Mahalo no kou kipa ʻana iā Nature.com. Ke hoʻohana nei ʻoe i kahi polokalamu kele pūnaewele me ke kākoʻo CSS palena ʻole. No ka ʻike maikaʻi loa, manaʻo mākou e hoʻohana i kahi polokalamu kele pūnaewele hou (a i ʻole e hoʻopau i ke ʻano Compatibility Mode ma Internet Explorer). Eia kekahi, e hōʻoia i ke kākoʻo mau, hōʻike mākou i ka pūnaewele me ka ʻole o nā styles a me JavaScript.
Hōʻike i kahi carousel o ʻekolu paheʻe i ka manawa hoʻokahi. E hoʻohana i nā pihi Mua a me Next no ka neʻe ʻana i ʻekolu paheʻe i ka manawa, a i ʻole e hoʻohana i nā pihi paheʻe ma ka hopena e neʻe i ʻekolu mau paheʻe i ka manawa.
Ua hoʻomākaukau ʻia nā ʻāpana silica porous e ke ʻano sol-gel me kekahi mau hoʻololi e loaʻa ai nā ʻāpana pore ākea. Ua lawe ʻia kēia mau ʻāpana me ka N-phenylmaleimide-methylvinyl isocyanate (PMI) a me ka styrene ma o ka hoʻololi ʻana i ke kaulahao hoʻoili-fragmentation (RAFT) polymerization e hana i nā polyamides intercalated N-phenylmaleimide. ʻO Styrene (PMP) pae paʻa. Ua hoʻopiha ʻia nā kolamu kila kuhili ʻole (100 × 1.8 mm anawaena o loko) me kahi paʻi slurry. Ua loiloi ʻia ka hana chromatographic o ke kolamu PMP e hoʻokaʻawale i ka hui ʻana o nā peptides synthetic me ʻelima mau peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) a me ka tryptic hydrolyzate o ke kanaka serum (HASrum albumin). Ma lalo o nā kūlana elution maikaʻi loa, hiki i ka helu theoretical o nā papa me kahi hui o nā peptides i 280,000 mau papa / sq.m. Ke hoʻohālikelike nei i ka hana hoʻokaʻawale ʻana o ke kolamu i hoʻomohala ʻia me ka kolamu Ascentis Express RP-Amide kalepa, ua ʻike ʻia ʻoi aku ka maikaʻi o ka hoʻokaʻawale ʻana o ka kolamu PMP ma mua o ke kolamu kalepa ma ke ʻano o ka hoʻokaʻawale ʻana a me ka hoʻonā.
Ua lilo ka ʻoihana biopharmaceutical i mea hoʻonui i ka mākeke honua me ka piʻi nui o ka māhele mākeke i nā makahiki i hala. Me ka ulu pahū o ka ʻoihana biopharmaceutical1,2,3 aia kahi pono nui no ka peptide a me ka nānā ʻana i ka protein. Ma kahi o ka peptide i hoʻopaʻa ʻia, ua hoʻokumu ʻia nā ʻano haumia i ka wā o ka peptide synthesis, no laila pono ka hoʻomaʻemaʻe chromatographic e loaʻa ai ka maʻemaʻe i makemake ʻia o ka peptide. He hana paʻakikī loa ka nānā ʻana a me ke ʻano o nā protein i loko o nā wai kino, nā ʻiʻo, a me nā cell ma muli o ka nui o nā ʻano mea i ʻike ʻia i loko o kahi laʻana. ʻOiai ʻo ka nui o ka spectrometry he mea hana pono no ka hoʻokaʻina ʻana i nā peptides a me nā protein, inā e hoʻokomo pololei ʻia ia mau laʻana i loko o ka spectrometer mass, ʻaʻole ʻoluʻolu ka kaʻawale ʻana. Hiki ke hoʻopau ʻia kēia pilikia ma ka hana ʻana i ka chromatography wai (LC) ma mua o ka hoʻopaʻa ʻana o MS, kahi e hoʻemi ai i ka nui o nā analytes e komo ana i ka spectrometer mass i ka manawa i hāʻawi ʻia4,5,6. Eia hou, hiki i nā analytes ke noʻonoʻo i kahi ʻāpana liʻiliʻi i ka wā o ka hoʻokaʻawale ʻana o ka wai, no laila e hoʻopaʻa i kēia mau analytes a hoʻonui i ka naʻau o ka ʻike MS. Ua holomua nui ka chromatography wai (LC) i nā makahiki he ʻumi i hala a ua lilo i ʻano hana hoʻohana nui no ka nānā ʻana proteomic7,8,9,10.
