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Porous silica particles tau npaj los ntawm txoj kev sol-gel nrog qee qhov kev hloov kho kom tau txais cov khoom siv macroporous.Cov khoom no tau muab los ntawm cov khoom siv rov qab ntxiv fragmentation saw hloov (RAFT) polymerization nrog N-phenylmaleimide-methylvinylisocyanate (PMI) thiab styrene los npaj N-phenylmaleimide theem ntawm steel borrown. (100 × 1.8 mm id) tau ntim los ntawm slurry packing.Kev ntsuam xyuas PMP kem kev sib cais ntawm cov peptide sib tov uas muaj tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine ntawm tib neeg albumskephalin kev ua haujlwm). der kev pom zoo elution tej yam kev mob, lub theoretical phaj suav ntawm cov peptide sib tov yog siab li 280,000 daim hlau / m².Piv qhov kev sib cais ntawm cov tsim kem nrog cov coj mus muag Ascentis Express RP-Amide kem, nws tau pom tias kev sib cais kev ua tau zoo ntawm PMP kem yog superior rau cov lag luam kem nyob rau hauv cov nqe lus ntawm kev sib cais efficiency thiab kev daws teeb meem.
Nyob rau hauv xyoo tas los no, kev lag luam biopharmaceutical tau dhau los ua kev lag luam thoob ntiaj teb nthuav dav nrog kev lag luam nce ntxiv. Nrog rau kev loj hlob ntawm biopharmaceutical kev lag luam1,2,3, kev tsom xam ntawm peptides thiab cov proteins yog qhov xav tau heev.Ntxiv rau lub hom phiaj peptide, ob peb impurities tau tsim thaum lub sij hawm peptide synthesis. thiab tus cwj pwm ntawm cov proteins hauv cov kua hauv lub cev, cov ntaub so ntswg thiab cov hlwb yog ib txoj haujlwm nyuaj heev vim muaj cov kab mob uas muaj peev xwm kuaj tau hauv ib qho qauv.Txawm hais tias huab hwm coj spectrometry yog ib qho cuab yeej zoo rau peptide thiab protein sequencing, yog tias cov qauv no tau txhaj rau hauv huab hwm coj spectrometer nyob rau hauv ib qho dhau los, kev sib cais yuav tsis yog qhov zoo tshaj plaws.Qhov teeb meem sib cais ua ntej LC yuav ua tau kom txo tau qhov kev soj ntsuam ua ntej. Tus naj npawb ntawm cov analytes nkag mus rau hauv huab hwm coj spectrometer ntawm ib lub sij hawm muab 4,5,6.Ntxiv rau, thaum lub sij hawm ua kua theem sib cais, cov analytes tuaj yeem tsom mus rau hauv cov cheeb tsam nqaim, yog li tsom rau cov analytes no thiab txhim kho MS nrhiav pom rhiab heev.Liquid chromatography (LC) tau nce siab heev nyob rau xyoo caum dhau los thiab tau dhau los ua cov txheej txheem nrov, 70 proteom.
Reversed-theem kua chromatography (RP-LC) yog dav siv rau kev ua kom huv thiab sib cais ntawm peptide sib tov siv octadecyl-hloov silica (ODS) raws li cov theem nyob ruaj ruaj 11,12,13.Txawm li cas los xij, RP nyob ruaj khov theem tsis muab kev txaus siab sib cais ntawm peptides thiab cov proteins vim lawv cov qauv tsim tshwj xeeb, theem 1 phij cuam 1. yuav tsum tau soj ntsuam peptides thiab cov proteins nrog cov polar thiab non-polar moieties kom cuam tshuam nrog thiab khaws cov analytes16.Mixed-mode chromatography, uas muab kev sib cuam tshuam ntau yam, tuaj yeem yog lwm txoj hauv kev rau RP-LC rau kev sib cais ntawm peptides, proteins, thiab lwm yam complex mixs. Ob peb mix-mode chromatography, uas muab cov kev sib txuas lus multimodal, tuaj yeem yog lwm txoj hauv kev rau RP-LC rau kev sib cais ntawm peptides, proteins, thiab lwm yam complex mixs. Ob peb mix-mode chromatography cov txheej txheem sib xyaw ua ke tau npaj rau theem 7 thiab cov protein sib cais. 18,19,20,21.Mixed-mode nyob ruaj ruaj theem (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) yog haum rau peptide thiab protein sib cais vim muaj ob pawg polar thiab non-polar pawg22,23,24,25,26,27,28 .Similaration intercalation cov pab pawg neeg zoo sib cais, cov kev sib txuas lus zoo sib txuas nrog cov polars sib cais cov theem. selectivity rau polar thiab non-polar analytes, raws li kev sib cais nyob ntawm kev sib cuam tshuam ntawm analytes thiab nyob ruaj ruaj theem.Multimodal kev sib cuam tshuam 29, 30, 31, 32. Tsis ntev los no, Zhang et al.30 tau npaj dodecyl-terminated polyamine nyob ruaj ruaj theem thiab ntse sib cais hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, thiab ob peb lwm yam analytes.Lub polar intercalator muaj ob pawg polar thiab non-polar, yog li nws tuaj yeem siv los cais cov peptides thiab cov proteins uas muaj ob qho tib si hydrophobic-embedded kem-polars. kab) muaj nyob rau hauv kev lag luam npe Ascentis Express RP-Amide kab, tab sis cov kab no yog siv rau kev tsom xam ntawm amine 33 nkaus xwb.
