu ye-PMP ekhonjiswe kahle ikholomu ye-PMP ephansi kakhulu. Amanani e-RSD, njengoba kukhonjisiwe kuThebula 3.
Ukuhlukaniswa kwengxube ye-peptide kukholomu ye-PMP (B) kanye nekholomu ye-Ascentis Express RP-Amide (A);isigaba seselula 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholomu ye-PMP (100 × 1.8 mm id);i-analytical I-elution order of the compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kanye 5 (leucine) acid enkephalin)).
Ikholomu ye-PMP (i-id engu-100 × 1.8 mm) yahlolelwa ukuhlukaniswa kokugaya kwe-tryptic kwe-albumin ye-serum yomuntu ekusebenzeni okuphezulu kwe-chromatography ye-liquid. I-chromatogram ekuMdwebo 6 ibonisa ukuthi isampula ihlukaniswe kahle futhi ukulungiswa kuhle kakhulu.Ukugaya kwe-HSA kuhlaziywe kusetshenziswa izinga lokugeleza elingu-100min/tole3/elihambayo elingu-100/tole3µ 1% TFA.Njengoba kubonisiwe ku-chromatogram (Umfanekiso 6), ukugaya kwe-HSA kuhlukaniswe kwaba iziqongo eziphakeme eziyi-17 ezihambisana nama-peptide angu-17. Ukusebenza kahle kokuhlukaniswa kwenani eliphakeme ngalinye kunhlabamkhosi ye-HSA kubaliwe futhi amanani anikezwa kuThebula lesi-5.
I-tryptic digest ye-HSA (100 × 1.8 mm id) yahlukaniswa kukholamu ye-PMP;izinga lokugeleza (100 µL/min), isigaba seselula 60/40 acetonitrile/amanzi ano-0.1% TFA.
lapho u-L engubude bekholomu, η yi-viscosity yesigaba seselula, i-ΔP ingcindezi yekholomu emuva, futhi u-u isivinini somugqa wesigaba esihambayo.Ukufinyeleleka kwekholomu ye-PMP kwakungu-2.5 × 10-14 m2, izinga lokugeleza lalingu-25 μL/min, futhi 60/40 v/v ikholomu engu-00 i-ACN/amanzi isetshenziswe i-ACN1 mm1 (ikholomu ngayinye ye-ACN1) isetshenziswe ikholomu ngayinye engu-1 mm. kwakufana nalokho esifundweni sethu sangaphambilini Ref.34.Ukuvuthwa kwekholomu egcwele izinhlayiya ezimboza kancane yilokhu: 1.7 × 10-15 yezinhlayiya ezingu-1.3 μm, 3.1 × 10-15 ngezinhlayiya ezingu-1.7 μm, 5.2 × 10-5 × 2 μm izinhlayiyana ezingu-2 × 2 × 2 m. μm izinhlayiya 43.Ngakho-ke, ukufinyeleleka kwesigaba se-PMP kufana nalokho kwezinhlayiya ze-core-shell ezingu-5 μm.
lapho i-Wx iyisisindo sekholomu egcwele i-chloroform, i-Wy isisindo sekholomu egcwele i-methanol, futhi u-ρ ukuminyana kwe-solvent.Ukuminyana kwe-methanol (ρ = 0.7866) ne-chloroform (ρ = 1.484).Isamba se-porosity yekholomu ye-SILICA PARTICLES 108 × 10 × 10 × 18 mm ikholomu i-108 x 1. Amakholomu e-Urea 31 esiwafunde ngaphambilini ayengu-0.63 kanye no-0.55, ngokulandelana.Lokhu kusho ukuthi ukuba khona kwe-urea ligand kunciphisa ukufinyeleleka kwesigaba esimile.Ngakolunye uhlangothi, ingqikithi ye-porosity yekholomu ye-PMP (i-id engu-100 × 1.8 mm) ingu-0.60.I-permeability yezinhlayiyana ze-C1 zekholomu ye-CcaMP iphansi kunekholomu ye-silili engu-C1 ye-CMP. Izigaba ezimile zohlobo oluyisi-8 ama-C18 ligands anamathiselwe ezinhlayiyeni ze-silica njengamaketango alayini, kuyilapho ezigabeni ezimile zohlobo lwe-polystyrene, kwakheka ungqimba lwe-polymer oluwugqinsi oluzungezile.