Hoʻohana nui ʻia ʻo Reverse-phase liquid chromatography (RP-LC) e hoʻomaʻemaʻe a hoʻokaʻawale i nā hui ʻana o nā peptides me ka hoʻohana ʻana i ka silica i hoʻololi ʻia ʻo octadecyl (ODS) ma ke ʻano o ka manawa paʻa11,12,13. Eia nō naʻe, ma muli o ko lākou ʻano paʻakikī a me ke ʻano amphoteric, ʻaʻole hiki i ka 14,15 RP stationary phases ke hāʻawi i kahi kaʻawale kūpono o nā peptides a me nā protein. No laila, ʻo ka nānā ʻana o nā peptides a me nā protein me nā ʻāpana polar a me nā ʻāpana polar ʻole e koi i nā ʻāpana kū i hoʻolālā kūikawā e launa a mālama i kēia mau analytes16. ʻO ka chromatography huikau, e hāʻawi ana i nā pilina multimodal, hiki ke lilo i mea ʻokoʻa i ka RP-LC no ka hoʻokaʻawale ʻana i nā peptides, nā protein, a me nā hui paʻakikī ʻē aʻe. Ua hoʻomākaukau ʻia kekahi mau ʻano ʻano ʻano like ʻole a ua hoʻohana ʻia nā kolamu i hoʻopiha ʻia me kēia mau ʻano paʻa e hoʻokaʻawale i nā peptides a me nā protein17,18,19,20,21. Ma muli o ka loaʻa ʻana o nā pūʻulu polar a me nā polar ʻole, ua kūpono nā ʻano hoʻohui ʻia (WAX / RPLC, HILIC / RPLC, polar intercalation / RPLC) no ka hoʻokaʻawale ʻana i nā peptides a me nā protein22,23,24,25,26,27,28. , Polar intercalated stationary phases with covalently bonded polar groups show good separation capability and unique selectivity for polar and non-polar analytes no ka mea, e pili ana ka hoʻokaʻawale ʻana i ka pilina ma waena o ka analyte a me ka manawa paʻa Multimodal interactions 29,30,31,32. I kēia mau lā, ʻo Zhang et al. 30 i loaʻa i ka behenyl-terminated stationary phases o polyamines a hoʻokaʻawale maikaʻi i nā hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, a me kekahi mau analytes. Loaʻa nā pūʻulu polar a me nā pūʻulu ʻole i ka polar, no laila hiki ke hoʻohana ʻia e hoʻokaʻawale i nā peptides a me nā protein i loko o nā ʻāpana hydrophobic a me nā hydrophilic. Loaʻa nā kolamu polar inline (e laʻa, nā kolamu C18 me ka laina amide) ma lalo o ka inoa kālepa ʻo Ascentis Express RP-Amide kolamu, akā ua hoʻohana wale ʻia kēia mau kolamu no ka nānā ʻana i ka amine 33.
