Ndatenda nekushanyira Nature.com. Uri kushandisa shanduro yebrowser ine tsigiro shoma yeCSS. Kuti uwane ruzivo rwakanyanya, tinokurudzira kuti ushandise browser yakagadziridzwa (kana kudzima Compatibility Mode muInternet Explorer). Pamusoro pezvo, kuti tive nechokwadi chekutsigira kunoenderera, tinoratidza saiti isina masitaera uye JavaScript.
Inoratidza carousel yemasiraidhi matatu panguva imwe chete. Shandisa mabhatani ePashure neAnotevera kufamba nemasiraidhi matatu panguva, kana kushandisa mabhatani anotsveyamisa ari kumagumo kufamba nemasiraidhi matatu panguva.
Porous silica particles yakagadzirwa ne sol-gel nzira ine mamwe magadzirirwo kuti awane yakafara-pore particles. Zvimedu izvi zvakatorwa neN-phenylmaleimide-methylvinyl isocyanate (PMI) uye styrene kuburikidza nereverse chain transfer-fragmentation (RAFT) polymerization kuburitsa N-phenylmaleimide intercalated polyamides. Styrene (PMP) yakamira chikamu. Narrow yakabereka Stainless mbiru mbiru (100 × 1.8 mm mukati dhayamita) akanga akazara pamwe slurry packing. Iko chromatographic performance yePMP column yakaongororwa kuti iparadzanise musanganiswa wemapeptide ekugadzira anosanganisira mashanu peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) uye tryptic humanS hydrolyzan album. Pasi pemamiriro ekunze akanakisa, nhamba yetioretical yemaplate ane musanganiswa wepeptides yakasvika 280,000 plates/sq.m. Kuenzanisa kupatsanurwa kwekuita kwekoramu yakagadziridzwa neyekutengesa Ascentis Express RP-Amide koramu, yakaonekwa kuti kupatsanurwa kwekoramu yePMP kwaive kwakakwirira kune yekoramu yekutengeserana maererano nekuparadzanisa kugona uye kugadzirisa.
Indasitiri yebiopharmaceutical yave musika uri kuwedzera wepasirese nekuwedzera kukuru kwemugove wemusika mumakore achangopfuura. Nekukura kuri kuita biopharmaceutical industry1,2,3 pane kudiwa kukuru kwepeptide neprotein analysis. Pamusoro pechinangwa chepeptide, kusvibiswa kwakasiyana kunoumbwa panguva yepeptide synthesis, saka kucheneswa kwechromatographic kunodiwa kuti uwane kuchena kwaunoda kwepeptide. Ongororo uye hunhu hwemapuroteni mumvura yemuviri, matishu, uye maseru ibasa rakaoma zvakanyanya nekuda kwenhamba huru yezvipenyu zvingango onekwa zviripo mune imwe sampuli. Kunyangwe misa spectrometry iri chishandiso chinoshanda chekuteedzera peptides nemapuroteni, kana masampuli akadai akaunzwa zvakananga muhukuru spectrometer, kupatsanurwa kunenge kusingagutsi. Dambudziko iri rinogona kugadziriswa nekuita liquid chromatography (LC) pamberi peMS kuongororwa, izvo zvinoderedza huwandu hweanalytes inopinda muhukuru spectrometer panguva yakatarwa4,5,6. Pamusoro pezvo, vaongorori vanogona kutarisisa munzvimbo yakamanikana panguva yekuparadzaniswa kwechikamu chemvura, nekudaro vachiisa pfungwa idzi uye nekuwedzera kunzwisiswa kwekuonekwa kweMS. Liquid chromatography (LC) yakafambira mberi zvakanyanya mumakore gumi apfuura uye yave nzira inoshandiswa zvakanyanya yekuongorora kweproteomic7,8,9,10.
Reverse-phase liquid chromatography (RP-LC) inoshandiswa zvakanyanya kuchenesa uye kupatsanura musanganiswa wemapeptide uchishandisa octadecyl-modified silica (ODS) seyakamira phase11,12,13. Nekudaro, nekuda kwechimiro chavo chakaomarara uye chimiro cheamphoteric, 14,15 RP yakamira nhanho haigone kupa kupatsanurwa kunogutsa kwepeptides nemapuroteni. Naizvozvo, kuongororwa kwepeptides uye mapuroteni ane polar uye asiri-polar zvimedu zvinoda zvakanyatsogadzirirwa zvakamira zvikamu kuti zvibatane uye kuchengetedza izvi analytes16. Yakasanganiswa chromatography, iyo inopa multimodal kupindirana, inogona kuve imwe nzira kune RP-LC yekuparadzanisa peptides, mapuroteni, uye mamwe akaomarara musanganiswa. Zvikamu zvakasanganiswa-mhando dzakamira dzakagadzirwa uye makoramu akazadzwa neaya akamira akashandiswa kupatsanura peptides nemapuroteni17,18,19,20,21. Nekuda kwekuvapo kwemapoka epolar uye asiri-polar, yakasanganiswa mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) inokodzera kupatsanurwa kwepeptides nemaprotein22,23,24,25,26,27,28. , polar intercalated stationary phases ane covalently bonded polar groups anoratidza kugona kupatsanura kwakanaka uye kusarudzika kwakasiyana kwepolar uye non-polar analytes nekuti kupatsanurwa kunoenderana nekudyidzana pakati peanalyte uye yakamira chikamu Multimodal interactions 29,30,31,32. Munguva pfupi yapfuura, Zhang et al. 30 yakawana behenyl-yakaguma yakamira zvikamu zve polyamines uye yakabudirira kupatsanura mahydrocarbon, antidepressants, flavonoids, nucleosides, estrogens, uye mamwe ma analytes. Iyo polar yakamisikidzwa yakamira zvinhu ine ese mapoka epolar uye asiri-polar, saka inogona kushandiswa kupatsanura peptides nemapuroteni kuita hydrophobic uye hydrophilic zvikamu. Polar inline columns (semuenzaniso, C18 makoramu ane amide inline) anowanikwa pasi pezita rekutengesa Ascentis Express RP-Amide makoramu, asi makoramu aya akashandiswa chete kuongororwa kweamine 33.
