Ndiyabulela ngokundwendwela i-Nature.com.Uguqulelo lwesikhangeli oyisebenzisayo lunenkxaso elinganiselweyo ye-CSS.Ukufumana amava angcono kakhulu, sincoma ukuba usebenzise i-browser ehlaziyiweyo (okanye ucime imodi yokuhambelana kwi-Internet Explorer). Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siya kubonisa isayithi ngaphandle kwezitayela kunye neJavaScript.
Iingqungquthela ze-silica ezinobuncwane zilungiswe yindlela ye-sol-gel kunye nokuguqulwa okuthile ukufumana i-macroporous particles.Ezi ngqungquthela zithathwe ngokubuyisela ukuguqulwa kwe-reversible fragmentation chain transfer (RAFT) i-polymerization kunye ne-N-phenylmaleimide-methylvinylisocyanate (PMI) kunye ne-styrene ukulungiselela i-N-phenylmaleide ye-polymaleimide yesitishi se-polymaleimide. iikholomu zensimbi ezingenasici (i-100 × 1.8 mm id) zipakishwe ngokupakishwa kwe-slurry.I-Vavaluated PMP ikholamu yokwahlulwa komxube we-peptide obandakanya iipeptide ezintlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, iTyr-Ile-Gly-Gly-Gly-Ser-Arg ye-Arg ye-Tryphan), i-try-Gly-Gly-Ser-Arg, i-Try-Gly-Gly-Ser-Arg-Arg, i-Gly-Tyr-Leu-Tyr. ukugaywa kwe-albumin ye-serum yabantu (HAS) .Ngaphantsi kweemeko ezifanelekileyo ze-lution, i-theoretical plate count yomxube we-peptide iphezulu njenge-280,000 plates/m².Ukuthelekisa ukusebenza kokwahlula kwekholamu ephuhlisiwe kunye nekholamu yorhwebo ye-Ascentis Express RP-Amide, kwabonwa ukuba ukusebenza kokwahlula kwekholamu ye-PMP yekholamu yorhwebo yayiphezulu kwikholamu ye-PMP.
Kwiminyaka yakutshanje, ishishini le-biopharmaceutical liye laba lurhwebo olukhulayo lwehlabathi kunye nokwanda okukhulu kwisabelo semarike.Ngokukhula okugqabhukileyo kweshishini le-biopharmaceutical1,2,3, uhlalutyo lweepeptides kunye neeproteni zinqweneleka kakhulu. ukubonakaliswa kweeprotheni kulwelo lomzimba, izicubu kunye neeseli ngumsebenzi onzima kakhulu ngenxa yenani elikhulu leentlobo ezinokubonwa kwisampulu enye.Nangona i-mass spectrometry sisixhobo esisebenzayo sokulandelelana kwe-peptide kunye neprotein, ukuba iisampulu ezinjalo zitofelwe kwi-spectrometer yobunzima kwipasi enye, ukwahlukana akuyi kuba yinto efanelekileyo.Le ngxaki inokuphunyezwa ngokunciphisa i-MS paragraphing (uhlalutyo lwe-MS) lunokunciphisa ulwelo lwe-LC. ukunciphisa inani labahlalutyi abangena kwi-spectrometer ye-mass spectrometer ngexesha elinikiweyo4,5,6.Ukongezelela, ngexesha lokuhlukana kwesigaba solwelo, abahlalutyi banokugxininiswa kwimimandla emxinwa, ngaloo ndlela bagxininise aba bahlalutyi kunye nokuphucula ukubonwa kwe-MS.
