Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv kev txhawb nqa rau CSS.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis kaw qhov kev sib raug zoo hauv Internet Explorer).
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 faib los ntawm kev rov qab sib txuas ntxiv fragmentation saw hloov (RAFT) polymerization nrog N-phenylmaleimide-methylvinylisocyanate (PMI) thiab styrene los npaj N-phenylmaleimide chaw nres tsheb (PMI) inter-phenylmaleimide. Phase.Narrow-bore stainless hlau kem (100 × 1.8 mm id) tau ntim los ntawm slurry packing.Kev ntsuam xyuas PMP kem sib cais ntawm cov peptide sib tov uas muaj tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Argly-Service) trypsin digestion ntawm tib neeg serum albumin (HAS).Raws li qhov zoo tshaj plaws 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 rau kev lag luam Ascentis Express RP-Amide kem, nws tau pom tias kev sib cais kev ua tau zoo ntawm cov kab ke kev lag luam thiab kev daws teeb meem ntawm PMP kem yog superior.
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 ntau ntxiv. Nrog rau kev loj hlob ntawm kev lag luam biopharmaceutical1,2,3, kev tsom xam ntawm peptides thiab cov proteins yog qhov xav tau heev.Ntxiv rau lub hom phiaj peptide, ob peb impurities yog generated thaum peptide synthesis, yog li kev xav tau ntawm peptides. purity.Qhov kev tsom xam thiab cov yam ntxwv 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 ntau 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 hauv ib qho dhau los, qhov kev sib cais yuav tsis yog qhov teeb meem zoo tshaj plaws (LC) Kev sib cais ua ntej MS tsom xam, uas yuav txo tus naj npawb ntawm cov analytes nkag mus rau hauv huab hwm coj spectrometer ntawm lub sijhawm 4,5,6.Ntxiv rau, thaum lub sijhawm ua kua theem sib cais, cov analytes tuaj yeem tsom mus rau hauv cov cheeb tsam nqaim, yog li kev tsom rau cov analytes thiab txhim kho MS nrhiav kom pom rhiab heev.Liquid chromatography (LC) tau nrov heev hauv cov txheej txheem dhau los. 7, 8, 9, 10.
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 qhov chaw nyob ruaj khov theem 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 tshwj xeeb 1, 12, 13. tsim nyob ruaj khov theem yuav tsum tau soj ntsuam peptides thiab cov proteins nrog 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 kev sib xyaw ua ke. Ob peb cov kab sib xyaw ua ke tau npaj cov theem thiab cov txheej txheem sib xyaw ua ke. peptide thiab protein sib cais17,18,19,20,21.Mixed-mode nyob ruaj ruaj theem (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) yog haum rau peptide thiab protein sib cais vim muaj cov polar thiab non-polar pawg 22,23,24,25,26,27 polar, covalently bonded polar pawg qhia zoo sib cais lub zog thiab tshwj xeeb xaiv rau polar thiab non-polar analytes, raws li kev sib cais nyob ntawm kev sib cuam tshuam ntawm cov tshuaj ntsuam xyuas thiab nyob ruaj khov 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 cais hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, thiab ob peb lwm yam analytes.Lub polar intercalator muaj ob pawg polar thiab tsis-polar, yog li nws tuaj yeem siv los cais cov peptides thiab cov proteins uas muaj ob qho tib si hydrophobic thiab hydrophilic kem. amide-embedded C18 kab) yog muag nyob rau hauv lub 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 cov tshuaj polyethylene. (PEG), TMOS, dej acetic acid tau hloov kho los npaj silica hais nrog qhov pore loj.Second, ib lub ligand tshiab, phenylmaleimide-methyl vinyl isocyanate, tau ua ke thiab siv los derivatize silica particles los npaj ib lub polar embedded nyob ruaj ruaj theem .Qhov tshwm sim rau hauv ib lub hli 10 stainless hlau packed. optimized packing scheme.Cov 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.Qhov kev sib cais ntawm PMP kem kev ua tau zoo. kem.Ob lub peptides thiab cov proteins tau pom tias yuav daws tau zoo thiab ua haujlwm tau zoo ntawm PMP kem, uas tau ua haujlwm 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 (MC4TEH), Steryroxy, (BPO), HPLC Qib 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 xyaw yog rhuab ntawm 40 ° C rau 6 teev thiab tom qab ntawd ntawm 120 ° C rau cov hlau tsis muaj dej thiab 8 teev. Cov khoom seem tau qhuav ntawm 70 ° C rau 12 xuab moos.Cov khoom muag muag qhuav yog cov av du hauv qhov cub thiab calcined ntawm 550 ° C rau 12 xuab moos.Peb cov khoom tau npaj thiab ua kom zoo los tshuaj xyuas cov khoom rov ua dua ntawm 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 (CPPM) 3. teev, lim thiab 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 rov funnel.Cov sib tov yog refluxed ntawm 180 ° C, lim dej thiab qhuav. 60 ° C rau 3 teev.Tom qab ntawd, PMCP-bonded silica hais (100 g) tau yaj hauv toluene (200 ml) thiab 4-hydroxy-TEMPO (2 mL) tau 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 ° C.