Nyob rau hauv txoj kev tshawb no tam sim no, lub polar-embedded nyob ruaj ruaj theem (N-phenylmaleimide-embedded polystyrene) tau npaj thiab soj ntsuam rau kev sib cais ntawm peptides thiab trypsin digests ntawm HSA.Lub sij hawm nyob ruaj ruaj tau npaj siv cov tswv yim hauv qab no.Porous silica hais tau npaj raws li cov txheej txheem muab hauv peb cov ntawv tshaj tawm yav dhau los nrog rau qee qhov kev hloov pauv ntawm polyethylene glycol . TMOS, dej acetic acid tau hloov kho los npaj silica hais nrog cov pore loj loj.Second, ib lub ligand tshiab, phenylmaleimide-methyl vinyl isocyanate, tau ua ke thiab siv los derivatize silica particles los npaj ib tug polar embedded nyob ruaj ruaj theem.Qhov tshwm sim nyob ruaj ruaj theem tau ntim rau hauv ib qho 10 hli stainless hlau. kem packing yog pab nrog cov neeg kho tshuab kev co los xyuas kom meej ib homogeneous txaj yog tsim nyob rau hauv lub kem.Tshaj packed kem sib cais ntawm peptide sib tov muaj tsib peptides;(Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) thiab Trypsin digest ntawm tib neeg serum albumin (HAS).Peptide sib tov thiab trypsin digest ntawm HSA raug soj ntsuam kom sib cais nrog zoo daws teeb meem thiab efficiency.P kem kev ua tau zoo. eptides thiab cov proteins tau pom tias yuav daws tau zoo thiab ua haujlwm tau zoo ntawm PMP kem, uas ua tau zoo dua li Ascentis Express RP-Amide kem.
PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Tib Neeg Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC4), Steyrene, Styrene, Acetonitrile (ACN), Methanol, 2-Propanol, thiab Acetone Purchased from Sigma-Aldrich (St. Louis, MO, USA).
Ib qho sib xyaw ntawm urea (8 g), polyethylene glycol (8 g), thiab 8 mL ntawm 0.01 N acetic acid tau nplawm rau 10 feeb, thiab tom qab ntawd 24 mL ntawm TMOS tau ntxiv rau hauv cov dej khov txias.Cov tshuaj tiv thaiv sib tov yog rhuab ntawm 40 ° C rau 6 teev thiab tom qab ntawd ntawm 120 ° C rau cov hlau tsis muaj dej thiab cov khoom siv tau qhuav rau 8 teev. 70 ° C rau 12 xuab moos.Cov qhuav mos mos yog cov hauv av du hauv qhov cub thiab calcined ntawm 550 ° C rau 12 teev.Peb cov khoom tau npaj thiab ua kom zoo los tshuaj xyuas cov khoom rov ua dua tshiab hauv qhov loj me, qhov pore loj thiab thaj chaw saum npoo.
Los ntawm kev hloov kho deg ntawm silica hais nrog pre-synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) ua raws li radial polymerization nrog styrene, ib pawg polar-muaj cov compound tau npaj.Nyob ruaj ruaj theem rau aggregates thiab polystyrene chains.Cov txheej txheem npaj tau piav qhia hauv qab no.
