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Iiparticles ze-silica ezinemibhobho zalungiswa ngendlela ye-sol-gel kunye notshintsho oluthile ukuze kufunyanwe iiparticles ezinemibhobho emikhulu. Ezi particles zafunyanwa nge-reversible addition fragmentation chain transfer (RAFT) polymerization nge-N-phenylmaleimide-methylvinylisocyanate (PMI) kunye ne-styrene ukulungiselela i-N-phenylmaleimide intercalation ye-polystyrene (PMP) stationary phase. Iicolumns zentsimbi engagqwaliyo ezibotshelelwe nge-Narrow-bore (100 × 1.8 mm id) zapakishwa nge-slurry packing. Ukwahlulwa kwekholamu ye-PMP okuvavanyiweyo komxube we-peptide oquka iipeptides ezintlanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) kunye ne-trypsin digestion ye-human serum albumin (HAS). Phantsi kweemeko ezifanelekileyo zokulungisa, inani leeplate zethiyori zomxube we-peptide liphezulu njengeeplates ezingama-280,000/m². Ukuthelekisa ukusebenza kokwahlulwa kwe Kwikholamu ephuhlisiweyo enekholamu yentengiso ye-Ascentis Express RP-Amide, kwabonwa ukuba ukusebenza kokwahlulwa kwekholamu ye-PMP kwakungcono kunekholamu yentengiso ngokubhekiselele ekusebenzeni kakuhle kokwahlulwa kunye nesisombululo.
Kwiminyaka yakutshanje, ishishini le-biopharmaceutical liye laba yimarike yehlabathi ekhulayo kunye nokwanda okukhulu kwesabelo semarike. Ngokukhula okukhulu kweshishini le-biopharmaceutical1,2,3, uhlalutyo lweepeptides kunye neeproteni lufunwa kakhulu. Ukongeza kwi-peptide ekujoliswe kuyo, ukungcola okuninzi kuveliswa ngexesha lokwenziwa kwe-peptide, ngaloo ndlela kufuna ukucocwa kwe-chromatographic ukuze kufunyanwe iipeptides zobumsulwa obufunekayo. Uhlalutyo kunye nokuchazwa kweeproteni 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 neeproteni, ukuba ezo sampuli zifakwa kwi-mass spectrometer ngexesha elinye, ukwahlukana akuyi kuba kuhle. Le ngxaki inokuncitshiswa ngokusebenzisa ukwahlukana kwe-liquid chromatography (LC) ngaphambi kohlalutyo lwe-MS, oluya kunciphisa inani lee-analytes ezingena kwi-mass spectrometer ngexesha elithile4,5,6. Ukongeza, ngexesha lokwahlukana kwesigaba solwelo, ii-analytes zinokujoliswa kwiindawo ezimxinwa, ngaloo ndlela zigxila ezi analytes kwaye ziphucule ukufunyanwa kwe-MS. Uvakalelo. I-chromatography yolwelo (LC) ihambele phambili kakhulu kwiminyaka elishumi edlulileyo kwaye iye yaba yindlela ethandwayo kuhlalutyo lwe-proteomic7,8,9,10.
I-Reversed-phase liquid chromatography (RP-LC) isetyenziswa kakhulu ekucoceni nasekuhlukaniseni imixube ye-peptide kusetyenziswa i-octadecyl-modified silica (ODS) njenge-stationary phase11,12,13. Nangona kunjalo, ii-RP stationary phases aziboneleli ngokwanelisayo ngokwahlukana kwee-peptides kunye neeproteni ngenxa yesakhiwo sazo esintsonkothileyo kunye nendalo yazo ye-amphiphilic 14,15. Ke ngoko, ii-stationary phases ezenzelwe ngokukodwa ziyafuneka ukuhlalutya ii-peptides kunye neeproteni ezine-polar kunye ne-non-polar moieties ukuze zisebenzisane kwaye zigcine ezi analytes16. I-Mixed-mode chromatography, ebonelela ngokusebenzisana kwe-multimodal, inokuba yindlela eyahlukileyo ye-RP-LC yokwahlula ii-peptides, iiproteni, kunye neminye imixube entsonkothileyo. Ii-mixed-mode stationary phases ezininzi zilungisiwe, kwaye iikholamu ezipakishwe ngezi zigaba zisetyenziselwe ukwahlulwa kwe-peptide kunye neeproteni17,18,19,20,21. Ii-mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zifanelekile ukwahlulwa kwe-peptide kunye neeproteni ngenxa ukubakho kwamaqela e-polar kunye namaqela angengawo e-polar22,23,24,25,26,27,28. Ngokufanayo, ii-polar intercalating stationary phases