Ma ke aʻo ʻana i kēia manawa, ua hoʻomākaukau ʻia a loiloi ʻia kahi pae hoʻopili polar (N-phenylmaleimide, embedding polystyrene) no ka hoʻokaʻawale peptide a me ka hoʻokaʻawale ʻana o ka HSA tryptic. Ua hoʻohana ʻia ka hoʻolālā ma lalo no ka hoʻomākaukau ʻana i ka pae paʻa. Ua hoʻomākaukauʻia nā'āpana silica porous e like me nā kaʻina hana i ho'ākākaʻia i loko o kā mākou mau puke i hala aku nei, me kekahi mau hoʻololi i nā papa hana hoʻomākaukau 31, 34, 35, 36, 37, 38, 39. Ua hoʻololiʻia nā lakio o ka urea, polyethylene glycol (PEG), TMOS a me ka wai-acetic acid e loaʻa ai nā mea silica nui me ka nui pore. ʻO ka lua, ua hoʻohui ʻia kahi ligand phenylmaleimide-methylvinyl isocyanate hou a ua hoʻohana ʻia kāna mau mea silica derivatized no ka hoʻomākaukau ʻana i nā ʻāpana paʻa polar i hoʻokomo ʻia. Hoʻopili ʻia ka pae paʻa i loaʻa i loko o ke kolamu kila kila (ka anawaena o loko 100 × 1.8 mm) e like me ka hoʻolālā packing i hoʻopaʻa ʻia. Hoʻopili ʻia ka paʻi ʻana o ke kolamu e ka haʻalulu mechanical e hōʻoia i kahi ʻāpana like i loko o ke kolamu. Ua loiloi ʻia ke kolamu i hoʻopaʻa ʻia no ka hoʻokaʻawale ʻana o ka hui ʻana o nā peptides i ʻelima mau peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). a me ka tryptic hydrolysates o ke kanaka serum albumin (HSA). Ua ʻike ʻia ua hoʻokaʻawale ʻia ka hui peptide a me ka HSA tryptic digest me ka hoʻonā maikaʻi a me ka pono. Ua hoʻohālikelike ʻia ka hoʻokaʻawale ʻana o ke kolamu PMP me ka kolamu Ascentis Express RP-Amide. Uaʻikeʻia he hopena maikaʻi nā peptides a me nā protein a me ka hoʻokaʻawale kiʻekiʻe ma ka kolamu PMP, a ʻoi aku ka kiʻekiʻe o ka hoʻokaʻawale ʻana o ka kolamu PMP ma mua o ka kolamu Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, albumin serum kanaka (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (TEMP4-BP), styrene, styrene, acetonitrile (ACN) no HPLC, methanol, 2-propanol a me acetone. ʻO Sigma-Aldrich Company (St. Louis, Missouri, USA).
Ua hoʻoulu ʻia kahi hui ʻana o ka urea (8 g), polyethylene glycol (8 g) a me 8 ml o 0.01 N. acetic acid no 10 mau minuke, a ua hoʻohui ʻia he 24 ml o TMOS ma lalo o ka hoʻoluʻu hau. Ua hoʻomehana ʻia ka hui ʻana i ka 40 ° C no 6 mau hola a laila ma 120 ° C no 8 mau hola i loko o kahi autoclave kila kila. Hoʻopau ʻia ka wai a hoʻomaloʻo ʻia ke koena ma 70 ° C no 12 mau hola. Hoʻopili ʻia nā poloka palupalu maloʻo a hoʻopaʻa ʻia i loko o ka umu ma 550 ° C no 12 mau hola. ʻEkolu mau pūʻulu i hoʻomākaukau ʻia a hoʻohālikelike ʻia e hoʻāʻo i ka hana hou ʻana o ka nui o nā ʻāpana, ka nui o ka pore a me ka ʻili.
ʻO ka hui polar a me ka pae paʻa no nā kaulahao polystyrene. Hōʻike ʻia ke kaʻina hana hoʻomākaukau ma lalo nei.