N-phenylmaleimide (200 mg) thiab methyl vinyl isocyanate (100 mg) tau yaj hauv cov toluene qhuav, thiab 0.1 mL ntawm 2,2′-azoisobutyronitrile (AIBN) tau ntxiv rau cov tshuaj tiv thaiv lub khob los npaj phenylmaleimide-methyl vinyl isocyanate CPPM ntawm 3 xuab moos sib tov thiab copolymer. qhuav hauv qhov cub ntawm 40 ° C rau 3 teev.
Qhuav silica hais (2 g) tau dispersed nyob rau hauv qhuav toluene (100 mL), stirred thiab sonicated nyob rau hauv ib tug 500 mL puag ncig hauv qab khob rau 10 min.PMCP (10 mg) yog yaj nyob rau hauv toluene thiab ntxiv dropwise rau cov tshuaj tiv thaiv hwj ntawm ib tug xa me nyuam funnel.Cov sib tov yog refluxed ntawm 180 ° C, lim thiab qhuav rau 6 ° C. Tom qab ntawd, PMCP-bonded silica particles (100 g) tau yaj nyob rau hauv toluene (200 ml) thiab 4-hydroxy-TEMPO (2 mL) ntxiv nyob rau hauv lub xub ntiag ntawm 100 µL ntawm dibutyltin dilaurate raws li ib tug catalyst.Cov sib tov yog stirred ntawm 50 ° C rau 8 teev, lim thiab qhuav rau 8 teev 3 teev.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), thiab TEMPO-PMCP-txuas nrog silica hais (1.5 g) tau dispersed nyob rau hauv toluene thiab purged nrog nitrogen.The polymerization ntawm styrene yog nqa tawm ntawm 100 ° C rau 12 teev.Cov khoom tshwm sim tau ntxuav nrog 60 ° C ntawm cov tshuaj tiv thaiv methanol.
Cov qauv no tau degassed ntawm 393 K rau 1 teev kom tau txais qhov seem seem ntawm tsawg dua 10-3 Torr.Tus nqi ntawm N2 adsorbed ntawm tus txheeb ze siab ntawm P / P0 = 0.99 tau siv los txiav txim siab tag nrho cov pore ntim.Lub morphology ntawm liab qab thiab ligand-bonded silica particles Tokyo tau soj ntsuam nrog cov khoom siv hluav taws xob siab, Nyiv P / P0 = 0.99. (pob liab silica thiab ligand-bonded silica particles) tau muab tso rau ntawm ib qho txhuas kem siv cov nplaum carbon daim kab xev.Gold tau plated rau cov qauv siv Q150T sputter coater, thiab 5 nm Au txheej tau muab tso rau hauv cov qauv.Qhov no txhim kho cov txheej txheem kev siv hluav taws xob tsawg thiab muab cov nplej zoo, txias sputtering.A Thermo 1 EA Electron, US Electron Electron. al analysis.A Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer tau siv los muab cov particle loj faib.Naked silica hais thiab ligand-bonded silica particles (5 mg txhua) tau dispersed nyob rau hauv 5 mL ntawm isopropanol, sonicated rau 10 min, 5 lub vortex ntawm kev tsom xam ntawm Masters thiab vortexed. Ua tau ntawm tus nqi ntawm 5 ° C ib feeb dhau qhov kub ntawm 30 txog 800 ° C.
Cov iav-lined stainless hlau nqaim-bore kab ntawm qhov ntev (100 × 1.8 mm id) tau ntim nrog cov txheej txheem slurry packing, siv tib txoj kev siv hauv Ref.31.A stainless hlau kem (iav-lined, 100 × 1.8 mm id) nrog lub qhov hluav taws xob haum uas muaj 1 µm frit tau txuas nrog slurry packer (Alltech Deerfield, IL, USA).Npaj theem nyob ruaj khov slurry los ntawm kev ncua 150 mg ntawm cov theem nyob ruaj khov rau hauv 1.2 mL ntawm cov khoom siv slurry. zoo li cov kuab tshuaj propelling.Fill rau kab ke ua ntu zus los ntawm kev siv lub zog ntawm 100 MP rau 10 feeb, 80 MP rau 15 feeb, thiab 60 MP rau 30 feeb.Thaum lub sijhawm ntim khoom, cov khoom siv vibration tau siv nrog ob lub GC kem shakers (Alltech, Deerfield, IL, USA) kom ntseeg tau tias cov khoom tsis sib haum ntawm cov kab sib txuas ntawm cov slurry. txuas lub kem los ntawm slurry packing unit thiab txuas lwm qhov haum rau lub inlet thiab mus rau LC system los xyuas nws cov kev ua tau zoo.