ezinamaqela e-polar adibeneyo kunye ne-covalently zibonisa amandla okwahlulahlula kunye nokukhetha okukhethekileyo kwii-polar kunye nee-non-polar analytes, njengoko ukwahlukana kuxhomekeke kunxibelelwano phakathi kwe-analyte kunye ne-stationary phase. Unxibelelwano lwe-Multimodal 29, 30, 31, 32. Kutshanje, uZhang et al. 30 balungiselele isigaba se-polyamine esingaguqukiyo esipheliswe yi-dodecyl kwaye bahlula ngempumelelo ii-hydrocarbons, ii-antidepressants, ii-flavonoids, ii-nucleosides, ii-estrogens, kunye nezinye ii-analytes ezininzi. I-polar intercalator inamaqela e-polar kunye nee-non-polar, ngoko ke ingasetyenziselwa ukwahlula ii-peptides kunye neeproteni ezine-hydrophobic kunye ne-hydrophilic moieties. Iikholamu ezifakwe kwi-polar (umz., iikholamu ze-C18 ezifakwe kwi-amide) ziyafumaneka kurhwebo phantsi kwegama lorhwebo elithi Ascentis Express RP-Amide columns, kodwa ezi kholamu zisetyenziselwa uhlalutyo lwe-amine 33 kuphela.
Kolu phononongo lwangoku, kulungiselelwe kwaye kwavavanywa isigaba esimileyo esifakwe kwi-polar (i-N-phenylmaleimide-embedded polystyrene) ukuze kwahlulwe iipeptides kunye ne-trypsin digests ze-HSA. Isigaba esimileyo silungiselelwe kusetyenziswa eli cebo lilandelayo. Iiparticles ze-silica ezineembobo zalungiswa ngokwenkqubo enikwe kwimpapasho yethu yangaphambili kunye notshintsho oluthile kwiprotokholi yokulungiselela. Umlinganiselo we-urea, i-polyethylene glycol (PEG), i-TMOS, i-acetic acid yamanzi yalungiswa ukuze kulungiswe iiparticles ze-silica ezinobukhulu obukhulu beepore. Okwesibini, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yenziwe kwaye yasetyenziswa ekufumaneni iiparticles ze-silica ukulungiselela isigaba esimileyo esifakwe kwi-polar. Isigaba esimileyo esivelileyo sapakishwa kwikholamu yentsimbi engagqwali (100 × 1.8 mm id) kusetyenziswa iskimu sokupakisha esilungisiweyo. Ukupakisha iikholamu kuncediswa ngokungcangcazela koomatshini ukuqinisekisa ukuba ibhedi efanayo yenziwa ngaphakathi kwikholamu. Vavanya ukwahlukana kweekholamu ezipakishiweyo zemixube ye-peptide equkethe iipeptides 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 yomntu (HAS). Umxube we-peptide kunye ne-trypsin digest ye-HSA zibonwe zahlukene ngesisombululo esihle kunye nokusebenza kakuhle. Ukusebenza kokwahlulwa kwekholamu ye-PMP kuthelekiswa noko kwekholamu ye-Ascentis Express RP-Amide. Zombini ii-peptides kunye neeproteni zibonwe zisonjululwe kakuhle kwaye zisebenza kakuhle kwikholamu ye-PMP, eyayisebenza ngcono kunekholamu ye-Ascentis Express RP-Amide.
I-PEG (iPolyethylene Glycol), iUrea, iAcetic Acid, iTrimethoxy Orthosilicate (TMOS), iTrimethyl Chlorosilane (TMCS), iTrypsin, iHuman Serum Albumin (HSA), iAmmonium Chloride, iUrea, iHexane Methyldisilazane (HMDS), iMethacryloyl Chloride (MC), iStyrene, i-4-Hydroxy-TEMPO, iBenzoyl Peroxide (BPO), iHPLC Grade Acetonitrile (ACN), iMethanol, i-2-Propanol, kunye neAcetone Zithengwe kwiSigma-Aldrich (St. 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 eli-10, kwaza kongezwa i-24 mL ye-TMOS kuyo phantsi kweemeko ezibandayo. Umxube we-reaction wafudunyezwa kwi-40°C kangangeeyure ezi-6 waza waphinda watshiswa kwi-120°C kangangeeyure ezi-8 kwi-autoclave yentsimbi engatyiwayo. Amanzi athululelwa kwaye izinto eziseleyo zomiswa kwi-70°C kangangeeyure ezili-12. Ubunzima obuthambileyo obomileyo bagutyungelwa kakuhle kwi-oven kwaye bafakwa kwi-calcium kwi-550°C kangangeeyure ezili-12. Kwalungiswa iibhetshi ezintathu kwaye zachazwa ukuze kuhlolwe ukuphinda kuvele ubungakanani beentwana, ubungakanani beembobo kunye nommandla womphezulu.