N-phenylmaleimide (200 mg) a me methyl vinyl isocyanate (100 mg) ua hoʻoheheʻe ʻia i loko o ka toluene anhydrous, a laila 0.1 ml o 2,2′-azoisobutyronitrile (AIBN) i hoʻohui ʻia i ka pahu hopena e loaʻa ai kahi copolymer o phenylmaleimide a me methylPMCP isocyanate (methylPMCP isocyanate). ) Ua hoʻomehanaʻia ka hui ma 60 ° C no 3 mau hola, kānana a maloʻo i loko o ka umu ma 40 ° C no 3 mau hola.
ʻO nā ʻāpana silica maloʻo (2 g) i hoʻopuehu ʻia i loko o ka toluene maloʻo (100 ml), hoʻoulu ʻia a sonicated no 10 min i loko o kahi pahu 500 ml a puni. Ua hoʻoheheʻe ʻia ʻo PMCP (10 mg) i toluene a hoʻohui ʻia i ka hāmole i ka pahu pane ma o kahi funnel hoʻohui. Hoʻopili ʻia ka hui ʻana ma 100 ° C no 8 mau hola, kānana, holoi ʻia me ka acetone a maloʻo ma 60 ° C no 3 mau hola. A laila, ua hoʻoheheʻe ʻia nā ʻāpana silica e pili ana me PMCP (100 g) i toluene (200 ml), a ua hoʻohui ʻia ʻo 4-hydroxy-TEMPO (2 ml) ma ke alo o 100 μl o dibutyltin dilaurate ma ke ʻano he catalyst. Hoʻopiliʻia ka huiʻana ma 50 ° C no 8 mau hola, kānana a maloʻo ma 50 ° C no 3 mau hola.
ʻO Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) a me nā ʻāpana silica i hoʻopili ʻia me TEMPO-PMCP (1.5 g) i hoʻopuehu ʻia i loko o ka toluene a holoi ʻia me ka nitrogen. Ua lawe ʻia ka polymerization o styrene ma 100 ° C no 12 mau hola. Holoi ʻia ka huahana me ka methanol a maloʻo i ka pō ma 60 ° C. Hōʻike ʻia ka hoʻolālā maʻamau o ka hopena ma ka fig. ʻekahi .
Hoʻopau ʻia nā laʻana ma 393 K no ka hola 1 a hiki i ka loaʻa ʻana o ke koena kaomi ma lalo o 10-3 Torr. Ua hoʻohana ʻia ka nui o N2 adsorbed ma ke kaomi pili P/P0 = 0.99 e hoʻoholo ai i ka nui o ka pore volume. Ua nānā ʻia ka morphology o nā ʻāpana silica maʻemaʻe a me ka ligand me ka hoʻohana ʻana i kahi microscope electron scanning (Hitachi High Technologies, Tokyo, Iapana). Ua kau ʻia nā laʻana maloʻo (silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia i ka ligand) ma luna o nā lāʻau alumini me ka hoʻohana ʻana i ka lipine kalapona. Ua waiho ʻia ke gula ma ka hāpana e hoʻohana ana i kahi mea sputtering Q150T, a ua waiho ʻia kahi papa Au 5 nm mānoanoa ma ka hāpana. Hoʻonui kēia i ka maikaʻi o ke kaʻina hana haʻahaʻa haʻahaʻa a hāʻawi i ka pulu anuanu maikaʻi. Ua hoʻohana ʻia ka hoʻohana ʻana i ka Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Ua ho'ohana 'ia ka Malvern nui particle size analyzer (Worcestershire, UK) Mastersizer 2000 no ka loa'a 'ana o ka pu'unaue nui. Uncoated silica particles a ligand-paʻa silica particles (5 mg kela mea keia mea) ua hoopuehuia ma 5 ml o ka isopropanol, sonicated no 10 minuke, agitated no 5 minuke, a kau ma luna o ka Mastersizer optical pae. Lawe ʻia ka nānā ʻana o Thermogravimetric ma kahi o 5 °C i kēlā me kēia minuke ma ka pae wela mai 30 a 800 °C.