Muchidzidzo chazvino, polar embedding stationary phase (N-phenylmaleimide, embedding polystyrene) yakagadzirirwa uye yakaongororwa kupatsanurwa kwepeptide uye tryptic HSA cleavage. Nzira inotevera yakashandiswa kugadzirira nhanho yakamira. Porous silica particles yakagadzirirwa maererano nemaitiro anotsanangurwa mumabhuku edu apfuura, pamwe nekumwe kuchinjwa kwezvirongwa zvekugadzirira 31, 34, 35, 36, 37, 38, 39. Izvo zviyero zve urea, polyethylene glycol (PEG), TMOS uye aqueous-acetic acid yakagadziridzwa kuti iwane mashizha makuru e-silica ane mashizha makuru. Kechipiri, itsva phenylmaleimide-methylvinyl isocyanate ligand yakagadzirwa uye yakabviswa silica particles yakashandiswa kugadzirira polar embedded stationary phases. Iyo yakawanikwa yakamira chikamu chakarongedzwa mukona yesimbi isina tsvina (yemukati dhayamita 100 × 1.8 mm) maererano neyakagadziridzwa kurongedza chirongwa. Kurongedzerwa kwekoramu kunobatsirwa ne mechanical vibration kuti ive nechokwadi cheunifomu layer mukati me column. Iyo column yakarongedzwa yakaongororwa kupatsanurwa kwemusanganiswa wemapeptides ane mashanu peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). uye tryptic hydrolysates yemunhu serum albumin (HSA). Zvakaonekwa kuti musanganiswa wepeptide uye HSA tryptic digest yakaparadzaniswa nekugadziriswa kwakanaka uye kushanda nesimba. Kupatsanurwa kwekoramu yePMP kwakaenzaniswa neiyo yeAscentis Express RP-Amide column. Yakaonekwa kuti peptides nemapuroteni ane kugadziriswa kwakanaka uye kubudirira kwepamusoro kwekuparadzanisa pamutsara wePMP, uye kupatsanurwa kwekoramu yePMP yakakwirira kudarika iyo yeAscentis Express RP-Amide column.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride, TPOxyrene, MC4, methacryloyl chloride, EM4 (BPO), acetonitrile (ACN) yeHPLC, methanol, 2-propanol uye acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Musanganiswa weurea (8 g), polyethylene glycol (8 g) uye 8 ml ye0.01 N. acetic acid yakamutswa kwemaminitsi gumi, uye 24 ml yeTMOS yakawedzerwa ipapo pasi pekutonhora kwechando. Musanganiswa wekuita wakapisa pa40 ° C kwemaawa matanhatu uye ipapo pa 120 ° C kwemaawa masere mune imwe simbi isina tsvina autoclave. Mvura yakasvibiswa uye yakasara yakaomeswa pa70 ° C kwemaawa gumi nemaviri. Mabhuroko akaomeswa akanyoroveswa akakuyiwa zvakanaka uye akabikwa muovheni pa550 ° C kwemaawa gumi nemaviri. Mabheji matatu akagadzirwa uye akaratidzirwa kuyedza kuberekana kwepadiki saizi, saizi yepore uye nzvimbo.
Polar boka uye yakamira chikamu che polystyrene cheni. Nzira yekugadzirira inotsanangurwa pasi apa.
N-phenylmaleimide (200 mg) uye methyl vinyl isocyanate (100 mg) yakanyungudutswa mu anhydrous toluene, uye ipapo 0.1 ml ye2,2'-azoisobutyronitrile (AIBN) yakawedzerwa kune reaction flask kuti iwane copolymer ye phenylmaleimide uye methylcyanate methylCP (PMM) isoso. ) Musanganiswa wacho wakapisa pa60 ° C kwemaawa matatu, yakasvibiswa uye yakaomeswa muchoto pa40 ° C kwemaawa matatu.