I-reversed-phase liquid chromatography (RP-LC) isetyenziselwa ngokubanzi ukuhlanjululwa kunye nokwahlula imixube ye-peptide usebenzisa i-silica e-octadecyl-modified (ODS) njengesigaba esimileyo11,12,13.Nangona kunjalo, izigaba ze-RP ezimileyo aziboneleli ngokwanelisayo ngokuhlukana kweepeptides kunye neeprotheni ngenxa ye-amphiphilic eyilwe ngaphambili, i-amphiphilic eyilelwe ubume bayo obukhethekileyo. izigaba ezimileyo ziyafuneka ukuhlalutya i-peptides kunye neeprotheni ezine-polar kunye ne-non-polar moieties ukusebenzisana kunye nokugcina ezi zihlalutyi16.I-Mixed-mode chromatography, ebonelela ngonxibelelwano lwe-multimodal, ingaba yinto engenye ye-RP-LC yokwahlula iipeptides, iiprotheni, kunye neminye imixube eyinkimbinkimbi. Ukwahlula i-peptide kunye neprotheyini17,18,19,20,21.Izigaba zokumisa ezixutyiweyo (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zifanelekile ukuhlukana kwe-peptide kunye neprotein ngenxa yobukho bamaqela amabini angama-polar kunye ne-non-polar22,23,24,25,26,26,26,26. Amaqela e-polar adityaniswe ngokudibeneyo abonisa amandla okuhlula okulungileyo kunye nokukhetha okukhethekileyo kwi-polar kunye ne-non-polar analytes, njengoko ukwahlukana kuxhomekeke kwintsebenziswano phakathi kwe-analyte kunye nesigaba esimileyo. Ukusebenzisana kwe-Multimodal 29, 30, 31, 32.Kutshanje, uZhang et al. I-30 ilungiselele i-dodecyl-terinated polyamine isigaba sokumisa kwaye yahlula ngempumelelo i-hydrocarbons, i-antidepressants, i-flavonoids, i-nucleosides, i-estrogens, kunye nezinye i-analytes ezininzi.I-polar intercalator inamaqela amabini angama-polar kunye nangekho-polar, ngoko ingasetyenziselwa ukwahlula i-peptides kunye neeprotheni ezine-hydrophobic kunye ne-hydrophilic-modiumeg hydrophilic (i-polar-polar-molumu) Iikholamu ze-amide ezifakwe kwi-C18) zifumaneka ngokurhweba phantsi kwegama lokuthengisa i-Ascentis Express RP-Amide ikholamu, kodwa ezi zikholomu zisetyenziselwa uhlalutyo lwe-amine 33 kuphela.
Kuphononongo lwangoku, i-polar-embedded stationary phase (i-N-phenylmaleimide-embedded polystyrene) ilungiselelwe kwaye ivavanywe ukuhlukana kwe-peptides kunye ne-trypsin digests ye-HSA.Isigaba sokumisa salungiswa ngokusebenzisa isicwangciso esilandelayo.Iincinci ze-silica ze-porous zalungiselelwa ngokwenkqubo enikwe kwipapasho lethu langaphambili kunye nolunye uhlengahlengiso, i-polyolPEG ye-protocol yokulungiswa kwe-polyperea, ukulungiswa kwe-polylyperea. I-TMOS, i-acetic acid yamanzi yahlengahlengiswa ukulungiselela i-silica particles kunye nobukhulu obukhulu be-pore.Okwesibini, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yaxutywa kwaye isetyenziselwa ukukhupha amaqhekeza e-silica ukulungiselela i-polar embedded stationary phase.Isiphumo sesigaba sokumisa sasipakishwe kwikholamu ye-stainless i-100 mm i-packing i-100 mm i-packing ye-stainless ×. iskimu.Ukupakishwa kwekholamu kuncediswa ngokungcangcazela komatshini ukuqinisekisa ukuba ibhedi elinganayo yenziwe ngaphakathi kwikholamu.Vavaluya ukwahlukana kwekholamu epakishweyo yemixube yepeptide ebandakanya iipeptide ezintlanu; (I-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leucine Enkephalin) kunye ne-Trypsin digest ye-albumin ye-serum yabantu (HAS). Ikholamu ye-RP-Amide.Zombini i-peptides kunye neeprotheni zabonwa ukuba zixazululwe kakuhle kwaye zisebenza kakuhle kwikholamu ye-PMP, eyayisebenza ngakumbi kunekholamu 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-Albumin ye-Human Serum (HSA), i-Ammonium Chloride, i-Urea, i-Hexane Methyldisilazane (HMDS), i-Methacryloyl Chloride, i-MCTEM, i-Perzoyl chloride, i-Benzoyldroksidi, i-Mc-TEM, i-Benzol. (BPO), i-HPLC yeBanga le-Acetonitrile (ACN), i-Methanol, i-2-Propanol, kunye ne-Acetone Ethengwe kwi-Sigma-Aldrich (eSt. Louis, MO, USA).
Umxube we-urea (8 g), i-polyethylene glycol (8 g), kunye ne-8 mL ye-0.01 N acetic acid yavuselelwa imizuzu eyi-10, kwaye i-24 mL ye-TMOS yongezwa phantsi kweemeko ezibandayo ze-ice. izinto eziseleyo zomiswa kwi-70 ° C kwiiyure ze-12. Ubunzima obomileyo obuthambileyo bungumhlaba ogudileyo kwi-oven kunye ne-calcined kwi-550 ° C kwiiyure ze-12. Iibhakethi ezintathu zilungiselelwe kwaye zibonakaliswe ukuba zihlolisise ukuveliswa kwakhona kwi-particle size, ubukhulu bepore kunye nommandla wendawo.