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 methanol thiab qhuav ntawm 60 ° C ntawm cov tshuaj tiv thaiv. Daim duab 1.
Cov qauv no tau degassed ntawm 393 K rau 1 teev kom tau txais qhov seem ntawm qhov siab ntawm tsawg dua 10-3 Torr.Tus nqi ntawm N2 adsorbed ntawm tus txheeb ze siab ntawm P / P0 = 0.99 yog siv los txiav txim siab tag nrho cov pore ntim.Lub morphology ntawm liab qab thiab ligand-bonded silica particles Tokyo electron soj ntsuam, Technologies, scans. Nyiv).Cov qauv qhuav (dawb 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 ntawm 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 nplua nuj, txias MA Wrapping E.A. EA1112 elemental analyzer tau siv rau kev tshuaj ntsuam elemental.A Malvern (Worcestershire, UK) Mastersizer 2000 particle loj 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 mining isopropanol, 10 exfoliate, thiab lwm yam. lub rooj ntev zaum kho qhov muag ntawm Mastersizer.Thermogravimetric tsom xam tau ua 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 lub slurry packer (Alltech Deerfield, IL, USA).Npaj theem nyob ruaj khov slurry los ntawm kev ncua 150 mg ntawm cov theem nyob ruaj khov hauv 1.2 mL ntawm cov khoom xa tuaj. Cov kuab tshuaj slurry zoo li cov kuab tshuaj propelling.Ua kem 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.Lub sijhawm ntim khoom, cov neeg kho tshuab kev co tau thov nrog ob lub GC kem shakers (Alltech, Deerfield, IL, USA) kom ntseeg tau tias cov khoom sib xws maj mam tiv thaiv kom tsis txhob muaj kev puas tsuaj nyob rau hauv kem.Disconnect kem los ntawm slurry packing unit thiab txuas lwm haum rau lub inlet thiab mus rau lub LC system los xyuas nws cov kev ua tau zoo.
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. broadning.Tom qab ntim, capillaries (50 μm id 365 thiab txo cov 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 Organic Analytes tau raug soj ntsuam los ntawm cov ntaub ntawv pov thawj UV. (Northampton, MA).
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 dej da dej ntawm 37 ° C, tom qab ntawd 1 mque ntawm 37 ° C. TFA. Lim 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 cov khoom uas yuav ua rau polystyrene particles. nto ntawm silica hais.