Umfanekiso 7A,B ubonisa ikholomu ye-PMP (i-id engu-100 × 1.8 mm) kanye nekholomu ye-Ascentis Express RP-Amide (i-id engu-100 × 1.8 mm) kusetshenziswa izimo ezifanayo zokugqama (okungukuthi, 60/40 ACN/H2O kanye no-0.1% TFA).) yesakhiwo sika-van Deemter.Izingxube ze-peptide ezikhethiwe (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leucine Enkephalin) ilungiswe ngo-20 µL/ Izinga lokugeleza eliphansi lawo womabili amakholomu ngu-800 µL/min. Ubuncane bekholomu ye-HETP 0min ukugeleza kwe-MPµµ ikholomu ye-Express RP-Amide ibingu-2.6 µm no-3.9 µm, ngokulandelanayo. Amanani e-HETP abonisa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP (100 × 1.8 mm id) kungcono kakhulu kunekholomu ye-Ascentis Express RP-Amide etholakalayo kwezohwebo (100 × 1.8 mm ikholomu ekhuphukayo ngenani le-N Deem 7). akubalulekile uma kuqhathaniswa nesifundo sethu sangaphambilini.Ukuphumelela okuphezulu kokuhlukaniswa kwekholomu ye-PMP (i-id engu-100 × 1.8 mm) uma kuqhathaniswa nekholomu ye-Ascentis Express RP-Amide isekelwe ekuthuthukisweni kokuma kwezinhlayiyana, usayizi, kanye nezinqubo zokupakisha zekholomu eziyinkimbinkimbi ezisetshenziswe emsebenzini wamanje34.
(A) icebo le-van Deemter (i-HETP uma iqhathaniswa nesivinini somugqa wesigaba seselula) itholwe kusetshenziswa ikholomu ye-PMP (100 × 1.8 mm id) ku-60/40 ACN/H2O ne-0.1% TFA.(B) isiqephu se-van Deemter (i-HETP iqhathaniswa nesivinini somugqa wesigaba seselula) itholwe kusetshenziswa ikholomu ye-Ascent0 × 1 i-Ascentis × 1 RP-Ascentis Express 0 ACN/H2O nge-0.1% TFA.
Isigaba sokuma se-polar-embedded polystyrene salungiswa futhi sahlolwa ukuze kuhlukaniswe izingxube ze-peptide zokwenziwa kanye nokugaya kwe-trypsin kwe-albumin ye-serum yomuntu (HAS) ekusebenzeni okuphezulu kwe-chromatography ye-liquid. izinhlayiya, ukuhlanganiswa okulawulwayo kwesigaba esimile, kanye nokupakishwa kwekholomu eyinkimbinkimbi.Ngaphezu kokusebenza kahle kokuhlukana okuphezulu, ukucindezela okuphansi kwekholomu emuva emazingeni aphezulu okugeleza kungenye inzuzo yalesi sigaba esimile.Amakholomu e-PMP abonisa ukukhiqiza okuhle futhi angasetshenziswa ekuhlaziyeni izingxube ze-peptide kanye nokugaya kwe-trypsin kwamaphrotheni ahlukahlukene. i-ography.Esikhathini esizayo, amakholomu e-PMP azophinde ahlolwe ukuze kuhlukaniswe amaprotheni kanye namasosha omzimba e-monoclonal.
I-Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Ucwaningo Lwezinhlelo Zokuhlukaniswa Kwe-Peptide NgeSigaba Esihlehlisiwe Se-Chromatography Ingxenye I: Ukuthuthukiswa Kwephrothokholi Yokubalingiswa Kwekholomu.J.I-Chromatography.1603, 113–129.https://doi.org/10.1016/j.chroma.2019.05.038 (2019).