Ngokuguqulwa komphezulu we-silica particles kunye ne-ligand yangaphambili ye-phenylmaleimide-methylvinylisocyanate (PCMP) elandelwa yi-polymerization ye-radial kunye ne-styrene, i-polar equkethe iqela eliqulethwe lilungiswe. Isigaba esimileyo se-aggregates kunye ne-polystyrene chain.Inkqubo yokulungiselela ichazwe ngezantsi.
I-N-phenylmaleimide (200 mg) kunye ne-methyl vinyl isocyanate (i-100 mg) yachithwa kwi-toluene eyomileyo, kwaye i-0.1 mL ye-2,2'-azoisobutyronitrile (AIBN) yongezwa kwi-flask yokuphendula ukulungiselela i-phenylmaleimide-methyl vinyl isocyanate copolymer ngeeyure ze-3, i-heart C ° 6. icwecwe kwaye yomiswe kwi-oven kwi-40 ° C ngeeyure ezi-3.
Iincinci ze-silica ezomileyo (i-2 g) zachithwa kwi-toluene eyomileyo (i-100 mL), ixutywe kwaye i-sonicated kwi-flask ye-500 mL ejikelezayo ephantsi kwe-10 min.PMCP (10 mg) yachithwa kwi-toluene kwaye yongezwa i-dropwise kwi-flask yokuphendula nge-funnel yokulahla. I-60 ° C kwiiyure ze-3. Emva koko, iinqununu ze-silica ezidibeneyo ze-PMCP (i-100 g) zachithwa kwi-toluene (200 ml) kunye ne-4-hydroxy-TEMPO (2 mL) yongezwa phambi kwe-100 µL ye-dibutyltin dilaurate njenge-catalyst. iiyure.
I-Styrene (1 mL), i-benzoyl peroxide BPO (0.5 mL), kunye ne-TEMPO-PMCP-eqhotyoshelweyo i-silica particles (1.5 g) yachithwa kwi-toluene kwaye yahlanjululwa nge-nitrogen.I-polymerization ye-styrene yenziwa kwi-100 ° C kwiiyure ze-12. Imveliso efunyenweyo yahlanjwa nge-methanol kwi-reactivity yobusuku kwi-methanol kunye ne-reactivity yomisiwe ebusuku. 1.
Iisampuli zachithwa kwi-393 K ngeyure ye-1 ukufumana uxinzelelo olushiyekileyo olungaphantsi kwe-10-3 Torr. Isixa se-N2 esibhengezwe kuxinzelelo olusondeleyo lwe-P / P0 = 0.99 yayisetyenziselwa ukugqiba umthamo we-pore. IJapan) .Iisampulu ezomileyo (i-silica engenanto kunye ne-ligand-bonded silica particles) zifakwe kwikholamu ye-aluminium usebenzisa i-adhesive carbon tape.Igolide yafakwa kwiisampuli usebenzisa i-Q150T i-sputter coater, kunye ne-5 nm Au layer ifakwe kwiisampuli.Oku kuphucula ukusebenza kakuhle kwenkqubo usebenzisa i-voltages ephantsi kwaye inika i-grain putter, i-USA Eleham (i-USA) i-elham ebandayo (i-USA) I-Flash EA1112 i-elemental analyzer yayisetyenziselwa uhlalutyo olusisiseko.I-Malvern (i-Worcestershire, e-UK) i-Mastersizer 2000 ye-particle size analyzer yasetyenziselwa ukufumana ukusabalalisa ubungakanani be-particle.Ii-silica ezinqunu kunye ne-ligand-bonded silica particles (5 mg nganye) zachithwa kwi-5 mL ye-isopropanol, i-mintical extical 5, i-min 10, i-sonicated kunye ne-min 5 ye-sonication ibhentshi ye-Mastersizer.Uhlalutyo lwe-Thermogravimetric lwenziwa ngesantya se-5 °C ngomzuzu phezu komgangatho wokushisa we-30 ukuya kwi-800 °C.
I-glass-lined stainless steel stainless-bore columns of dimensions (100 × 1.8 mm id) yayipakishwe kusetyenziswa indlela yokupakisha i-slurry, isebenzisa inkqubo efanayo esetyenziswe kwi-Ref. I-31.Ikholamu yensimbi engenasici (i-glass-lined, i-100 × 1.8 mm id) kunye ne-outlet fitting ene-1 µm frit yayiqhagamshelwe kwi-slurry packer (Alltech Deerfield, IL, USA) Lungiselela i-slurry yesigaba esimileyo ngokumisa i-150 mg yesigaba esimileyo se-mL ye-stationary mL kwi-menotha yokugcina i-menotha kwi-1. isetyenziswe njenge-solvent slurry kunye ne-solvent ejikelezayo.Gcwalisa ikholamu ngokulandelelana ngokufaka uxinzelelo lwe-MP ye-100 imizuzu ye-10, i-80 MP yemizuzu ye-15, kunye ne-60 MP yemizuzu ye-30. Ngexesha lokupakisha, ukungcangcazela komatshini kusetyenziswe kunye neengqungquthela ezimbini zekholomu ze-GC (Alltech, Deerfield) ukuqinisekisa i-column IL, i-U. kwaye ukhulule uxinzelelo ngokukhawuleza ukukhusela nawuphi na umonakalo ngaphakathi kwikholomu.Nqamula ikholomu kwiyunithi yokupakisha i-slurry kwaye udibanise enye into efanelekileyo kwi-inlet kunye nenkqubo ye-LC ukujonga ukusebenza kwayo.