Ngokutshintsha umphezulu wee-particles ze-silica nge-ligand phenylmaleimide-methylvinylisocyanate (PCMP) esele yenziwe ngaphambili, kulandele i-radial polymerization nge-styrene, kwalungiselelwa i-compound equlathe iqela le-polar. Isigaba esizinzileyo see-aggregates kunye nee-polystyrene chains. Inkqubo yokulungiselela ichazwe ngezantsi.
I-N-phenylmaleimide (200 mg) kunye ne-methyl vinyl isocyanate (100 mg) zanyibilikiswa kwi-toluene eyomileyo, kwaye i-0.1 mL ye-2,2′-azoisobutyronitrile (AIBN) yongezwa kwi-reaction flask ukulungiselela i-phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Lo mxube ufudunyezwe kwi-60°C iiyure ezi-3, wahluzwa waza womiswa kwi-oven kwi-40°C iiyure ezi-3.
Iisuntswana ze-silica ezomileyo (2 g) zasasazwa kwi-toluene eyomileyo (100 mL), zaxutywa zaza zafakwa kwi-flask esezantsi eyi-500 mL kangangemizuzu eli-10. I-PMCP (10 mg) yanyibilikiswa kwi-toluene yaza yongezwa kwi-flask ye-reaction nge-funnel ewisayo. Umxube wahluzwa kwi-100°C kangangeeyure ezi-8, wahluzwa waza wahlanjwa nge-acetone waza womiswa kwi-60°C kangangeeyure ezi-3. Emva koko, iisuntswana ze-silica ezibotshelelweyo ze-PMCP (100 g) zanyibilikiswa kwi-toluene (200 ml) kwaye i-4-hydroxy-TEMPO (2 mL) yongezwa phambi kwe-100 µL ye-dibutyltin dilaurate njenge-catalyst. Umxube washukunyiswa kwi-50°C kangangeeyure ezi-8, wahluzwa waza womiswa kwi-50°C kangangeeyure ezi-3.
I-Styrene (1 mL), i-benzoyl peroxide BPO (0.5 mL), kunye nee-TEMPO-PMCP-attached silica particles (1.5 g) zasasazwa kwi-toluene zaza zacocwa nge-nitrogen. I-polymerization ye-styrene yenziwe kwi-100°C kangangeeyure ezili-12. Imveliso ephumayo yahlanjwa nge-methanol yaza yomiswa kwi-60°C ubusuku bonke. Inkqubo yokusabela iyonke iboniswe kuMfanekiso 1.
Iisampulu zasuswa igesi kwi-393 K kangangeyure e-1 ukuze kufunyanwe uxinzelelo olushiyekileyo olungaphantsi kwe-10-3 Torr. Isixa se-N2 esifakwe kuxinzelelo oluhambelanayo lwe-P/P0 = 0.99 sisetyenzisiwe ukumisela umthamo we-pore iyonke. Imo ye-bare and ligand-bonded silica particles ihlolwe nge-scanning electron microscopy (Hitachi High Technologies, Tokyo, Japan). Iisampulu ezomileyo (bare silica and ligand-bonded silica particles) zibekwe kwikholamu ye-aluminiyam kusetyenziswa i-adhesive carbon tape. Igolide yabekwa kwiisampulu kusetyenziswa i-Q150T sputter coater, kwaye umaleko we-5 nm Au wafakwa kwiisampulu. Oku kuphucula ukusebenza kakuhle kwenkqubo kusetyenziswa ii-voltages eziphantsi kwaye kubonelela nge-fine grain, cold sputtering. I-Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer yasetyenziswa kuhlalutyo lwe-elemental. I-Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer yasetyenziswa ukufumana usasazo lobungakanani be-particle. I-Naked silica Iinxalenye kunye neenxalenye ze-ligand-bonded silica (5 mg nganye) zasasazwa kwi-5 mL ye-isopropanol, zafakwa kwi-sonicated imizuzu eli-10, zaza zafakwa kwi-vortex imizuzu emi-5, zaza zabekwa kwi-optical bench yeMastersizer. Uhlalutyo lwe-thermogravimetric lwenziwe ngesantya se-5 °C ngomzuzu kuluhlu lobushushu oluphakathi kwama-30 ukuya kuma-800 °C.