Ib lub twj tso kua mis LC (10AD Shimadzu, Nyiv), injector (Valco (USA) C14 W.05) nrog 50nL txhaj tshuaj voj, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary qhov rais tau tsim tshwj xeeb µLC ntaus ntawv ntes (UV-2075) thiab iav-lined heev microcolumns txuas rau kab txuas ntxiv. Tom qab ntim, capillaries (50 μm id 365 thiab txo lub union capillaries (50 μm) tau muab ntsia rau ntawm 1/16 "qhov hluav taws xob ntawm lub union txo. Cov ntaub ntawv sau thiab chromatographic ua tau ua tiav siv Multichro 2000 software. Kev soj ntsuam ntawm 254 nm Analytes raug kuaj rau UV cov ntaub ntawv absorbance, Chromat 8.
Albumin los ntawm tib neeg cov ntshav, lyophilized hmoov, ≥ 96% (agarose gel electrophoresis) 3 mg tov nrog trypsin (1.5 mg), 4.0 M urea (1 mL), thiab 0.2 M ammonium bicarbonate (1 mL).Cov tshuaj tau nplawm rau 10 feeb thiab khaws cia rau hauv ib da dej ntawm 37 ° F, ces 1 mque ntawm 37 ° C. ter cov tshuaj thiab khaws cia hauv qab 4 ° C.
Kev sib cais ntawm peptide mixs thiab HSA trypsin digests tau soj ntsuam nyias ntawm PMP kab.Kuaj xyuas qhov sib cais ntawm peptide sib tov thiab trypsin digest ntawm HSA los ntawm PMP kem thiab sib piv cov txiaj ntsig rau Ascentis Express RP-Amide kem.Theoretical phaj tooj yog xam raws li hauv qab no:
SEM dluab ntawm liab qab silica hais thiab ligand-bonded silica hais yog qhia nyob rau hauv FIG.2 .SEM dluab ntawm liab qab silica hais (A, B) qhia tau hais tias, nyob rau hauv sib piv rau peb yav dhau los kev tshawb fawb, cov hais no yog spherical nyob rau hauv uas cov hais yog elongated los yog muaj irregular symmetry.Qhov saum npoo ntawm ligand-bonded silica hais (C, D) yog smoother tshaj li ntawm cov liab qab silica saw ntawm cov ntaub ntawv uas tej zaum yuav smoother. ib particle.
Scanning electron microscope dluab ntawm liab qab silica hais (A, B) thiab ligand-bonded silica hais (C, D).
Cov particle loj faib ntawm cov liab qab silica hais thiab ligand-bonded silica hais yog qhia nyob rau hauv daim duab 3(A).Volume-based particle size distribution curves pom tias qhov loj ntawm cov silica hais tau nce tom qab kev hloov tshuaj (Fig. 3A).Qhov particle loj faib cov ntaub ntawv ntawm cov silica particles los ntawm qhov kev kawm tam sim no 0. 5), ntawm PMP yog 3.36 μm, piv rau peb txoj kev kawm yav dhau los nrog ad(0.5) tus nqi ntawm 3.05 μm (polystyrene-bound silica particles) 34.Cov batch no muaj qhov nqaim particle loj dua piv rau peb txoj kev kawm yav dhau los vim qhov sib txawv ntawm PEG, urea, TMOS, thiab cov tshuaj tiv thaiv me ntsis ntawm cov ntsiab lus ntawm acetic acid. silica particle theem peb yav dhau los tau kawm.Qhov no txhais tau hais tias nto functionalization ntawm silica hais nrog styrene tsuas yog tso ib txheej polystyrene (0.97 µm) ntawm silica nto, whereas nyob rau hauv lub PMP theem txheej thickness yog 1.38 µm.
Particle loj faib (A) thiab pore loj faib (B) ntawm liab qab silica hais thiab ligand-bound silica hais.