Impompo ye-LC (10AD Shimadzu, Japan), i-injector (Valco (USA) C14 W.05) ene-50nL injection loop, i-membrane degasser (Shimadzu DGU-14A), i-UV-VIS ifestile ye-capillary yakhiwe I-Special µLC isixhobo somtshini (UV-2075) kunye ne-glass-lined yokudibanisa i-microcolbins encinci ye-minize edibanisa i-microcobins emfutshane kakhulu. Emva kokupakishwa, ii-capillaries (50 μm id 365 kunye nokunciphisa i-union capillaries (50 μm) zifakwe kwi-1/16″ yokuphuma yomanyano lokunciphisa.Ukuqokelelwa kwedatha kunye nokulungiswa kwechromatographic kwenziwa kusetyenziswa isoftware ye-Multichro 2000. Ukubeka iliso kwi-UV yedatha ye-UV 25. ihlalutywe ngu-OriginPro8 (Northampton, MA).
I-albumin esuka kwiserum yabantu, i-lyophilized powder, ≥ 96% (i-agarose gel electrophoresis) 3 mg ixutywe ne-trypsin (1.5 mg), i-4.0 M urea (1 mL), kunye ne-0.2 M ye-ammonium bicarbonate (1 mL) .Isisombululo saxutywa ngemizuzu eyi-10 kwaye sigcinwe kwindawo yokuhlambela amanzi ngeeyure ezingama-67, ° C emva koko nge-67 inch, ° C. 0.1% TFA.Hlunga isisombululo kwaye ugcine ngaphantsi kwe-4 °C.
Ukwahlulwa kwemixube ye-peptide kunye ne-HSA ye-trypsin digests yavavanywa ngokwahlukileyo kwiikholamu ze-PMP.Jonga ukuhlukana komxube we-peptide kunye ne-trypsin digest ye-HSA ngekholomu ye-PMP kwaye uthelekise iziphumo kwikholamu ye-Ascentis Express RP-Amide.Inombolo yeplate yethiyori ibalwa ngolu hlobo lulandelayo:
Imifanekiso ye-SEM yeengqungquthela ze-silica ezingenanto kunye ne-ligand-bonded silica particles iboniswe kwi-FIG. I-2 .Imifanekiso ye-SEM yeengqungquthela ze-silica ezingenanto (A, B) zibonisa ukuba, ngokungafaniyo nezifundo zethu zangaphambili, ezi ngqungquthela ziyi-spherical apho iinqununu zilinganiswe okanye zine-symmetry engaqhelekanga. amasuntswana e-silica.
Ukuskena imifanekiso ye-electron microscope ye-silica engenanto (A, B) kunye ne-ligand-bonded silica particles (C, D).
Ukusabalalisa ubungakanani bemilinganiselo ye-silica engenanto kunye ne-ligand-bonded silica particles kuboniswe kwi-Figure 3 (A) i-Volume-based based curves yokusabalalisa i-curves ibonise ukuba ubukhulu be-silica particles nyuka emva kokuguqulwa kweekhemikhali (umzobo 3A) . d (0.5), ye-PMP yi-3.36 μm, xa kuthelekiswa nesifundo sethu sangaphambili kunye ne-ad (0.5) yexabiso le-3.05 μm (i-polystyrene-bound silica particles) 34.Le bhetshi yayinokusabalalisa ubungakanani be-particle encinci xa kuthelekiswa nesifundo sethu sangaphambili ngenxa yemilinganiselo eyahlukayo ye-PEG, urea, i-TMOS kwi-particle enkulu ye-acidity ye-reaction yesigaba esincinci, kunye nobukhulu bomxube we-asidi ye-PMOS. leyo ye-polystyrene-bound silica particle phase esikhe sayifunda ngaphambili.Oku kuthetha ukuba ukusebenza komphezulu we-silica particles kunye ne-styrene ifake kuphela umaleko we-polystyrene (0.97 µm) kumphezulu we-silica, kanti kwisigaba se-PMP ubukhulu be-maleko yi-1.38 µm.