Iikholamu zentsimbi engagqwaliyo eneglasi enobukhulu obuncinci (100 × 1.8 mm id) zapakishwa kusetyenziswa indlela yokupakisha ngodaka, kusetyenziswa inkqubo efanayo esetyenzisiweyo kwiRef. 31. Ikholamu yentsimbi engagqwaliyo (efakwe iglasi, eyi-100 × 1.8 mm id) ene-outlet fitting equlethe i-1 µm frit iqhagamshelwe kwi-slurry packer (Alltech Deerfield, IL, USA). Lungisa i-slurry yesigaba esingaguqukiyo ngokuxhoma i-150 mg yesigaba esingaguqukiyo kwi-1.2 mL ye-methanol uze uyithumele kwikholamu yokugcina. I-Methanol isetyenziswe njenge-slurry solvent kunye ne-propelling solvent. Zalisa ikholamu ngokulandelelana ngokusebenzisa uxinzelelo lwe-100 MP imizuzu eli-10, i-80 MP imizuzu eli-15, kunye ne-60 MP imizuzu engama-30. Ngexesha lokupakisha, ukungcangcazela koomatshini kusetyenziswe ii-GC column shakers ezimbini (Alltech, Deerfield, IL, USA) ukuqinisekisa ukupakishwa okufanayo kwekholamu. Vala i-slurry packer kwaye ukhulule uxinzelelo kancinci ukuthintela nawuphi na umonakalo ngaphakathi kwikholamu. Nqamla ikholamu kwiyunithi yokupakisha i-slurry kwaye uqhagamshele enye i-fitting kwindawo yokungena nakwinkqubo ye-LC ukuze ujonge ukuba iyasebenza. ukusebenza.
Ipompo ye-LC (10AD Shimadzu, eJapan), i-injector (Valco (USA) C14 W.05) ene-50nL injection loop, i-membrane degasser (Shimadzu DGU-14A), i-UV-VIS capillary window yenziwe ngeSpecial µLC device detector (UV-2075) kunye nee-microcolumns ezineglasi. Sebenzisa ityhubhu yokudibanisa emxinwa kakhulu nemfutshane ukunciphisa isiphumo sokwandiswa kwebhendi eyongezelelweyo. Emva kokupakisha, ii-capillaries (50 μm id 365 kunye ne-reducing union capillaries (50 μm) zifakwe kwindawo yokuphuma ye-1/16″ ye-reducing union. Ukuqokelelwa kwedatha kunye nokucubungula i-chromatographic kwenziwe kusetyenziswa isoftware ye-Multicchro 2000. Ukubeka esweni kwi-254 nm Analytes kuvavanywe ukufunxwa kwe-UV. Idatha ye-Chromatographic ihlalutywe yi-OriginPro8 (Northampton, MA).
I-Albumin evela kwi-serum yomntu, umgubo we-lyophilized, ≥ 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 ammonium bicarbonate (1 mL). Isisombululo sivuselelwe imizuzu eli-10 saza sagcinwa emanzini kwi-37°C kangangeeyure ezi-6, emva koko sacinywa nge-1 mL ye-0.1% TFA. Hluza isisombululo kwaye ugcine ngaphantsi kwe-4 °C.
Ukwahlulwa kwengxubevange ye-peptide kunye ne-HSA trypsin digests kuhlolwe ngokwahlukeneyo kwiikholamu ze-PMP. Jonga ukwahlulwa komxube we-peptide kunye ne-trypsin digest ye-HSA ngekholamu ye-PMP kwaye uthelekise iziphumo kwikholamu ye-Ascentis Express RP-Amide. Inombolo yeplate yethiyori ibalwa ngolu hlobo lulandelayo:
Imifanekiso ye-SEM yee-particles ze-silica ezingenanto kunye nee-particles ze-ligand-bonded silica iboniswe kwiFIG. 2. Imifanekiso ye-SEM yee-particles ze-silica ezingenanto (A, B) ibonisa ukuba, ngokungafaniyo nezifundo zethu zangaphambili, ezi particles ziyi-spherical apho ii-particles zinde okanye zino-symmetry engacwangciswanga. Umphezulu wee-particles ze-silica ezinamathele kwi-ligand (C, D) uthambile kunowee-particles ze-silica ezingenanto, ezinokubangelwa kukugqunywa kwee-polystyrene chains kumphezulu wee-particles ze-silica.