Qhov pore loj, pore ntim thiab thaj tsam ntawm silica hais ntawm txoj kev kawm tam sim no tau muab rau hauv Table 1(B).PSD profiles ntawm liab qab silica hais thiab ligand-bonded silica particles yog qhia nyob rau hauv daim duab 3(B).Cov txiaj ntsig tau muab piv rau peb txoj kev tshawb fawb yav dhau los.Qhov pore qhov loj me ntawm cov liab qab silica hais thiab ligand-bonded silica particles yog qhia nyob rau hauv daim duab 3(B). qhov pore loj zuj zus los ntawm 69 tom qab kev hloov kho tshuaj, raws li qhia hauv Table 1(B), thiab qhov kev hloov ntawm qhov nkhaus yog qhia hauv daim duab 3(B) .Ib yam li ntawd, qhov pore ntim ntawm silica particles txo los ntawm 0.67 mus rau 0.58 cm3 / g tom qab tshuaj hloov kho.Qhov tshwj xeeb ntawm thaj chaw ntawm silica particles yog tam sim no kawm txog 1 g ntawm peb cov ntaub ntawv. (124 m2/g).Raws li pom nyob rau hauv Table 1(B), qhov chaw nto (m2/g) ntawm cov silica hais kuj txo los ntawm 116 m2/g mus rau 105 m2/g tom qab tshuaj hloov kho.
Cov txiaj ntsig ntawm kev txheeb xyuas cov theem nyob ruaj khov yog pom nyob rau hauv Table 2.Cov pa roj carbon loading ntawm lub sijhawm tam sim no nyob ruaj khov yog 6.35%, uas yog qis dua cov pa roj carbon loading ntawm peb txoj kev tshawb fawb dhau los (polystyrene bonded silica particles, 7.93% 35 thiab 10.21%, feem) 42. Vim tias cov pa roj carbon loading ntawm cov khoom siv tam sim no nyob rau hauv qhov chaw ruaj khov, qee qhov kev npaj ntawm SP lige tam sim no yog qis. s xws li phenylmaleimide-methylvinylisocyanate (PCMP) thiab 4-hydroxy-TEMPO tau siv.The nitrogen hnyav feem pua ​​​​ntawm cov theem tam sim no nyob ruaj khov yog 2.21%, piv rau 0.1735 thiab 0.85% los ntawm qhov hnyav ntawm nitrogen hauv cov kev tshawb fawb yav dhau los, feem.Qhov no txhais tau hais tias wt % ntawm nitrogen chaw nres tsheb yog siab dua ntawm cov khoom tam sim no ntawm cov khoom siv phenyl. ) thiab (5) yog 2.7% thiab 2.9%, feem, thaum cov pa roj carbon loading ntawm cov khoom kawg (6) yog 6.35%, raws li qhia nyob rau hauv Table 2.Qhov hnyav poob tau soj ntsuam nrog PMP nyob ruaj khov theem, thiab TGA nkhaus yog qhia nyob rau hauv daim duab 4.TGA nkhaus qhia tau hais tias qhov hnyav poob ntawm 8.6%, cov pa roj carbon monoxide tsuas yog nyob rau hauv kev pom zoo, tab sis 6% yog tsis zoo, tab sis 6% ntawm cov khoom tsis zoo, thiab 6% yog tsis muaj kev pom zoo. H.