Ukusabalalisa ubungakanani be-particle (A) kunye nokusabalalisa ubungakanani bepore (B) yeengqungquthela ze-silica ezingenanto kunye ne-ligand-bound bound silica particles.
Ubungakanani be-pore, umthamo we-pore kunye nommandla wendawo yeengqungquthela ze-silica zophando lwangoku zinikezelwa kwiThebhile 1 (B) .Iiprofayili ze-PSD ze-silica ezingenanto kunye ne-ligand-bonded silica particles ziboniswe kuMzobo 3 (B) .Iziphumo zifaniswa nesifundo sethu sangaphambili. ibonisa ukuba ubukhulu be-pore buyancipha nge-69 emva kokuguqulwa kweekhemikhali, njengoko kuboniswe kwiThebhile 1 (B), kwaye ukuguqulwa kwejika kuboniswe kwi-Fig. 3 (B) . Ngokufanayo, umthamo we-pore we-silica particles wehla ukusuka kwi-0.67 ukuya kwi-0.58 cm3 / g emva kokuguqulwa kweekhemikhali. isifundo sethu sangaphambili (i-124 m2 / g) .Njengoko kuboniswe kwiThebhile 1 (B), indawo engaphezulu (m2 / g) ye-silica particles nayo yehla ukusuka kwi-116 m2 / g ukuya kwi-105 m2 / g emva kokuguqulwa kweekhemikhali.
Iziphumo zohlalutyo lwe-elemental yesigaba esimileyo ziboniswa kwiThebhile 2. Ukulayishwa kwekhabhoni yesigaba esimileyo sangoku yi-6.35%, ephantsi kwekhabhoni yokulayishwa kwesifundo sethu sangaphambili (i-polystyrene edibeneyo ye-silica particles, i-7.93% 35 kunye ne-10.21%, ngokulandelanayo) 42. Ukulayishwa kwekhabhoni ye-carbonation yesigxina, i-SP yangoku, ukulungiswa kwe-SP, i-SP ezinye i-polar ligands ezifana ne-phenylmaleimide-methylvinylisocyanate (PCMP) kunye ne-4-hydroxy-TEMPO zisetyenzisiwe.Ipesenti ye-nitrogen yesisindo senqanaba elikhoyo lokumisa i-2.21%, xa kuthelekiswa ne-0.1735 kunye ne-0.85% ngesisindo se-nitrogen kwizifundo zangaphambili, ngokulandelanayo.Oku kuthetha ukuba i-nitrogen wt inqanaba eliphezulu kwisigxina sangoku se-nitrogen i-phenylmaleimide.Ngokufanayo, ukulayishwa kwekhabhoni yeemveliso (4) kunye (5) yi-2.7% kunye ne-2.9%, ngokulandelanayo, ngelixa ukulayishwa kwekhabhoni yemveliso yokugqibela (6) yayingu-6.35%, njengoko kuboniswe kwiThebhile 2. Ukulahlekelwa kwesisindo kuhlolwe kunye nesigaba sokumisa se-PMP, kwaye i-curve ye-TGA iboniswe kwi-4 ye-Figure ye-TGA. iyavumelana kakuhle nokulayishwa kwekhabhoni (6.35%) kuba iiligand aziqulathanga i-C kuphela kodwa ne-N, O, kunye ne-H.
I-phenylmaleimide-methylvinylisocyanate ligand yakhethwa ukuguqulwa kwendawo yeengqungquthela ze-silica ngenxa yokuba inamaqela e-polar phenylmaleimide kunye namaqela e-vinylisocyanate. I-phenylmaleimide moiety ayinayo intlawulo ebonakalayo kwi-pH eqhelekileyo.I-polarity yesigaba esimileyo sinokulawulwa ngumlinganiselo ochanekileyo we-styrene kunye nexesha lokuphendula le-polymerization ye-radical yamahhala.Isinyathelo sokugqibela sokuphendula (i-polymerization yamahhala) ibaluleke kakhulu kwaye inokutshintsha i-polarity yesigaba esimileyo.Uhlalutyo lwe-Elemental lwenziwa ukujonga ukuba isixa se-carbon enyukayo kwaye sandiswa ixesha lokulayishwa kwekhabhoni. Ukulayishwa kwekhabhoni yesigaba sokumisa kunye ne-vice versa.SPs ezilungiselelwe ngeengxininiso ezahlukeneyo ze-styrene zine-carbon loadings ezahlukeneyo.Kwakhona, layisha ezi zigaba ezimileyo kwiintsika zetsimbi ezingenasici kwaye ukhangele ukusebenza kwazo kwe-chromatographic (ukukhethwa, isisombululo, ixabiso le-N, njl.) .Ngokusekelwe kwezi zilingo, ukuqulunqwa okuphuculweyo kukhethwe ukulungiselela isigaba se-PMP esilungisiweyo sokulawulwa kwesigxina kunye nokuqinisekisa ukuba i-PMP ilawulwa kakuhle.