Ukuskena imifanekiso ye-electron microscope yee-particles ze-silica ezingenanto (A, B) kunye nee-particles ze-silica ezibotshelelwe yi-ligand (C, D).
Ukusasazwa kobukhulu be-particle yee-particle ze-silica ezingenanto kunye nee-particle ze-silica ezibotshelelweyo kwi-ligand kuboniswe kuMfanekiso 3(A). Ii-curves zokusasazwa kobukhulu be-particle ezisekelwe kumthamo zibonise ukuba ubungakanani bee-particle ze-silica bunyukile emva kokuguqulwa kweekhemikhali (Umzobo 3A). Idatha yokusasazwa kobukhulu be-particle yee-particle ze-silica ezivela kuphononongo lwangoku kunye nophononongo lwangaphambili ithelekiswa kwiTheyibhile 1(A). Ubungakanani be-particle obusekelwe kumthamo, d(0.5), ye-PMP yi-3.36 μm, xa kuthelekiswa nophando lwethu lwangaphambili olunexabiso le-ad(0.5) le-3.05 μm (ii-particle ze-silica ezibotshelelweyo kwi-polystyrene)34. Le batch yayinokusasazwa kobukhulu be-particle obuncinci xa kuthelekiswa nophando lwethu lwangaphambili ngenxa yomlinganiselo owahlukileyo we-PEG, urea, TMOS, kunye ne-acetic acid kumxube we-reaction. Ubungakanani be-particle be-PMP bukhulu kancinci kunobo be-particle ye-silica ebotshelelweyo kwi-polystyrene esasiyifundile ngaphambili. Oku kuthetha ukuba ukusebenza komphezulu wee-particle ze-silica ezine-styrene kubeke kuphela umaleko we-polystyrene (0.97 µm) kwi umphezulu we-silica, ngelixa kwisigaba se-PMP ubukhulu beleya yayiyi-1.38 µm.
Ukusasazwa kobungakanani bamasuntswana (A) kunye nokusasazwa kobungakanani bemingxuma (B) yamasuntswana e-silica angenanto kunye namasuntswana e-silica aboshwe yi-ligand.
Ubungakanani bembobo, umthamo wembobo kunye nommandla womphezulu wee-silica particles zolu phando lwangoku zinikwe kwiTheyibhile 1(B). Iiprofayili zePSD zee-silica particles ezingenanto kunye nee-ligand-bonded silica particles ziboniswe kwiFigure 3(B). Iziphumo ziyafana nophando lwethu lwangaphambili. Ubungakanani bee-pore zee-silica particles ezingenanto kunye nee-ligand-bound particles yi-310 kunye ne-241, ngokulandelanayo, nto leyo ebonisa ukuba ubungakanani bee-pore buyancipha ngama-69 emva kokuguqulwa kweekhemikhali, njengoko kubonisiwe kwiTheyibhile 1(B), kwaye utshintsho lwe-curve luboniswe kwiFig. 3(B). Ngokufanayo, umthamo wee-pore zee-silica particles wehlile ukusuka kwi-0.67 ukuya kwi-0.58 cm3/g emva kokuguqulwa kweekhemikhali. Ummandla othile womphezulu wee-silica particles ezifundwayo ngoku yi-116 m2/g, nto leyo efaniswa nophando lwethu lwangaphambili (124 m2/g). Njengoko kubonisiwe kwiTheyibhile 1(B), indawo yomphezulu (m2/g) yee-silica particles nayo yehlile ukusuka kwi-116 m2/g ukuya kwi-105 m2/g emva kokuguqulwa kweekhemikhali.