Yakaomeswa silica particles (2 g) yakaparadzirwa mu toluene yakaoma (100 ml), yakavhenganiswa uye sonicated kwemaminitsi gumi mu 500 ml yakapoteredza pasi flask. PMCP (10 mg) yakanyungudutswa mu toluene uye yakawedzera kudonha kune iyo reaction flask kuburikidza nekuwedzera funnel. Iko kusanganiswa kwakagadziriswa pa100 ° C kwemaawa masere, yakasvibiswa, yakashambidzwa neacetone uye yakaomeswa pa60 ° C kwemaawa matatu. Zvadaro, iyo silica particles yakabatana nePMCP (100 g) yakanyungudutswa mu toluene (200 ml), uye 4-hydroxy-TEMPO (2 ml) yakawedzerwa ipapo pamberi pe100 μl ye dibutyltin dilaurate sechinhu chinokonzera. Iko kusanganiswa kwakakonzerwa pa50 ° C kwemaawa masere, yakasvibiswa uye yakaomeswa pa50 ° C kwemaawa matatu.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) uye silica particles zvakabatanidzwa neTEMPO-PMCP (1.5 g) zvakaparadzirwa mu toluene uye zvakacheneswa nenitrogen. Iyo polymerization ye styrene yakaitwa pa100 ° C kwemaawa gumi nemaviri. Chigadzirwa chacho chakagezwa nemethanol ndokuomeswa usiku hwose pa60°C. The general scheme of the reaction inoratidzwa mufig. Poshi .
Iwo masampuli akacheneswa pa 393 K kwe1 h kusvikira yakasara yekumanikidza isingasviki 10-3 Torr yakawanikwa. Huwandu hweN2 adsorbed pakumanikidzwa kwehukama P/P0 = 0.99 yakashandiswa kuona iyo yakazara pore vhoriyamu. The morphology of pure and ligand-bound silica particles akaongororwa pachishandiswa scanning electron maikorosikopu (Hitachi High Technologies, Tokyo, Japan). Dry samples (pure silica uye ligand bound silica particles) yakaiswa paaluminium tsvimbo vachishandisa kabhoni tape. Goridhe yakaiswa pamuenzaniso uchishandisa Q150T sputtering mudziyo, uye 5 nm gobvu Au layer yakaiswa pane iyo sampuli. Izvi zvinovandudza kushanda kweiyo low voltage process uye inopa yakanaka inotonhora spraying. Elemental ongororo yakaitwa pachishandiswa Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Iyo Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 yakashandiswa kuwana iyo particle size kugovera. Uncoated silica particles uye ligand-bound silica particles (5 mg imwe neimwe) yakaparadzirwa mu 5 ml ye isopropanol, sonicated kwemaminitsi gumi, yakashungurudzwa kwemaminitsi mashanu, uye yakaiswa pa Mastersizer optical bench. Ongororo yeThermogravimetric inoitwa pamwero we5 °C paminiti mutembiricha kubva pa30 kusvika 800 °C.
Girazi fiber lined yakamanikana yakabereka Stainless simbi makoramu ane zviyero (ID 100 × 1.8 mm) yakanga yakazara nenzira yekuzadza slurry ichitevera nzira imwechete sereferensi 31. Stainless simbi mbiru (girazi lined, ID 100 × 1 .8 mm) uye chibudisi chine 1 µm fritfield fritter, All yakabatana ne tech fritter. IL, USA). Gadzirira kumiswa kwechikamu chakamira nekumisa 150 mg yechikamu chakamira mu 1.2 ml yemethanol uye kuidyisa muchikamu chemvura. Methanol yakashandiswa se slurry solvent uye control solvent. Rongedza koramu nekushandisa kutevedzana kwekumanikidza kwe100 MP kwemaminetsi gumi, 80 MP ye15 min, uye 60 MP ye30 min. Maitiro ekurongedza akashandisa maviri gasi chromatography column vibrators (Alltech, Deerfield, IL, USA) kune mechanic vibration kuti ive nechokwadi cheyunifomu kurongedza. Vhara iyo slurry packer uye kusunungura kumanikidza zvishoma nezvishoma kudzivirira kukanganisa kune tambo. Iyo column yakabviswa kubva kune slurry nozzle uye imwe yekukodzera yakanamirwa kune inopinza uye yakabatana neLC system kuyedza kushanda kwayo.
MLC yetsika yakagadzirwa uchishandisa LC pombi (10AD Shimadzu, Japan), sampler ine 50 nL jekiseni loop (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), uye UV-VIS capillary window. Detector mudziyo (UV-2075) uye enamelled microcolumn. Shandisa machubhu akamanikana uye mapfupi ekubatanidza kuderedza mhedzisiro yekuwedzera koramu yekuwedzera. Mushure mekuzadza koramu, isa capillary (50 µm id 365) panzvimbo yekubuda kwe1/16 ″ yekudzikisa junction uye isa capillary (50 µm) yekudzikisa junction. Kuunganidzwa kwedata uye kugadzirisa kwechromatogram kunoitwa uchishandisa Multichro 2000 software. Pa 254 nm, iyo UV absorbance yezvidzidzo analytes yakatariswa pa 0. Chromatographic data yakaongororwa uchishandisa OriginPro8 (Northampton, MA).