Imixube emihlanu ye-peptide (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nayo yavavanywa ngokusebenzisa ikholomu ye-PMP usebenzisa isigaba seselula; 60/40 (v/v) i-acetonitrile/amanzi (0.1% TFA) ngesantya sokuqukuqela esingama-80 μL/min.Phantsi kweemeko ezizezona zilungileyo ze-lution, inombolo yepleyiti yethiyori (N) ngekholamu nganye (100 × 1.8 mm id) ngama-20,000 ± 100 (iipleyiti ezi-200,00 ze-N² zinika i-200,000 inika i-3000 yeepleyiti ezine-3 inika i-0). Iikholamu ze-PMP kunye neechromatograms ziboniswe kuMzobo 5A. Uhlalutyo olukhawulezayo kwikholamu ye-PMP ngesantya esiphezulu sokuhamba (700 μL/min), iipeptides ezintlanu zaye zakhutshwa ngomzuzu omnye, amaxabiso e-N ayelungile kakhulu, 13,500 ± 330 ngekholamu nganye (100 × 1.8 mm id), i-13 ihambelana ne-0, i-0 ihambelana ne-05m, i-13 ihambelana ne-0 (i-05m I-5B) .Iikholomu ezintathu ezilinganayo ezifanayo (i-100 × 1.8 mm id) zixutywe ngamaqashiso amathathu ahlukeneyo esigaba esimileyo se-PMP ukujonga ukuveliswa kwakhona.I-concentration ye-analyte yekholomu nganye irekhodwe ngokusebenzisa iimeko ezifanelekileyo ze-lution kunye nenani leeplati zethiyori N kunye nexesha lokugcinwa ukuhlukanisa umxube ofanayo wokuvavanya kwikholamu ye-reproduci eboniswe kwi-data ye-MP kwikholam nganye. ukuveliswa kwakhona kwekholamu ye-PMP ihambelana kakuhle namaxabiso aphantsi kakhulu e-%RSD, njengoko kubonisiwe kwiThebhile 3.
Ukwahlula umxube we-peptide kwikholamu ye-PMP (B) kunye ne-Ascentis Express RP-Amide ikholamu (A); isigaba seselula 60/40 ACN / H2O (TFA 0.1%), imilinganiselo yekholomu ye-PMP (100 × 1.8 mm id); uhlalutyo Umyalelo we-lution we-compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kunye ne-5 (leucine) acid enkephalin)).
Ikholamu ye-PMP (i-100 × 1.8 mm id) yavavanyelwa ukwahlulwa kwe-tryptic digests ye-albumin ye-serum yabantu kwi-chromatography yolwelo oluphezulu. Ichromatogram kuMfanekiso 6 ibonisa ukuba isampulu yahlulwe kakuhle kwaye isisombululo sihle kakhulu. I-HSA digests yahlalutywa kusetyenziswa isantya sokuhamba se-100/3 µ I-acetonitrile / amanzi kunye ne-0.1% ye-TFA. Njengoko kuboniswe kwi-chromatogram (Umfanekiso 6), ukugaya kwe-HSA kuye kwahlulahlulwe kwiincopho ze-17 ezihambelana ne-peptides ye-17. Ukusebenza kokuhlukana kwencopho nganye kwi-digest ye-HSA kubalwe kwaye amaxabiso anikezelwa kwiThebhile 5.
I-tryptic digest ye-HSA (i-100 × 1.8 mm id) yahlulwe kwikholamu ye-PMP; izinga lokuhamba (100 µL/min), isigaba esihambayo 60/40 acetonitrile/amanzi ane-0.1% TFA.