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).Cov ntaub ntawv particle loj faib cov ntaub ntawv ntawm cov silica particles los ntawm cov ntaub ntawv tam sim no piv rau cov ntaub ntawv 1. d (0.5), ntawm PMP yog 3.36 μm, piv rau peb txoj kev tshawb fawb 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 faib piv rau peb txoj kev kawm dhau los vim qhov sib txawv ntawm PEG, urea, ticarticle particles ntawm cov tshuaj tiv thaiv me ntsis. Qhov loj dua li ntawm polystyrene-bound silica particle theem peb tau kawm yav dhau los.Qhov no txhais tau hais tias qhov chaw ua haujlwm ntawm silica hais nrog styrene tsuas yog tso ib txheej polystyrene (0.97 µm) ntawm silica nto, whereas nyob rau hauv 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 cov silica hais ntawm txoj kev tshawb fawb tam sim no tau muab nyob 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 yog 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 pom nyob rau hauv daim duab 3(B). Raws li, uas qhia tau hais tias qhov pore loj txo los ntawm 69 tom qab tshuaj hloov kho, raws li qhia nyob rau hauv Table 1(B), thiab qhov kev hloov ntawm lub nkhaus yog qhia nyob rau hauv daim duab 3(B) .Ib yam li ntawd, lub pore ntim ntawm cov silica particles txo los ntawm 0.67 mus rau 0.58 cm3 / g tom qab tshuaj kho qhov chaw ntawm silica 1. m2 / g, uas yog piv rau peb txoj kev kawm yav dhau los (124 m2 / g).Raws li pom nyob rau hauv Table 1 (B), thaj tsam ntawm cov silica particles kuj txo los ntawm 116 m2 / g mus rau 105 m2 / g tom qab hloov tshuaj.
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 kawm yav dhau los (polystyrene bonded silica particles, 7.93% 35 thiab 10.21%, feem) 42. Vim tias cov pa roj carbon loading ntawm qhov tam sim no nyob ruaj ruaj theem ntawm kev npaj yog tsawg, SP. Qee qhov polar ligands xws li phenylmaleimide-methylvinylisocyanate (PCMP) thiab 4-hydroxy-TEMPO tau siv.Qhov hnyav nitrogen 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. phenylmaleimide.Ib yam li ntawd, cov pa roj carbon loading ntawm cov khoom (4) 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 poob phaus tau soj ntsuam nrog PMP nyob ruaj ruaj theem, thiab TGA nkhaus yog qhia nyob rau hauv TGA qhov hnyav ntawm 4.6% kev pom zoo. nrog cov pa roj carbon loading (6.35%) vim tias cov ligands tsis tsuas yog C tab sis kuj N, O, thiab 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 kev cuam tshuam nruab nrab ntawm qhov chaw nres tsheb electrostatic tsis muaj kev cuam tshuam nrog cov theem ntawm analytes thiab kev sib cuam tshuam. phenylmaleimide moiety tsis muaj nqi virtual ntawm ib txwm pH.Qhov polarity ntawm lub sijhawm nyob ruaj khov tuaj yeem tswj tau los ntawm qhov zoo ntawm styrene thiab lub sijhawm tshuaj tiv thaiv ntawm cov 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 khov.Elemental tsom xam tau ua los xyuas cov pa roj carbon monoxide loading theem ntawm qhov chaw nres tsheb. Cov tshuaj tiv thaiv lub sij hawm nce cov pa roj carbon loading ntawm cov theem nyob ruaj khov thiab vice versa.SPs npaj nrog ntau ntau ntawm styrene muaj cov pa roj carbon sib txawv loadings.Thiab, thauj cov theem nyob ruaj khov rau hauv cov kab hlau stainless thiab xyuas lawv cov chromatographic kev ua tau zoo (xaiv, daws teeb meem, N tus nqi, thiab lwm yam).Raws li cov kev sim no, ib qho kev ua kom zoo tau raug xaiv los npaj PMP polaryte retentional theem kom zoo.