Human serum albumin, lyophilized powder, ≥ 96% (agarose gel electrophoresis) 3 mg yakasanganiswa netrypsin (1.5 mg), 4.0 M urea (1 ml) uye 0.2 M ammonium bicarbonate (1 ml) . Mushonga wacho wakakurudzirwa kwemaminitsi gumi uye wakachengetwa mumvura yekugeza pa 37 ° C kwe6 h, ndokudzima ne 1 ml ye 0.1% TFA. Sefa mushonga wochengeta pasi pe4°C.
Kupatsanurwa kwemusanganiswa wemapeptides uye tryptic digest HSA pane PMP column yakaongororwa zvakasiyana. Tarisa tryptic hydrolysis yemusanganiswa wemapeptides uye HSA yakapatsanurwa nePMP column uye enzanisa zvawanikwa neAscentis Express RP-Amide column. Nhamba ye theoretical plates inoverengerwa uchishandisa inotevera equation:
SEM mifananidzo ye pure silica particles uye ligand bound silica particles inoratidzwa muMufananidzo 2. SEM mifananidzo ye pure silica particles (A, B) inoratidza chimiro che spherical umo mazamu akareba kana kuti ane symmetry isina kukwana kana ichienzaniswa nezvidzidzo zvedu zvekare. Iyo yepamusoro ye silica particles yakasungwa ne ligand (C, D) yakapfava kupfuura iyo yakachena silica particles, iyo inogona kunge yakakonzerwa nemaketani epolystyrene akavhara pamusoro pemasikisi e silica.
Kuongorora erekitironi micrographs e pure silica particles (A, B) uye ligand yakasungwa silica particles (C, D).
Iyo particle size distribution ye pure silica particles uye ligand-bound silica particles inoratidzwa mumufananidzo 2. 3 (A). Volumetric particle size distribution curves yakaratidza kuti silica particle size yakawedzera mushure mekugadzirisa kemikari (Fig. 3A). Iyo silica particle size yekugovera data kubva kuchidzidzo chezvino uye chidzidzo chekare chinofananidzwa muTafura 1 (A). Iyo volumetric particle size d (0.5) yePMP yaive 3.36 µm, ichienzaniswa nekushambadzira (0.5) kukosha kwe3.05 µm muchidzidzo chedu chekare (polystyrene bonded silica particles)34. Nekuda kwekuchinja kwereshiyo yePEG, urea, TMOS uye acetic acid mumusanganiswa wekuita, kugoverwa kwehukuru hwechikamu chebatch iyi kwaive kudiki kana kuchienzaniswa nekudzidza kwedu kwekare. Saizi yechikamu chePMP yakakura zvishoma pane iyo yepolystyrene yakasungwa silica particle chikamu chatakadzidza kare. Izvi zvinoreva kuti kushanda kwepamusoro kwe silica particles ne styrene kwakaisa chete polystyrene layer (0.97 µm) pa silica pamusoro, nepo muchikamu chePMP ukobvu hwemazambiringa hwaive 1.38 µm.
Particle size distribution (A) uye pore size distribution (B) ye pure silica particles uye ligand bound silica particles.
Saizi yepore, vhoriyamu yepore, uye nzvimbo yepamusoro yesilica masisitimu anoshandiswa muchidzidzo ichi anoratidzwa muTafura 1 (B). Iyo PSD profiles ye pure silica particles uye ligand-yakasungwa silica particles inoratidzwa muFig. 3(B). Mhedzisiro yacho yakaenzana neyedu kudzidza34 yapfuura. Iyo pore saizi ye pure uye ligand-yakasungwa silica particles yaive 310 Å uye 241 Å, zvichiteerana, zvichiratidza kuti mushure mekushandurwa kwemakemikari, saizi yepore yakaderera ne69 Å, sezvakaratidzwa muTebhurari 1 (B), uye shanduko yekuchinja inoratidzwa muFig. (124 m2/g). Sezvinoratidzwa muTebhurari 1 (B), nzvimbo yepamusoro (m2 / g) ye silica particles mushure mekugadziriswa kwemakemikari zvakare yakaderera kubva 116 m2 / g kusvika 105 m2 / g.
Mhedzisiro yekuongororwa kwekutanga kweiyo stationary phase inounzwa muTafura. 2. Kabhoni yekabhoni yezvino inomira yechikamu ndeye 6.35%, iyo yakaderera kupfuura yekudzidza kwedu kwekare (silica particles inobatanidza ne polystyrene, 7.93% 35 uye 10.21%, maererano) 42. Kabhoni yekabhoni yezvino yakamira pasi pasi, sezvo mamwe mapolar ligands akadai phenylmaleimide methyl-POPC ane methyl-POPC akashandiswa. kuwedzera kune styrene mukugadzirira kweSP. Huremu muzana yenitrogen muchikamu chazvino chakamira i2.21% zvichienzaniswa ne0.1735 uye 0.85% muzvidzidzo zvakapfuura42. Izvi zvinoreva kuti ikozvino yakamira chikamu ine huremu hwepamusoro muzana hwenitrogen nekuda kwephenylmaleimide. Saizvozvo, zvigadzirwa (4) uye (5) zvine kabhoni ye2.7% uye 2.9%, zvichiteerana, nepo yekupedzisira chigadzirwa (6) ine kabhoni ye6.35%, sezvakaratidzwa muTable 2. Thermogravimetric analysis (TGA) yakashandiswa pane yakamira chikamu chePMP kuti iedze kurasikirwa nekurema, uye iyo TGA Figuve yehuremu inoratidzwa mu GA4 Figuve inoratidzwa. 8.6%, iyo inobvumirana zvakanaka nekabhoni yemukati (6.35%), sezvo ligands ine kwete C chete, asiwo N, O uye H.