Cov phenylmaleimide-methylvinylisocyanate ligand raug xaiv rau kev hloov kho saum npoo ntawm silica hais vim nws muaj cov polar phenylmaleimide pawg thiab vinylisocyanate pawg.Vinyl isocyanate pawg tuaj yeem ua rau muaj zog ntxiv nrog styrene los ntawm kev ua neej nyob radical polymerization.Qhov thib ob yog vim li cas los ntxig ib pab pawg uas muaj qhov nruab nrab ntawm qhov chaw nres tsheb electrostatic tsis muaj kev cuam tshuam nrog cov theem ntawm anallet, vim li ntawd, phenylmaleimide. imide moiety tsis muaj nqi virtual ntawm ib txwm pH.Qhov polarity ntawm cov theem nyob ruaj khov tuaj yeem tswj tau los ntawm qhov zoo tshaj plaws ntawm styrene thiab lub sij hawm cov tshuaj tiv thaiv dawb radical polymerization.Cov kauj ruam kawg ntawm cov tshuaj tiv thaiv (dawb-radical polymerization) yog qhov tseem ceeb thiab tuaj yeem hloov lub polarity ntawm cov theem nyob ruaj ruaj.Elemental tsom xam tau ua los xyuas cov pa roj carbon loading ntawm cov tshuaj tiv thaiv lub sij hawm ntawm qhov chaw nres tsheb nce ntxiv. ary theem thiab vice versa.SPs npaj nrog sib txawv concentrations ntawm styrene muaj sib txawv cov pa roj carbon loadings.Ntxiv, thauj cov theem nyob ruaj ruaj rau hauv stainless hlau txhua thiab xyuas lawv cov chromatographic kev ua tau zoo (selectivity, daws teeb meem, N tus nqi, thiab lwm yam).Raws li cov kev sim no, ib tug optimized formulation raug xaiv los npaj lub PMP nyob ruaj ruaj theem kom tswj polarity retention thiab zoo analyst.
Tsib peptide sib tov (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) kuj tau soj ntsuam siv PMP kem siv lub sijhawm txawb;60/40 (v/v) acetonitrile/dej (0.1% TFA) ntawm tus nqi ntws ntawm 80 μL / min.Raws li qhov zoo tshaj plaws elution tej yam kev mob, lub theoretical phaj tooj (N) ib kem (100 × 1.8 mm id) yog 20,000 ± 100 (200,000 T 3 pcs / m² tus nqi). thiab cov chromatograms tau pom nyob rau hauv daim duab 5A.Kev tsom xam ceev ceev ntawm PMP kem ntawm cov dej ntws siab (700 μL / min), tsib peptides tau eluted nyob rau hauv ib feeb, N qhov tseem ceeb tau zoo heev, 13,500 ± 330 ib kem (100 × 1.8 mm id), T Correspond 0 500 × 1.8 mm (1h30) entically sized columns (100 × 1.8 mm id) tau ntim nrog peb ntau qhov sib txawv ntawm PMP nyob ruaj khov theem los xyuas txog kev rov tsim dua tshiab.Cov tshuaj ntsuam xyuas cov ntsiab lus rau txhua kem tau sau tseg siv cov kev pom zoo elution thiab tus naj npawb ntawm theoretical daim hlau N thiab lub sij hawm khaws cia kom cais tib qhov kev sim sib tov ntawm txhua kab.Cov ntaub ntawv rov ua dua tshiab ntawm TMP kem zoo heev. % RSD tus nqi, raws li qhia hauv Table 3.
Kev sib cais ntawm peptide sib tov ntawm PMP kem (B) thiab Ascentis Express RP-Amide kem (A);mobile theem 60/40 ACN/H2O (TFA 0.1%), PMP kem qhov ntev (100 × 1.8 mm id);analytical Qhov kev txiav txim elution ntawm cov tebchaw: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) thiab 5 (leucine) acid enkephalin)).
Ib kem PMP (100 × 1.8 mm id) raug soj ntsuam rau kev sib cais ntawm tryptic digests ntawm tib neeg cov ntshav albumin nyob rau hauv kev ua tau zoo kua chromatography.Lub chromatogram hauv daim duab 6 qhia tau hais tias tus qauv yog zoo sib cais thiab cov kev daws teeb meem yog zoo heev.HSA digests tau soj ntsuam siv ib tug txaus tus nqi ntawm 100 µL / 7 ton.A 3 theem, mobile theem. s qhia nyob rau hauv cov chromatogram (Daim duab 6), HSA digestion tau muab faib ua 17 peaks sib haum mus rau 17 peptides.Qhov kev sib cais efficiency ntawm txhua lub ncov hauv HSA digest yog xam thiab cov txiaj ntsig tau muab rau hauv Table 5.