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, theoretical phaj tooj (N) ib kem (100 × 1.8 mm id) yog 20,000 ± 100 (200,000 T) tus nqi rau cov phaj. peb kab PMP thiab cov chromatograms tau pom nyob rau hauv daim duab 5A. Kev soj ntsuam 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 yog qhov zoo heev, 13,500 ± 330 ib kem (100 × 1.5mm / 0) phaj (Daim duab 5B).Peb cov kab uas zoo ib yam (100 × 1.8 mm id) tau ntim nrog peb ntau ntau ntawm PMP nyob ruaj ruaj theem los xyuas txog kev rov tsim dua tshiab.Cov tshuaj ntsuam xyuas rau txhua kem tau sau tseg siv qhov zoo tshaj plaws elution tej yam kev mob thiab tus naj npawb ntawm theoretical daim hlau N thiab tuav lub sij hawm kom cais cov tib cov ntaub ntawv reproduc sib tov rau txhua kem. 4.Qhov kev rov tsim dua tshiab ntawm PMP kem sib raug zoo nrog % RSD qhov tseem ceeb tsawg heev, 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)).
PMP kem (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 nyob rau hauv daim duab 6 qhia tau hais tias tus qauv yog zoo sib cais thiab cov kev daws teeb meem zoo heev.HSA digests tau soj ntsuam siv ib tug txaus tus nqi ntawm 100 µL 70 ton / min, mobile theem. 0.1% TFA.Raws li pom nyob rau hauv chromatogram (Daim duab 6), HSA digestion tau muab faib ua 17 peaks coj 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 kab ntev, η yog qhov viscosity ntawm lub sijhawm txawb, ΔP yog kab rov qab siab, thiab u yog cov kab nrawm nrawm 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 18 kem. mm id) zoo ib yam li peb txoj kev kawm yav dhau los Ref.34.Cov 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 × 10.5-15 m particles, 5.2 × 10.5-15 m thiab μm particles Rau 5 μm hais 43.Yog li ntawd, qhov permeability ntawm PMP theem zoo ib yam li cov 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 10 mm (10 mmol). thiab C18-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 PMP ntawm kem ntawm permeability. C18-bonded silica particles vim hais tias nyob rau hauv C18-hom nyob twj ywm theem cov C18 ligands txuas rau silica hais raws li cov kab chains, thaum nyob rau hauv polystyrene-hom nyob ruaj khov theem, ib tug 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 hauv 20 µL / Qhov tsawg kawg nkaus tus nqi rau ob kab ntawv yog 800 µL / min.Qhov tsawg kawg nkaus HETP tus nqi μL (L) 8. thiab Ascentis Express 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 ntawm PMP kem (100 × 1.8 mm id) zoo dua li cov khoom lag luam Ascentis Express RP-Amide kem (100 × 7). Qhov txo qis ntawm N tus nqi nrog nce ntws tsis tseem ceeb piv rau peb txoj kev tshawb fawb 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 raws li kev txhim kho hauv cov duab particle, qhov loj me, thiab cov txheej txheem txheej txheem ntim cov khoom 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 x 1.0-8 mm Express (RPA) 60/40 ACN / H2O nrog 0.1% TFA.
Lub polar-embedded polystyrene nyob ruaj khov theem tau npaj thiab tshuaj xyuas rau kev sib cais ntawm cov khoom sib xyaw peptide 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 cov kab ntau yam yog vim li cas, qhov loj me thiab qhov loj ntawm qhov sib txawv. silica particles, tswj synthesis ntawm cov theem nyob ruaj ruaj, thiab complex kem packing.Ntxiv rau kev sib cais siab efficiency, qis qis rov qab siab ntawm tus nqi ntws siab yog lwm qhov zoo ntawm no nyob ruaj ruaj theem.PMP kab qhia zoo reproducibility thiab yuav siv tau rau kev soj ntsuam ntawm peptide mixtures thiab trypsin digestion ntawm ntau yam proteins. Ua kua chromatography.Nyob rau yav tom ntej, PMP kab tseem yuav raug soj ntsuam rau kev sib cais ntawm cov proteins thiab monoclonal antibodies.
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. Cov yam ntxwv hloov pauv loj thiab kev ua tau zoo ntawm kev ua haujlwm siab chromatographic kab ntim nrog C18 sub-2 μm tag nrho thiab superficially ntxeem tau hais tau raug tshawb xyuas.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).