Iyo ligand phenylmaleimide-methylvinyl isocyanate yakasarudzwa kuti ichinje pamusoro peiyo silica particles nekuda kwayo polar phenylmaleimide uye vinylisocyanate mapoka. Vinyl isocyanate mapoka anogona kuwedzera kuita ne styrene nekurarama radical polymerization. Chikonzero chechipiri kuisa boka rine kudyidzana kuri pakati nepakati neanalyte uye pasina simba re electrostatic interactions pakati pe analyte ne stationary phase, sezvo phenylmaleimide moiety isina virtual charge at normal pH. Iyo polarity yechikamu chakamira inogona kudzorwa neyakakwana huwandu hwe styrene uye yekuita nguva yemahara radical polymerization. Danho rekupedzisira rekuita (yemahara radical polymerization) yakakosha sezvo ichishandura polarity yechikamu chakamira. Elemental ongororo yakaitwa kuti itarise iyo kabhoni yemukati mune izvi zvakamira zvikamu. Izvo zvakacherechedzwa kuti kuwedzera huwandu hwe styrene uye nguva yekupindura inowedzera kabhoni yemukati meiyo yakamira chikamu uye zvinopesana. MaSPs akagadzirirwa neakasiyana mazinga e styrene ane akasiyana kabhoni mitoro. Saizvozvowo, idzi nhanho dzakamira dzakaiswa pambiru dzesimbi uye hunhu hwadzo hwechromatographic (selectivity, resolution, N value, etc.) dzakatariswa. Zvichienderana nezviyedzo izvi, chimiro chakagadziridzwa chegadziriro yePMP yakamira chikamu chakasarudzwa kuti chipe inodzorwa polarity uye kuchengetwa kwakanaka kweanoongorora.
Iyo PMP column yakaongororwawo kuti iongororwe misanganiswa mishanu yepeptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) vachishandisa kugona kwechikamu chenhare. 60/40 (v/v) ACN/mvura (0.1% TFA) pamwero wekuyerera we80 µl/min. Pasi pemamiriro akakwana erution (200,000 plates/m), nhamba ye theoretical plates (N) per column (100 × 1.8 mm) i20,000 ± 100. MaN values ​​​​etatu PMP columns anoratidzwa muTafura 3 uye chromatograms inoratidzwa muFigure 5A. Kuongorora nekukurumidza pakuyerera kwepamusoro (700 µl/min) pachikamu chePMP, mapeptide mashanu akaburitswa mukati meminiti imwe, yakanakisa N kukosha kwe13,500 ± 330 pakoramu (100 x 1.8 mm dhayamita), yakaenzana ne135,000 mahwendefa/ m (Fig. 5B). Makoramu matatu ehukuru hwakafanana (yemukati dhayamita 100 x 1.8 mm) akazadzwa nemabhechi matatu akasiyana ePMP yakamira chikamu chekuyedza kuberekazve. Vaongorori vakarekodhwa pakoramu yega yega nekupatsanura musanganiswa wakafanana webvunzo pakoramu yega yega uchishandisa akanakisa elution mamiriro, nhamba yetioretical mahwendefa N, uye nguva yekuchengeta. Iyo data reproducibility yemakoramu ePMP inoratidzwa muTafura 4. Kugadzirwazve kwekoramu yePMP kwakabatana zvakanaka nekushomeka kwe% RSD kukosha sezvakaratidzwa muTable 3.
Kuparadzaniswa kwepeptide musanganiswa paPMP column (B) uye Ascentis Express RP-Amide column (A), mobile phase 60/40 ACN/H2O (TFA 0.1%), PMP column dimensions (100 x 1.8 mm id) , ongororo Elution order yemakomponi: 1 (Gly-Tyr-Leu), 2 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) uye 5 (leucic acid enkephalin).
A PMP column (yemukati dhayamita 100 x 1.8 mm) yakaongororwa kupatsanurwa kwe tryptic hydrolyzate ye human serum albumin neHPLC. Iyo chromatogram muMufananidzo 6 inoratidza kuti masampuli akapatsanurwa zvakanaka nekugadziriswa kwakanaka kwazvo. HSA mhinduro dzakaongororwa pachishandiswa chiyero chekuyerera che100 μl/min, nhanho nhanho ye70/30 acetonitrile/mvura uye 0.1% TFA. Cleavage yeHSA yakakamurwa kuva 17 peaks, sezvakaratidzwa muchromatogram (Fig. 6), inoenderana ne17 peptides. Kuparadzaniswa kwekuita kwepamusoro pepamusoro kubva kuHSA hydrolyzate kwakaverengerwa uye kukosha kunoratidzwa muTafura 5.