Ua hoʻopili ʻia nā kolamu aniani kuhili ʻole me nā ana (ID 100 × 1.8 mm) ma ke kaʻina hana hoʻopihapiha ma muli o ke kaʻina hana e like me ka kuhikuhi 31. Koluma kila kila (ke aniani laina, ID 100 × 1 .8 mm) a me kahi puka i loaʻa kahi 1 µm frit i hoʻopili ʻia i kahi mīkini pahu slurry De USA). E hoʻomākaukau i ka hoʻokuʻu ʻana o ka manawa paʻa ma ka hoʻokuʻu ʻana i 150 mg o ka manawa paʻa i 1.2 ml o ka methanol a hānai iā ia i loko o kahi kolamu waihona. Ua hoʻohana ʻia ka Methanol ma ke ʻano he slurry solvent a me ka mea hoʻomalu. E hoʻopaʻa i ke kolamu ma ka hoʻohana ʻana i ke kaʻina kaomi o 100 MP no 10 min, 80 MP no 15 min, a me 60 MP no 30 min. Ua hoʻohana ke kaʻina hana paʻi ʻana i ʻelua mau mea hoʻoheheʻe kolamu chromatography (Alltech, Deerfield, IL, USA) no ka haʻalulu mechanical e hōʻoia i ka paʻi ʻana o ke kolamu like ʻole. E pani i ka slurry packer a hoʻokuʻu mālie i ke kaomi e pale i ka pōʻino i ke kaula. Ua hoʻokuʻu ʻia ke kolamu mai ka nozzle slurry a ua hoʻopili ʻia kekahi mea pono i ka puka komo a hoʻopili ʻia i ka ʻōnaehana LC e hoʻāʻo ai i kāna hana.
Ua kūkulu ʻia kahi MLC maʻamau me ka hoʻohana ʻana i ka LC pump (10AD Shimadzu, Iapana), kahi mea hoʻohālike me ka 50 nL injection loop (Valco (USA) C14 W.05), kahi membrane degasser (Shimadzu DGU-14A), a me ka puka makani capillary UV-VIS. Mea ʻike maka (UV-2075) a me ka microcolumn enamelled. E hoʻohana i nā paipu hoʻohui haiki a pōkole loa e hōʻemi i ka hopena o ka hoʻonui ʻia ʻana o ke kolamu. Ma hope o ka hoʻopiha ʻana i ke kolamu, e hoʻokomo i kahi capillary (50 µm id 365) ma ka puka o ka hui hōʻemi 1/16″ a hoʻokomo i kahi capillary (50 µm) o ka hui hōʻemi. Hana ʻia ka hōʻiliʻili ʻikepili a me ka hana chromatogram me ka polokalamu Multichro 2000. Ma ka 254 nm, ua nānāʻia ka UV absorbance o nā kumuhana analytes ma 0. Ua nānāʻia kaʻikepili Chromatographic me OriginPro8 (Northampton, MA).
Albumin serum kanaka, lyophilized pauka, ≥ 96% (agarose gel electrophoresis) 3 mg i hui pū ʻia me trypsin (1.5 mg), 4.0 M urea (1 ml) a me 0.2 M ammonium bicarbonate (1 ml). Ua hoʻonāukiʻia ka hopena no 10 min a mālamaʻia i loko o ka waiʻauʻau ma 37 ° C no 6 mau hola, a laila hoʻopauʻia me 1 ml o 0.1% TFA. E kānana i ka hopena a mālama i lalo o 4°C.