Iziphumo zohlalutyo lwezinto ezisisiseko zesigaba esisisigxina ziboniswe kwiTheyibhile 2. Umthwalo wekhabhoni wesigaba esisisigxina sangoku yi-6.35%, engaphantsi kunomthwalo wekhabhoni wesifundo sethu sangaphambili (ii-polystyrene bonded silica particles, 7.93%35 kunye ne-10.21%, ngokulandelanayo) 42. Umthwalo wekhabhoni wesigaba esisisigxina sangoku uphantsi, Kuba ekulungiseleleni i-SP yangoku, ukongeza kwi-styrene, kusetyenziswe ezinye ii-ligands ze-polar ezifana ne-phenylmaleimide-methylvinylisocyanate (PCMP) kunye ne-4-hydroxy-TEMPO. Ipesenti yobunzima be-nitrogen yesigaba esisisigxina sangoku yi-2.21%, xa kuthelekiswa ne-0.1735 kunye ne-0.85% ngobunzima be-nitrogen kwizifundo zangaphambili, ngokulandelanayo. Oku kuthetha ukuba i-wt % ye-nitrogen iphezulu kwisigaba esisisigxina sangoku ngenxa ye-phenylmaleimide. Ngokufanayo, umthwalo wekhabhoni weemveliso (4) kunye (5) yayiyi-2.7% kunye ne-2.9%, ngokulandelanayo, ngelixa Umthwalo wekhabhoni wemveliso yokugqibela (6) yayiyi-6.35%, njengoko kubonisiwe kwiTheyibhile 2. Ukulahleka kobunzima kujongiwe nge-PMP stationary phase, kwaye i-TGA curve iboniswe kuMfanekiso 4. I-TGA curve ibonisa ukulahleka kobunzima be-8.6%, nto leyo evumelana kakuhle nomthwalo wekhabhoni (6.35%) kuba ii-ligands aziqulathanga kuphela i-C kodwa ne-N, O, kunye ne-H.
I-ligand ye-phenylmaleimide-methylvinylisocyanate ikhethwe ukuze kulungiswe umphezulu wee-particles ze-silica kuba inamaqela e-polar phenylmaleimide kunye namaqela e-vinylisocyanate. Amaqela e-Vinyl isocyanate anokusabela ngakumbi kwi-styrene ngokuphinda-phinda okuphilayo. Isizathu sesibini kukufaka iqela elinonxibelelwano oluphakathi ne-analyte kwaye akukho nxibelelwano oluqinileyo lwe-electrostatic phakathi kwe-analyte kunye nesigaba esimileyo, kuba indawo ye-phenylmaleimide ayinayo itshaja ebonakalayo kwi-pH eqhelekileyo. I-polarity yesigaba esimileyo inokulawulwa sisixa esifanelekileyo se-styrene kunye nexesha lokuphendula le-free radical polymerization. Inyathelo lokugqibela le-reaction (i-free-radical polymerization) libalulekile kwaye linokutshintsha i-polarity yesigaba esimileyo. Uhlalutyo lwezinto lwenziwe ukujonga umthwalo we-carbon kwezi zigaba ezimileyo. Kwabonwa ukuba ukwandisa ubungakanani be-styrene kunye nexesha lokuphendula kwandisa umthwalo we-carbon wesigaba esimileyo kwaye ngokuchaseneyo. Ii-SP ezilungiselelwe ngamanani ahlukeneyo e-styrene zinemithwalo eyahlukeneyo ye-carbon. Kwakhona, faka ezi zigaba ezimileyo kwintsimbi engagqwali. iikholamu kwaye ujonge ukusebenza kwazo kwe-chromatographic (ukukhetha, isisombululo, ixabiso le-N, njl.njl.). Ngokusekelwe kwezi mvavanyo, kukhethwe indlela elungiselelweyo yokulungiselela isigaba se-PMP esisisigxina ukuqinisekisa i-polarity elawulwayo kunye nokugcinwa okuhle kwe-analyte.