Ib qho tryptic digest ntawm HSA (100 × 1.8 mm id) tau muab cais rau ntawm PMP kem;ntws tus nqi (100 µL / min), mobile theem 60/40 acetonitrile / dej nrog 0.1% TFA.
qhov twg L yog kem ntev, η yog qhov viscosity ntawm lub sijhawm txawb, ΔP yog kab rov qab siab, thiab u yog linear tshaj tawm ntawm lub xov tooj ntawm tes.Qhov permeability ntawm PMP kem yog 2.5 × 10-14 m2, tus nqi ntws yog 25 μL / min, thiab 60/40 v / v / v. ACN permeability kem yog 1.0 m. zoo ib yam li peb txoj kev kawm yav dhau los Ref.34.Qhov permeability ntawm kem packed nrog superficially ntxeem tau hais yog: 1.7 × 10-15 rau 1.3 μm hais, 3.1 × 10-15 rau 1.7 μm hais, 5.2 × 10-15 m particles 2.4 × 2.5 m particles 43.Yog li ntawd, qhov permeability ntawm PMP theem zoo ib yam li ntawm 5 μm core-plhaub hais.
qhov twg Wx yog qhov hnyav ntawm kem ntim nrog chloroform, Wy yog qhov hnyav ntawm kem ntim nrog methanol, thiab ρ yog qhov ntom ntawm cov kuab tshuaj.Densities ntawm methanol (ρ = 0.7866) thiab chloroform (ρ = 1.484).The tag nrho porosity ntawm SILICA PARTICLES-18 mm-10. Urea kab 31 uas peb tau kawm yav dhau los yog 0.63 thiab 0.55, ntsig txog.Qhov no txhais tau hais tias muaj cov urea ligands txo cov permeability ntawm cov theem nyob ruaj ruaj.Ntawm qhov tod tes, tag nrho cov porosity ntawm PMP kem (100 × 1.8 mm id) yog 0.60.Qhov permeability ntawm 1 pawg ntawm PMP bons yog qis dua li PMP kab. C18-hom nyob ruaj khov theem C18 ligands txuas rau cov silica hais raws li cov kab sib txuas, thaum nyob rau hauv polystyrene-hom nyob ruaj ruaj theem, A kuj tuab polymer txheej yog tsim nyob ib ncig ntawm nws.
Daim duab 7A, B qhia PMP kem (100 × 1.8 mm id) thiab Ascentis Express RP-Amide kem (100 × 1.8 mm id) siv tib yam elution (ie, 60/40 ACN / H2O thiab 0.1% TFA).) ntawm van Deemter zaj dab neeg.Xaiv cov peptide sib tov (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) tau npaj rau hauv 20 µL / Qhov tsawg kawg nkaus tus nqi rau ob kab yog 800 µL / min.Qhov tsawg kawg nkaus HETP tus nqi yog µMP (L) centigrade 8. Kev nthuav qhia RP-Amide kem yog 2.6 µm thiab 3.9 µm, ntsig txog.Qhov HETP qhov tseem ceeb qhia tau hais tias kev sib cais ua haujlwm ntawm PMP kem (100 × 1.8 mm id) yog qhov zoo dua li cov khoom lag luam Ascentis Express RP-Amide kem (100 × 1.8 mm ter) uas tsis muaj nqi ntawm van de 7. qhov tseem ceeb piv rau peb txoj kev kawm yav dhau los.Qhov kev sib cais ntau dua ntawm PMP kem (100 × 1.8 mm id) piv rau Ascentis Express RP-Amide kem yog ua raws li kev txhim kho hauv cov duab particle, qhov loj me, thiab cov txheej txheem ntim cov txheej txheem siv hauv kev ua haujlwm tam sim no34.
(A) van Deemter zaj duab xis (HETP piv rau theem xov tooj ntawm tes kab nrawm) tau siv PMP kem (100 × 1.8 mm id) hauv 60/40 ACN / H2O nrog 0.1% TFA.(B) van Deemter plot (HETP piv rau theem xov tooj ntawm tes linear tshaj tawm) tau siv Ascentis Kem 10 × 0.0A RP8 / 0.0A CN / H2O nrog 0.1% TFA.