HSA tryptic hydrolysates yakapatsanurwa pane PMP column (yemukati dhayamita 100 x 1.8 mm), kuyerera (100 μl/min), mobile phase 60/40 acetonitrile/mvura, uye 0.1% TFA.
apo L ndiyo kureba kwekoramu, η ndiyo viscosity yechikamu chefoni, ΔP ndiyo inodzvinyirira shure kwekoramu, uye u ndiyo mutsara wevheji yechikamu chefoni. Kupinda kwePMP column kwaiva 2.5 × 10-14 m2, kuyerera kwe25 µl / min, 60 / 40 v / v yakashandiswa. ACN/mvura. Kupinda kwePMP column (ID 100 × 1.8 mm) yakanga yakafanana neyeyedu yapfuura Ref.34 yekudzidza. Kupinda kwembiru yakazadzwa netumwe tunhu tudikidiki i1.7×10 .6 µm, 2.5×10-14 m2 ye5 µm zvidimbu43. Nokudaro, kupindira kwechikamu chePMP kwakafanana nekukwanisa kwepakati-shell particles nehukuru hwe5 μm.
uko Wx ndiko kuwanda kwekoramu yakazadzwa nechloroform, Wy ndiko kuwanda kwekoramu yakazadzwa nemethanol, uye ρ ndiko kuwanda kwezvinonyungudutsa. Kuwanda kwemethanol (ρ = 0.7866) uye chloroform (ρ = 1.484). Iyo yakazara porosity ye silica-C18 particle column (100 × 1.8 mm ID) 34 uye yedu yakambodzidza C18-urea31 column yaiva 0.63 uye 0.55, maererano. Izvi zvinoreva kuti kuvapo kweurea ligands kunoderedza kupera kwechikamu chekumira. Kune rimwe divi, iyo yakazara porosity yePMP column (yemukati dhayamita 100 × 1.8 mm) ndeye 0.60. Makoramu ePMP haapindike kupfuura makoramu akatakurwa neC18 yakasungwa silica particles nekuti muC18 mhando yakamira nhanho maC18 ligands anonamirwa kune silica particles mumaketani akatsetseka, nepo mumhando yepolystyrene yakamira nhanho polima yakakora inoumbwa yakatenderedza zvimedu. layer A. Muchiyedzo chakajairika, column porosity inoverengerwa sezvizvi:
Pamusoro pemuonde. 7A, B inoratidza Van Deemter mapurani echikamu chePMP (id 100 x 1.8 mm) uye Ascentis Express RP-Amide mbiru (id 100 x 1.8 mm) pasi pemamiriro akafanana eelution, 60/40 ACN/H2O uye 0 .1% TFA 20 µl/min pa 8/0 min kusvika 8/min. Iwo mashoma HETP makoshero payakanyanya kuyerera mwero (80 µl/min) aive 2.6 µm uye 3.9 µm yePMP column uye Ascentis Express RP-Amide column, zvichiteerana. Huwandu hweHETP hunoratidza kuti kupatsanurwa kwekoramu yePMP (100 x 1.8 mm id) yakakwira zvakanyanya kupfuura iyo inotengeswa Ascentis Express RP-Amide column (100 x 1.8 mm id). Iyo van Deemter graph muFig. 7 (A) inoratidza kuti kuderera kweN kukosha hakusi kwakakwirira zvikuru nekuwedzera kuyerera kuchienzaniswa nekudzidza kwedu kwekare. Iyo yepamusoro yekuparadzanisa kugona kweiyo PMP column (id 100 × 1.8 mm) ichienzaniswa neiyo Ascentis Express RP-Amide koramu yakavakirwa pane yakagadziridzwa chidimbu chimiro uye saizi uye yakaomesesa koramu yekurongedza nzira inoshandiswa mubasa razvino34.
(A) Van Deemter plot (HETP vs. mobile phase linear velocity) inowanikwa paPMP column (id 100 x 1.8 mm) mu 60/40 ACN/H2O ne 0.1% TFA. (B) Van Deemter plot (HETP versus mobile phase linear velocity) inowanikwa paAscentis Express RP-Amide column (id 100 x 1.8 mm) mu 60/40 ACN/H2O ine 0.1% TFA.
A polar stationary phase ye intercalated polystyrene yakagadzirwa uye yakaongororwa kupatsanurwa kwemusanganiswa wesynthetic peptides uye tryptic hydrolyzate ye human serum albumin (HSA) mune yakakwira performance liquid chromatography. Iko chromatographic kuita kwePMP makoramu emusanganiswa wepeptide yakanakisa maererano nekuparadzanisa kugona uye kugadzirisa. Iyo yakagadziridzwa yekuparadzanisa kugona kwePMP makoramu imhaka yezvikonzero zvakati wandei senge silica particle saizi uye pore saizi, inodzorwa synthesis yematanho akamira, uye yakaoma makoramu kurongedza zvinhu. Pamusoro peiyo yepamusoro yekuparadzanisa kugona, imwe mukana weiyi yakamira chikamu ndeye yakaderera mbiru kumashure kudzvanywa pamazinga ekuyerera. PMP mbiru dzinogadzirwa zvakanyanya uye dzinogona kushandiswa kuongorora musanganiswa wepeptides uye tryptic digestion yemapuroteni akasiyana. Isu tinoda kushandisa koramu iyi kupatsanurwa kweiyo bioactive komisheni kubva kune zvakasikwa zvigadzirwa, zvakatorwa zvemiti yemishonga uye howa mune yemvura chromatography. Mune ramangwana, makoramu ePMP achaongororwawo kuparadzaniswa kweprotein uye monoclonal antibodies.