Iingxube ezintlanu zepeptide (iGly-Tyr, iGly-Leu-Tyr, iGly-Gly-Tyr-Arg, iTyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nazo zavavanywa kusetyenziswa ikholamu ye-PMP kusetyenziswa isigaba esihambayo; I-60/40 (v/v) i-acetonitrile/amanzi (0.1% TFA) ngesantya sokuhamba se-80 μL/min. Phantsi kweemeko ezifanelekileyo zokululwa, inombolo yeplate yethiyori (N) ngekholamu nganye (100 × 1.8 mm id) yi-20,000 ± 100 (200,000 plates/m²). Itheyibhile 3 inika amaxabiso e-N kwiikholamu ezintathu ze-PMP kwaye ii-chromatograms ziboniswe kuMfanekiso 5A. Uhlalutyo olukhawulezileyo kwikholamu ye-PMP ngesantya sokuhamba esiphezulu (700 μL/min), iipeptide ezintlanu zalunyulwa kumzuzu omnye, amaxabiso e-N ayelungile kakhulu, i-13,500 ± 330 ngekholamu nganye (100 × 1.8 mm id), Ihambelana neeplates eziyi-135,000/m (Umfanekiso 5B). Iikholamu ezintathu ezinobukhulu obufanayo (100 × 1.8 mm id) zazigcwele amaqela amathathu ahlukeneyo e-PMP stationary phase ukuze zihlolwe. ukuphinda-phinda. Uxinaniso lwe-analyte kwikholamu nganye lurekhodwe kusetyenziswa iimeko ezifanelekileyo zokulungisa kunye nenani leeplate zethiyori u-N kunye nexesha lokugcina ukwahlula umxube wovavanyo ofanayo kwikholamu nganye. Idatha yokuphinda-phinda kwikholamu ze-PMP iboniswe kwiTheyibhile 4. Ukuphinda-phinda kwikholamu ye-PMP kuhambelana kakuhle namaxabiso aphantsi kakhulu e-%RSD, njengoko kuboniswe kwiTheyibhile 3.
Ukwahlulwa komxube we-peptide kwikholamu ye-PMP (B) kunye nekholamu ye-Ascentis Express RP-Amide (A); isigaba esiphathwayo 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholamu ye-PMP (100 × 1.8 mm id); uhlalutyo Ulandelelwano lwe-elution lwee-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 (100 × 1.8 mm id) ihlolwe ukwahlulwa kwe-tryptic digests ye-albumin ye-serum yomntu kwi-chromatography yolwelo esebenza kakuhle. I-chromatogram ekuMfanekiso 6 ibonisa ukuba isampuli yahlulwe kakuhle kwaye isisombululo silungile kakhulu. I-HSA digests ihlalutywe kusetyenziswa isantya sokuhamba se-100 µL/min, i-mobile phase 70/30 acetonitrile/water kunye ne-0.1% TFA. Njengoko kubonisiwe kwi-chromatogram (Umfanekiso 6), i-HSA digest yahlulwe yaba ziincopho ezili-17 ezihambelana neepeptide ezili-17. Ukusebenza kakuhle kokwahlulwa kwencopho nganye kwi-HSA digest kubalwe kwaye amaxabiso anikwe kwiTheyibhile 5.
I-tryptic digest ye-HSA (100 × 1.8 mm id) yahlulwe kwikholamu ye-PMP; izinga lokuhamba (100 µL/min), i-mobile phase 60/40 acetonitrile/water ene-0.1% TFA.
apho u-L bubude bekholamu, u-η bubukrakra besigaba esihambayo, u-ΔP luxinzelelo lwekholamu ebuyela umva, kwaye u lisantya esithe ngqo sesigaba esihambayo. Ukuvuleka kwekholamu ye-PMP yayiyi-2.5 × 10-14 m2, izinga lokuhamba laliyi-25 μL/min, kwaye kwasetyenziswa i-60/40 v/v ACN/water. Ukuvuleka kwekholamu ye-PMP (100 × 1.8 mm id) kwakufana noko kophando lwethu lwangaphambili. Ref.34. Ukuvuleka kwekholamu ezaliswe ziinxalenye ezingenanto ngaphandle kokuqulatha yi: 1.7 × 10-15 kwiinxalenye eziyi-1.3 μm, 3.1 × 10-15 kwiinxalenye eziyi-1.7 μm, 5.2 × 10-15 kunye no-2.5 ​​× 10-14 m2 kwiinxalenye eziyi-2.6 μm. Kwiinxalenye eziyi-5 μm. 43. Ke ngoko, ukuvuleka kwesigaba se-PMP kufana noko kweenxalenye eziyi-5 μm ze-core-shell.