Gomez, B. et al.Ama-peptide asebenzayo athuthukisiwe adizayinelwe ukwelashwa kwezifo ezithathelwanayo.Biotechnology.Advanced.36(2), 415-429.https://doi.org/10.1016/j.biotechadv.2018.01.004 (2018).
Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic peptides zokwelapha: isayensi kanye nemakethe.ukutholakala kwezidakamizwa.15 (1-2) namuhla, 40-56.https://doi.org/10.1016/j.drudis.2009.10.109.10.0).
U-Xie, F., Smith, RD & Shen, Y. Advanced Proteomic Liquid Chromatography.J.I-Chromatography.A 1261, 78–90 (2012).
U-Liu, W. et al.Izimboni ezithuthukisiwe ze-liquid chromatography-mass spectrometry zinika amandla ukuhlanganiswa kwe-metabolomics eqondiswe kabanzi kanye ne-proteomics.anus.Chim.Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Indima ye-UHPLC ekuthuthukisweni kwezidakamizwa.J.Sep. Sci.30(8), 1183-1190 (2007).
I-Wu, N. & Clausen, AM Izici eziyisisekelo nezisebenzayo ze-ultrahigh pressure liquid chromatography ukuze kuhlukaniswe ngokushesha.J.Sep. Sci.30(8), 1167-1182.https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitchef, P. Ukusetshenziswa kwe-ultra-high performance liquid chromatography ekuthuthukisweni kwezidakamizwa.J.I-Chromatography.1119(1-2), 140-146.https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al.Monolithic macroporous hydrogels alungiswe kusukela kuwoyela emanzini aphezulu emulsions yesigaba sangaphakathi ukuze kuhlanzwe ngempumelelo ama-enteroviruses.Chemical.Britain.J.401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Indima ye-chromatography ewuketshezi ku-proteomics.J.I-Chromatography.A 1053(1-2), 27-36 (2004).
Fekete, S., Veuthey, J.-L.& Guillarme, D. Amathrendi asafufusayo ekwehlukanisweni kwe-reversed-phase liquid chromatography yama-peptide okwelapha namaprotheni: ithiyori kanye nokusetshenziswa.J.Ikhemisi.Biomedical Science.anus.69, 9-27 (2012).
U-Gilar, M., Olivova, P., Daly, AE & Gebler, JC Ukuhlukaniswa kwezinhlangothi ezimbili kwama-peptide kusetshenziswa uhlelo lwe-RP-RP-HPLC kusetshenziswa amanani ahlukene we-pH kubukhulu bokuhlukaniswa bokuqala nokwesibili.Sep. Sci.28(14), 1694-1703 (2005).
Feletti, S. et al.Izici zokudluliswa kwenqwaba nokusebenza kwe-kinetic kwamakholomu echromatographic asebenza kahle kakhulu apakishwe nge-C18 sub-2 μm ngokugcwele futhi izinhlayiya ezinezimbotshana ezikha phezulu kuye kwaphenywa.Sep. Sci.43 (9-10), 1737-1745 (2020).
Piovesana, S. et al.Amathrendi akamuva nezinselele zokuhlaziya ekuhlukaniseni, ukuhlonza kanye nokuqinisekiswa kwe-plant bioactive peptides.anus.biological anus.Chemical.410(15), 3425–3444.https://doi.org/10.1007/s00216-02-018-08-08).
Mueller, JB et al.I-proteomic landscape yombuso wokuphila.Imvelo 582(7813), 592-596.https://doi.org/10.1038/s41586-020-2402-x (2020).
DeLuca, C. et al.Downstream processing of therapeutic peptides by preparative liquid chromatography.Molecule (Basel, Switzerland) 26(15), 4688(2021).
I-Yang, Y. & Geng, X. I-chromatography yemodi exubile kanye nokusebenza kwayo kuma-biopolymers.J.I-Chromatography.A 1218(49), 8813–8825 (2011).
I-Zhao, G., Dong, X.-Y.& Sun, Y. Ligands ye-protein chromatography yemodi exubile: umgomo, ukwakheka kwezinhlamvu, nokuklama.J.I-Biotechnology.144(1), 3-11 (2009).


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