Munda, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Kuongorora mune yakadzoserwa chikamu chromatography peptide kupatsanurwa masisitimu chikamu I: Kugadzirwa kweprotocol yekolamu maitiro. Munda, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Kuongorora mune reversed phase chromatography peptide separation systems chikamu I: Kugadzirwa kweprotocol yekolamu characterization.Munda, JK, Owerby, MR, Lau, J., Togersen, H., uye Petersson, P. Kuongorora kwePeptide Separation Systems neReverse-Phase Chromatography, Chikamu I: Kugadzira Protocol yeColumn Characterization. Munda, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Kuongorora mune reversed phase chromatography peptide separation systems chikamu I: Kugadzirwa kweprotocol yezvikamu zvekoramu. Munda, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Kuongorora mune reversed phase chromatography peptide separation systems chikamu I: Kugadzirwa kweprotocol yezvikamu zvekoramu.Munda, JK, Owerby, MR, Lau, J., Togersen, H., uye Petersson, P. Kuongorora kwePeptide Separation Systems neReverse-Phase Chromatography, Chikamu I: Kugadzira Protocol yeColumn Characterization.J.色谱法。 1603，113-129。 https://doi.org/10.1016/j.chroma.2019.05.038(2019).
Gomez, B. nevamwe. Nzira dzekugadzira dzakavandudzwa dzinoshanda peptides pakurapa kwezvirwere zvinotapukira. Biotechnology. Zvakabudirira 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: Sayenzi nemusika. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeutic peptides: Sayenzi nemusika.Vliege P, Lisowski V, Martinez J uye Chreschatyski M. Synthetic therapeutic peptides: sainzi uye musika.Vliege P, Lisowski V, Martinez J uye Khreschatsky M. Synthetic therapeutic peptides: sainzi uye musika. kuwanikwa kwezvinodhaka. Nhasi 15 (1–2), 40–56. https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Advanced proteomic liquid chromatography. Xie, F., Smith, RD & Shen, Y. Advanced proteomic liquid chromatography.Ona F., Smith RD naShen Yu. Yepamberi yeproteomic liquid chromatography. Xie, F., Smith, RD & Shen, Y. 高级蛋白质组液相色谱. Xie, F., Smith, RD & Shen, Y. Advanced protein composition 液相色谱.Ona F., Smith RD naShen Yu. Yepamberi yeproteomic liquid chromatography.J. Chromatography. A 1261, 78–90 (2012).
Liu, W. nevamwe. Yepamberi liquid chromatography-mass spectrometry inokwanisa kusanganisa yakafara-based metabolomics uye proteomics. anus. Chim. Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Basa re UHPLC mukusimudzira mishonga. Chesnut, SM & Salisbury, JJ Basa re UHPLC mukusimudzira mishonga.Chesnut, SM uye Salisbury, JJ Basa re UHPLC mukusimudzira mishonga.Chesnut, SM uye Salisbury, JJ Basa re UHPLC mukuvandudza zvinodhaka. J. Sept Sayenzi. 30(8), 1183–1190 (2007).
Wu, N. & Clausen, AM Yakakosha uye inoshanda maficha eiyo Ultrahigh pressure fluid chromatography yekuparadzanisa nekukurumidza. Wu, N. & Clausen, AM Yakakosha uye inoshanda maficha eiyo Ultrahigh pressure fluid chromatography yekuparadzanisa nekukurumidza.Wu, N. naClausen, AM Yakakosha uye inoshanda maficha eiyo high pressure liquid chromatography yekuparadzana nekukurumidza. Wu, N. & Clausen, AM 用于快速分离的超高压液相色谱的基础和实践方面. Wu, N. & Clausen, AM Basic uye inoshanda maficha e-ultra-high pressure liquid chromatography yekuparadzana nekukurumidza.Wu, N. naClausen, AM Yakakosha uye inoshanda maficha eiyo high pressure liquid chromatography yekuparadzana nekukurumidza.J. Sept. Sainzi. 30(8), 1167–1182. https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitchef, P. Kushandiswa kwe-ultra-performance liquid chromatography mukugadzirwa kwemishonga. Wren, SA & Tchelitchef, P. Kushandiswa kwe-ultra-performance liquid chromatography mukugadzirwa kwemishonga.Ren, SA uye Chelischeff, P. Kushandiswa kwe Ultra high performance liquid chromatography mukugadzirwa kwemishonga. Wren, SA & Tchelitchef, P. 超高效液相色谱在药物开发中的应用。 Wren, SA & Tchelitchef, P.Ren, SA uye Chelischeff, P. Kushandiswa kwe-ultra-performance liquid chromatography mukugadzirwa kwezvinodhaka.J. Chromatography. 1119(1-2), 140-146. https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al. A monolithic macroporous hydrogel yakabva kune emulsion yemafuta-mu-mvura ine chikamu chepamusoro chemukati chekuchenesa kwakanaka kwe enterovirus 71. Kemikari. project. Journal 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Basa remvura chromatography muproteomics. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Basa remvura chromatography muproteomics.Shi, Y., Xiang, R., Horvath, C. uye Wilkins, JA Basa remvura chromatography muproteomics. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA 液相色谱在蛋白质组学中的作用. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JAShi, Y., Xiang, R., Horvath, C. uye Wilkins, JA Basa remvura chromatography muproteomics.J. Chromatography. A 1053 (1-2), 27-36 (2004).