ʻO ka hoʻokaʻawale ʻana o ka hui ʻana o nā peptides a me ka tryptic digest HSA ma kahi kolamu PMP ua loiloi ʻokoʻa. E nānā i ka tryptic hydrolysis o ka hui ʻana o nā peptides a me HSA i hoʻokaʻawale ʻia e kahi kolamu PMP a hoʻohālikelike i nā hopena me kahi kolamu Ascentis Express RP-Amide. Ua helu ʻia ka helu o nā papa theoretical me ka hoʻohana ʻana i ka hoohalike penei:
Hōʻike ʻia nā kiʻi SEM o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia i ka ligand. ʻOi aku ka maʻalahi o ka ʻili o nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand (C, D) ma mua o nā ʻāpana silica maʻemaʻe, ʻo ia paha ke kumu o nā kaulahao polystyrene e uhi ana i ka ʻili o nā ʻāpana silica.
E nānā ana i nā micrographs electron o nā ʻāpana silica maʻemaʻe (A, B) a me nā ʻāpana silica i hoʻopaʻa ʻia i ka ligand (C, D).
Hōʻike ʻia ka puʻunaue nui o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia i ka ligand ma Fig. 2. 3(A). Ua hōʻike ʻia ka nui o ka nui o ka silica ma hope o ka hoʻololi kemika (Fig. 3A). Hoʻohālikelike ʻia ka ʻikepili puunaue silica particle mai ka haʻawina o kēia manawa a me ka haʻawina mua ma ka Papa 1(A). ʻO 3.36 µm ka nui o ka ʻāpana volumetric d(0.5) o PMP, i hoʻohālikelike ʻia me ka waiwai ad(0.5) o 3.05 µm i kā mākou noiʻi mua (polystyrene bonded silica particles)34. Ma muli o ka hoʻololi ʻana i ka ratio o PEG, urea, TMOS a me ka acetic acid i loko o ka hui ʻana, ʻoi aku ka liʻiliʻi o ka hāʻawi ʻana i ka nui o kēia pūʻulu i kā mākou noiʻi mua. ʻOi aku ka nui o ka nui o ka māhele PMP ma mua o ka polystyrene bound silica particle phase a mākou i aʻo mua ai. ʻO ia ke ʻano o ka hana ʻana o ka ili o nā ʻāpana silica me ka styrene i waiho wale ʻia i kahi papa polystyrene (0.97 µm) ma ka ʻili silica, ʻoiai ma ka pae PMP he 1.38 µm ka mānoanoa o ka papa.
Ka puunaue ana i ka nui o ka palaki (A) a me ka puunaue ana i ka nui o ka pore (B) o na mea silika maemae a me na mea silica i paa i ka ligand.
Hōʻike ʻia ka nui o ka pore, ka nui pore, a me ka ʻili o nā ʻāpana silica i hoʻohana ʻia ma kēia haʻawina ma ka Papa 1 (B). Hōʻike ʻia nā moʻolelo PSD o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia i ka ligand ma Fig. 3(B). Ua like nā hopena me kā mākou noiʻi mua34. He 310 Å a me 241 Å ka nui o ka pore o na mea silica maemae a me ka ligand, e hoike ana ma hope o ka hoololi kemika, ua emi ka nui o ka pore e 69 Å, e like me ia i hoikeia ma ka Papa 1 (B), a ua hoikeia ka huina hoololi ma Fig. m2/g). E like me ka mea i hōʻike ʻia ma ka Papa 1(B), ua emi iho ka ʻilikai (m2/g) o nā ʻāpana silica ma hope o ka hoʻololi kemika mai 116 m2/g a i 105 m2/g.