apho i-Wx bubunzima bekholam ezaliswe yi-chloroform, i-Wy bubunzima bekholam ezaliswe yi-methanol, kwaye i-ρ bubunzima be-solvent. Ubuninzi be-methanol (ρ = 0.7866) kunye ne-chloroform (ρ = 1.484). I-porosity iyonke yeekholam ze-SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 kunye neekholam ze-C18-Urea 31 esizifundileyo ngaphambili yayiyi-0.63 kunye ne-0.55, ngokulandelelana. Oku kuthetha ukuba ubukho bee-urea ligands kunciphisa ukuvuleka kwesigaba esimileyo. Kwelinye icala, i-porosity iyonke yekholam ye-PMP (100 × 1.8 mm id) yi-0.60. Ukuvuleka kweekholam ze-PMP kuphantsi kunokweekholam ezizaliswe zii-silica particles ze-C18-bonded kuba kwizigaba ze-C18-type stationary, ii-C18 ligands zinamathele kwii-silica particles njengee-linear chains, ngelixa kwizigaba ze-polystyrene-type stationary, i-A ityebile kakhulu. Umaleko wepolymer wenziwa ngeenxa zonke kuwo. Kwisilingo esiqhelekileyo, i-porosity yekholamu ibalwa ngolu hlobo:
Umfanekiso 7A,B ubonisa ikholamu ye-PMP (100 × 1.8 mm id) kunye nekholamu ye-Ascentis Express RP-Amide (100 × 1.8 mm id) kusetyenziswa iimeko ezifanayo zolution (oko kukuthi, 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) ilungiselelwe kwi-20 µL/ Isantya esincinci sokuhamba kweekholamu zombini yi-800 µL/min. Amaxabiso aphantsi e-HETP kwisantya sokuhamba esifanelekileyo (80 µL/min) kwikholamu ye-PMP kunye nekholamu ye-Ascentis Express RP-Amide yayiyi-2.6 µm kunye ne-3.9 µm, ngokulandelelana. Amaxabiso e-HETP abonisa ukuba ukusebenza kakuhle kokwahlulwa kwekholamu ye-PMP (100 × 1.8 mm id) kungcono kakhulu kunekholamu ye-Ascentis Express RP-Amide ethengiswayo (100 × 1.8 mm id). Iploti ye-van Deemter kwiFig. 7(A) ibonisa ukuba ukwehla kwexabiso le-N ngokunyuka kokuhamba akubalulekile xa kuthelekiswa nophando lwethu lwangaphambili. Ukusebenza okuphezulu kokwahlulwa kwekholamu ye-PMP (100 × 1.8 mm id) xa kuthelekiswa nekholamu ye-Ascentis Express RP-Amide isekelwe kuphuculo kwimo yamasuntswana, ubungakanani, kunye neenkqubo zokupakisha iikholamu ezintsonkothileyo ezisetyenziswa kumsebenzi wangoku34.
(A) i-van Deemter plot (i-HETP ngokuchasene nesantya esijikelezayo sephase) efunyenwe kusetyenziswa ikholamu ye-PMP (100 × 1.8 mm id) kwi-60/40 ACN/H2O ene-0.1% TFA. (B) i-van Deemter plot (i-HETP ngokuchasene nesantya esijikelezayo sephase) efunyenwe kusetyenziswa ikholamu ye-Ascentis Express RP-Amide (100 × 1.8 mm id) kwi-60/40 ACN/H2O ene-0.1% TFA.
Isigaba esimileyo se-polystyrene esifakwe kwi-polar salungiswa saza savavanywa ukuze kwahlulwe imixube ye-peptide eyenziweyo kunye nokugaywa kwe-trypsin ye-albumin ye-human serum (HAS) kwi-chromatography yolwelo esebenza kakhulu. Ukusebenza kwe-chromatographic yeekholamu ze-PMP zemixube ye-peptide kugqwesileyo ekusebenzeni kakuhle nasekusombululeni ukwahlukana. Ukusebenza okuphuculweyo kokwahlukana kweekholamu ze-PMP kungenxa yezizathu ezahlukeneyo, ezinje ngobukhulu bee-particle kunye nobukhulu bee-pore zee-particles ze-silica, ukwenziwa okulawulwayo kwesigaba esimileyo, kunye nokupakisha okuntsonkothileyo kweekholamu. Ukongeza ekusebenzeni kakuhle kokwahlukana, uxinzelelo oluphantsi lwekholamu engasemva kwizinga eliphezulu lokuhamba kwamanzi yenye inzuzo yale sigaba esimileyo. Iikholamu ze-PMP zibonisa ukuphinda-phinda okuhle kwaye zingasetyenziselwa uhlalutyo lwemixube ye-peptide kunye nokugaywa kwe-trypsin yeeproteni ezahlukeneyo. Siceba ukusebenzisa le kholamu ukwahlula iimveliso zendalo, iikhompawundi ezisebenzayo kwizityalo zonyango kunye nezicatshulwa zefungus kwi-chromatography yolwelo. Kwixesha elizayo, iikholamu ze-PMP ziya kuvavanywa kwakhona ukuze kwahlulwe iiproteni kunye nee-antibodies ze-monoclonal.
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