Fekete, S., Vutey, J.-L. & Guillarme, D. Maitiro matsva mune yakadzoserwa-chikamu chemvura chromatographic kupatsanurwa kwekurapa peptides uye mapuroteni: Theory uye mashandisirwo. & Guillarme, D. Maitiro matsva mune yakadzoserwa-chikamu chemvura chromatographic kupatsanurwa kwekurapa peptides uye mapuroteni: Theory uye mashandisirwo. & Guillarme, D. Новые тенденции в разделении терапевтических пептидов и белков с помощью жидкостной хроматографии в разделении терапевтических пептидов и белков с помощью жидкостной хроматографии с обратевтических пептидов. & Guillarme, D. Maitiro matsva mukuparadzaniswa kwepeptides yekurapa uye mapuroteni nereverse phase liquid chromatography: dzidziso uye mashandisirwo. & Guillarme, D. 治疗性肽和蛋白质的反相液相色谱分离的新趋势：理论和应用. uye Guillarme, D.uye Guillarmé, D. Maitiro matsva mukuparadzaniswa kwepeptides yekurapa uye mapuroteni nereverse phase liquid chromatography: dzidziso uye mashandisirwo.J. Pharm. Biomedical Science. anus. 69, 9–27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Maviri-dimensional kupatsanurwa kwepeptides achishandisa RP-RP-HPLC system ine pH yakasiyana mukutanga uye yechipiri kupatsanurwa zviyero. Gilar, M., Olivova, P., Daly, AE & Gebler, JC Maviri-dimensional kupatsanurwa kwepeptides achishandisa RP-RP-HPLC system ine pH yakasiyana mukutanga uye yechipiri kupatsanurwa zviyero.Gilar M., Olivova P., Dali AE uye Gebler JK Maviri-dimensional kupatsanurwa kwepeptides vachishandisa RP-RP-HPLC system ine pH yakasiyana mukutanga uye yechipiri kupatsanura zviyero.Gilar M., Olivova P., Dali AE uye Gebler JK Maviri-dimensional kupatsanurwa kwepeptides uchishandisa akasiyana pH kukosha mune yekutanga neyechipiri kupatsanurwa zviyero uchishandisa iyo RP-RP-HPLC system. J. Sept Sayenzi. 28 (14), 1694–1703 (2005).
Fellitti, S. et al. Kuongorora kwehuwandu hwekufambisa uye kinetic hunhu hwepamusoro-inoshanda chromatography makoramu akazara neakazara porous uye pamusoro pepamusoro porous C18 zvimedu zvidiki pane 2 µm. J. Sept Sayenzi. 43 (9–10), 1737–1745 (2020).
Piovesana, S. et al. Zvichangoburwa maitiro uye matambudziko ekuongorora mukuzviparadzanisa nevamwe, kuzivikanwa, uye kusimbiswa kwechirimwa bioactive peptides. anus. Chisikwa anal. Chemical. 410(15), 3425-3444. https://doi.org/10.1007/s00216-018-0852-x (2018).
Muller, JB nevamwe. Proteomic landscape yehumambo hwehupenyu. Zvisikwa 582 (7813), 592–596. https://doi.org/10.1038/s41586-020-2402-x (2020).
De Luca, K. nevamwe. Kurapa-kurapwa kwepeptides yekurapa nekugadzirira mvura chromatography. Mamorekuru (Basel, Switzerland) 26(15), 4688 (2021).
Yang, Y. & Geng, X. Mixed-mode chromatography uye mashandisirwo ayo kune biopolymers. Yang, Y. & Geng, X. Mixed-mode chromatography uye mashandisirwo ayo kune biopolymers.Yang, Yu. uye Geng, X. Mixed mode chromatography uye kushandiswa kwayo kune biopolymers. Yang, Y. & Geng, X. 混合模式色谱及其在生物聚合物中的应用. Yang, Y. & Geng, X. Mixed mode chromatography uye kushandiswa kwayo mune biopolymers.Yang, Yu. uye Gene, X. Mixed mode chromatography uye kushandiswa kwayo kune biopolymers.J. Chromatography. A 1218(49), 8813–8825 (2011).