Lub polar-embedded polystyrene nyob ruaj ruaj theem tau npaj thiab tshuaj xyuas rau kev sib cais ntawm cov hluavtaws peptide sib tov thiab trypsin digests ntawm tib neeg cov ntshav albumin (HAS) hauv kev ua haujlwm siab ua kua chromatography.The chromatographic kev ua tau zoo ntawm PMP kab rau peptide mixtures yog zoo heev nyob rau hauv kev sib cais efficiency thiab kev daws teeb meem.Qhov kev txhim kho kev sib cais ntawm PMP particles ntawm ntau yam yog vim li cas, qhov loj ntawm particles yog qhov loj. , tswj synthesis ntawm cov theem nyob ruaj ruaj, thiab cov kab ke nyuaj. Ntxiv rau qhov kev sib cais siab, qis qis rov qab siab ntawm tus nqi ntws siab yog lwm qhov kom zoo dua ntawm qhov chaw nyob ruaj khov no.PMP kem nthuav tawm zoo rov tsim dua tshiab thiab tuaj yeem siv rau kev tshuaj xyuas peptide mixtures thiab trypsin digestion ntawm ntau cov proteins.Peb npaj siab yuav siv cov kab no rau kev sib cais ntawm cov khoom siv ntuj tsim tawm, PMP cov khoom lag luam zoo, thiab cov khoom lag luam zoo nyob rau yav tom ntej. MP kab tseem yuav raug soj ntsuam rau kev sib cais ntawm cov proteins thiab cov tshuaj tiv thaiv monoclonal.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Kev tshawb fawb ntawm Peptide Separation Systems los ntawm Reversed Phase Chromatography Tshooj I: Kev Txhim Kho ntawm Kab Cuam Tshuam Tus Qauv Txheej Txheem.J.Chromatography.1603, 113–129.https://doi.org/10.1016/j.chroma.2019.05.038 (2019).
Gomez, B. et al.Improved active peptides tsim los kho cov kab mob sib kis.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 therapeutic peptides: science and the market.drug discovery.15 (1-2) hnub no, 40-56.https://doi.org/10.1016/j.drudis.2009.10.
Xie, F., Smith, RD & Shen, Y. Advanced Proteomic Liquid Chromatography.J.Chromatography.A 1261, 78–90 (2012).
Liu, W. et al.Advanced kua chromatography-mass spectrometry enables kev koom ua ke ntawm broadly targeted metabolomics thiab proteomics.anus.Chim.Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Lub luag haujlwm ntawm UHPLC hauv kev tsim tshuaj.J.Sep. Sci.30(8), 1183-1190 (2007).
Wu, N. & Clausen, AM Fundamental and practicalities of ultrahigh pressure kua chromatography rau ceev sib cais.Sep. Sci.30(8), 1167-1182.https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Daim ntawv thov ntawm kev ua haujlwm siab ua kua chromatography hauv kev tsim tshuaj.J.Chromatography.1119(1-2), 140-146.https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al.Monolithic macroporous hydrogels npaj los ntawm roj-hauv-dej siab sab hauv theem emulsions kom huv si ntawm enteroviruses.Chemical.Britain.J.401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Lub luag haujlwm ntawm kua chromatography hauv proteomics.J.Chromatography.A 1053(1-2), 27-36 (2004).
Fekete, S., Veuthey, J.-L.&Guillarme, D. Cov kev tshwm sim nyob rau hauv reversed-theem kua chromatography sib cais ntawm kev kho peptides thiab proteins: txoj kev xav thiab kev siv.J.Pharmacy.Biomedical Science.anus.69, 9-27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Ob-dimensional sib cais ntawm peptides siv RP-RP-HPLC system siv cov pH sib txawv nyob rau hauv thawj thiab thib ob sib cais qhov ntev.J.Sep. Sci.28(14), 1694-1703 (2005).
Feletti, S. et al.Qhov loj hloov cov yam ntxwv thiab kinetic kev ua tau zoo ntawm high-efficiency chromatographic txhua packed nrog C18 sub-2 μm tag nrho thiab superficially ntxeem tau hais raug soj ntsuam.J.Sep. Sci.43 (9-10), 1737-1745 (2020).
Piovesana, S. et al. Cov ncauj lus tsis ntev los no thiab cov teeb meem kev tshuaj ntsuam xyuas hauv kev sib cais, kev txheeb xyuas thiab kev siv tau ntawm cov nroj tsuag bioactive peptides.anus.biological anus.Chemical.410(15), 3425–3444.https://doi.org/10.1007/s0018x16-10.
Mueller, JB et al.The proteomic toj roob hauv pes ntawm lub nceeg vaj ntawm lub neej.Nature 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).
Yang, Y. & Geng, X. Mixed-mode chromatography thiab nws daim ntawv thov rau biopolymers.J.Chromatography.A 1218(49), 8813–8825 (2011).
Zhao, G., Dong, X.Biotechnology.144(1), 3-11 (2009).