apho uL ubude bekholomu, η yi-viscosity yesigaba esihambahambayo, i-ΔP yikholomu yoxinzelelo lwangasemva, kwaye u-uyisantya somgca wesigaba esihambayo.I-permeability yekholomu ye-PMP yayiyi-2.5 × 10-14 m2, izinga lokuhamba laliyi-25 μL / min, kunye ne-60/40 v / v. I-1.8 mm id) yayifana neyokufunda kwethu kwangaphambili i-Ref.34.I-permeability yekholomu epakishwe ngamasuntswana angama-porous angaphezulu: 1.7 × 10-15 kwi-1.3 μm particles, 3.1 × 10-15 kwi-1.7 μm particles, 5.0-1 × 2 × 2 × 1 × 2 × 1 × 2 1 × 2 5 × 1 × 2 × 2 × 1 × 1 × 2 × 2 × 2 × 1 × 2 × 1 × 2 × 1 × 2 × 1 × 2 × 1 × 1 × 2 5 × 1 × 2 × 2 × 1 × 2 5 × 1 5 × 1 × 2 × 2 × 2 × 2 × 1. I-2.6 μm iincinci ze-5 μm 43. Ngoko ke, ukugqithiswa kwesigaba se-PMP kufana ne-5 μm ye-core-shell particles.
apho i-Wx bubunzima bekholamu epakishwe ngeklorofomu, iWy bubunzima bekholamu epakishwe ngemethanol, kwaye ρ bubuninzi bento enyibilikayo.Uxinaniso lwemethanol (ρ = 0.7866) kunye nechloroform (ρ = 1.484).Itotali yeporosity yeSILICA PARTICLES 108 × 4 × 10 × 10 × 1.8 mm kunye ne-C18-Urea iikholomu ze-31 esizifunde ngaphambili ziyi-0.63 kunye ne-0.55, ngokulandelanayo.Oku kuthetha ukuba ubukho be-urea ligands kunciphisa ukunyanzeliswa kwesigaba sokumisa.Ngakolunye uhlangothi, i-porosity iyonke yekholomu ye-PMP (i-100 × 1.8 mm id) yi-0.60 ephantsi kwekholamu ye-P. I-C18-bonded silica particles kuba kwi-C18-type-standard phases i-C18 ligands ifakwe kwii-silica particles njengamatyathanga anqamlekileyo, ngelixa kwizigaba ze-polystyrene-type stationary, i-Alary ye-polymer engqingqwa iqulunqwe ngeenxa zonke.
Umzobo 7A, B ubonisa ikholomu ye-PMP (i-100 × 1.8 mm id) kunye nekholamu ye-Ascentis Express RP-Amide (i-100 × 1.8 mm id) isebenzisa iimeko ezifanayo ze-lution (okt, 60/40 ACN / H2O kunye ne-0.1% TFA). ) yeploti yevan Deemter. Imixube yepeptide ekhethiweyo (iGly-Tyr, iGly-Leu-Tyr, iGly-Gly-Tyr-Arg, iTyr-Ile-Gly-Ser-Arg, iLeucine Enkephalin) yalungiswa nge-20 µL/ Ubuncinane besantya sokuhamba kuzo zombini iikholamu yi-800 µL/min. Ubuncinane bexabiso le-HETP µmin. ikholamu kunye nekholomu ye-Ascentis Express RP-Amide yayiyi-2.6 µm kunye ne-3.9 µm, ngokulandelanayo. Amaxabiso e-HETP abonisa ukuba ukwahlukana kakuhle kwekholamu ye-PMP (100 × 1.8 mm id) ingcono kakhulu kunekholamu ye-Ascentis Express RP-Amide ekhoyo (100 × id . 7 mm) ibonisa i-PMP (i-100 × id . ukuba ukuncipha kwexabiso le-N ngokunyuka kokuhamba okunyukayo akubalulekanga xa kuthelekiswa nesifundo sethu sangaphambili.Ukuphumelela okuphezulu kokuhlukana kwekholamu ye-PMP (i-100 × 1.8 mm id) xa kuthelekiswa nekholamu ye-Ascentis Express RP-Amide isekelwe ekuphuculeni i-particle shape, ubukhulu, kunye neenkqubo eziyinkimbinkimbi zokupakisha ikholamu ezisetyenziswa kumsebenzi wangoku34.
(A) i-van Deemter plot (i-HETP ngokubhekiselele kwi-mobile phase ye-linear velocity) ifunyenwe ngokusebenzisa ikholomu ye-PMP (100 × 1.8 mm id) kwi-60/40 ACN / H2O kunye ne-0.1% TFA. (B) i-van Deemter plot (HETP versus mobile phase linear velocity) ifunyenwe ngokusebenzisa i-Ascents × 1 ikholomu ye-Ascentis × i-1 RP-Ascentis Express. 60/40 ACN/H2O kunye 0.1% TFA.
I-polar-embedded i-polystyrene isigaba sokumisa salungiswa kwaye savavanywa ukwahlula imixube ye-peptide eyenziweyo kunye ne-trypsin digests ye-albumin ye-serum yabantu (HAS) kwi-chromatography yolwelo oluphezulu. Ubungakanani beengqungquthela ze-silica, i-synthesis elawulwayo yesigaba sokumisa, kunye nokupakishwa kwekholomu eyinkimbinkimbi.Ukongezelela kwintsebenzo ephezulu yokuhlukana, uxinzelelo oluphantsi lwekholomu ephantsi kwizinga eliphezulu lokuhamba kunye nenye inzuzo yesi sigaba esimileyo.Iikholomu ze-PMP zibonisa ukuveliswa kakuhle kwaye zingasetyenziselwa uhlalutyo lwemixube ye-peptide kunye ne-trypsin digestion yeeprotheni ezahlukeneyo. Iingcaphuno zefungal kwi-chromatography yolwelo.Kwixesha elizayo, iikholamu ze-PMP ziya kuphinda zivavanywe ukuhlukana kweeprotheni kunye ne-monoclonal antibodies.
I-Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. UPhando kwiiNkqubo zoKwahlula iPeptide ngeSigaba esiHlehlisiwe seChromatography Icandelo I: Uphuhliso lweProtokholi yeKholamu yoMfanekiso.J. I-Chromatography.1603, 113–129.https://doi.org/10.1016/j.chroma.2019.05.038 (2019).
Gomez, B. et al.Ukuphuculwa kweepeptides ezisebenzayo ezenzelwe unyango lwezifo ezosulelayo.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 therapeutic: isayensi kunye ne-market.drug discovery.15 (1-2) namhlanje, 40-56.https://doi.org/10.1016/j.drudis.2009.10.109.10.10).
Xie, F., Smith, RD & Shen, Y. Advanced Proteomic Liquid Chromatography.J. I-Chromatography.A 1261, 78-90 (2012).
Liu, W. et al.Ulwelo oluphucukileyo lwechromatography-mass spectrometry yenza ukuba kudityaniswe i-metabolomics ejoliswe ngokubanzi kunye neproteomics.anus.Chim.Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Indima ye-UHPLC kuphuhliso lwamachiza.J. Sep. Sci.30 (8), 1183-1190 (2007).
I-Wu, N. & Clausen, AM Imiba esisiseko kunye nesebenzayo ye-ultrahigh pressure liquid chromatography yokwahlula ngokukhawuleza.J. Sep. Sci.30(8), 1167-1182.https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitchef, P. Ukusetyenziswa kolwelo oluphezulu lwechromatography ekuphuhlisweni kweziyobisi.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 elungiselelwe ukusuka kwi-oli-in-water high emulsions yesigaba sangaphakathi sokucoca ngokufanelekileyo kwe-enteroviruses.Chemical.Britania.J. 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Indima yolwelo lwechromatography kwiproteomics.J. I-Chromatography.A 1053 (1-2), 27-36 (2004).
Fekete, S., Veuthey, J.-L.&Guillarme, D. Iindlela ezisakhulayo kwi-reverse-phase liquid chromatography ukwahlulwa kweepeptides zonyango kunye neeprotheni: ithiyori kunye nezicelo.J. Pharmacy.Biomedical Science.anus.69, 9-27 (2012).
I-Gilar, M., Olivova, P., Daly, AE & Gebler, JC Ukuhlukana kwe-two-dimensional ye-peptides usebenzisa inkqubo ye-RP-RP-HPLC esebenzisa amaxabiso ahlukeneyo e-pH kwimilinganiselo yokuqala kunye neyesibini yokuhlukana. Sep. Sci.28 (14), 1694-1703 (2005).
I-Feletti, i-S. et al. Iimpawu zokudlulisa ubunzima kunye nokusebenza kwe-kinetic ye-high-effective chromatographic columns epakishwe nge-C18 sub-2 μm ngokupheleleyo kunye namasuntswana angama-porous aphandwayo.J. Sep. Sci.43 (9-10), 1737-1745 (2020).
I-Piovesana, S. et al. Iintsingiselo zamva nje kunye nemingeni yohlalutyo kwindawo yodwa, ukuchongwa kunye nokuqinisekiswa kwe-plant bioactive peptides.anus.biological anus.Chemical.410(15), 3425–3444.https://doi.org/10.1007/s00216-02-018-x08).
Mueller, JB kunye nabanye
DeLuca, C. et al.Downstream processing of peptides zonyango ngokulungiselela ulwelo chromatography.Molecule (Basel, Switzerland) 26(15), 4688(2021).
Yang, Y. & Geng, X. Imixed-mode chromatography kunye nokusetyenziswa kwayo kwi-biopolymers.J. I-Chromatography.A 1218(49), 8813-8825 (2011).
Zhao, G., Dong, X.-Y.&Sun, Y. Ligands ye-protein-mode exubileyo yechromatography: umgaqo, ukulinganisa, kunye noyilo.J. I-Biotechnology.144 (1), 3-11 (2009).