Hōʻike ʻia nā hopena o ka loiloi elemental o ka pae paʻa ma ka Papa. 2. ʻO 6.35% ka maʻiʻo kalapona o ka pae paʻa o kēia manawa, ʻo ia ka mea haʻahaʻa ma mua o kā mākou noiʻi mua (nā ʻāpana silica e pili ana me ka polystyrene, 7.93%35 a me 10.21%, kēlā me kēia) 42. ʻO ka maʻiʻo kalapona o ka pae paʻa o kēia manawa ma lalo, no ka mea, ua hoʻohana ʻia kekahi mau ligand polar e like me phenylmaleimide to methylMP isocyanate (hydro-PC vinyl-MP isocyanate) styrene i ka hoomakaukau ana o SP. ʻO ka pākēneka paona o ka nitrogen i ka manawa paʻa i kēia manawa he 2.21% ke hoʻohālikelike ʻia me 0.1735 a me 0.85% ma nā haʻawina mua42. 'O ia ho'i, he ki'eki'e ka pākēneka kaumaha o ka naikokene ma muli o ka phenylmaleimide i ka manawa pa'a. Pēlā nō, loaʻa i nā huahana (4) a me (5) kahi kalapona maʻiʻo o 2.7% a me 2.9%, ʻoiai ka huahana hope (6) he 6.35% ka kalapona, e like me ka mea i hōʻike ʻia ma ka Papa 2. Ua hoʻohana ʻia ka Thermogravimetric analysis (TGA) ma ke kahua paʻa o PMP e hoʻāʻo ai no ka pohō kaumaha, a ua hōʻike ʻia ka pihi TGA 4. ʻO ia ka mea i ʻaelike maikaʻi me ka ʻike kalapona (6.35%), no ka mea, ʻaʻole i loko o nā ligands C wale nō, akā ʻo N, O a me H.
Ua koho ʻia ka ligand phenylmaleimide-methylvinyl isocyanate e hoʻololi i ka ʻili o nā ʻāpana silica ma muli o kāna mau hui polar phenylmaleimide a me vinylisocyanate. Hiki i nā hui isocyanate Vinyl ke hana hou me ka styrene ma o ka polymerization radical ola. ʻO ke kumu ʻelua, ʻo ia ka hoʻokomo ʻana i kahi pūʻulu i loaʻa nā pilina haʻahaʻa me ka analyte a ʻaʻohe pilina electrostatic ikaika ma waena o ka analyte a me ka pae paʻa, no ka mea, ʻaʻohe o ka phenylmaleimide moiety i ka pH maʻamau. Hiki ke hoʻomalu ʻia ka polarity o ka manawa paʻa e ka nui o ka styrene a me ka manawa pane o ka polymerization radical manuahi. He mea koʻikoʻi ka ʻanuʻu hope o ka pane (polymerization radical manuahi) i ka hoʻololi ʻana i ka polarity o ka pae paʻa. Ua hoʻokō ʻia ka hoʻopaʻa ʻana i ka ʻeleu no ka nānā ʻana i ka ʻike kalapona i loko o kēia mau ʻāpana kū. Ua ʻike ʻia ʻo ka hoʻonui ʻana i ka nui o ka styrene a me ka manawa pane e hoʻonui ai i ka ʻike kalapona o ka pae paʻa a me ka hope. ʻO nā SP i hoʻomākaukau ʻia me nā manaʻo like ʻole o ka styrene he mau ukana kalapona ʻokoʻa. Pēlā nō, ua kau ʻia kēia mau ʻāpana kū ma luna o nā kolamu kila kila a ua nānā ʻia ko lākou mau hiʻohiʻona chromatographic (selectivity, resolution, N value, etc.). Ma muli o kēia mau hoʻokolohua, ua koho ʻia kahi haku mele no ka hoʻomākaukau ʻana i ka PMP stationary phase e hāʻawi i ka polarity hoʻomalu a me ka mālama maikaʻi ʻana o ka analyte.
Ua loiloi pū ʻia ke kolamu PMP no ka nānā ʻana o ʻelima hui ʻana o nā peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) me ka hoʻohana ʻana i ka hiki o ka pae kelepona. 60/40 (v/v) ACN/wai (0.1% TFA) ma kahi kahe kahe o 80 µl/min. Ma lalo o nā kūlana elution maikaʻi loa (200,000 papa / m), ʻo ka helu o nā papa theoretical (N) no kēlā me kēia kolamu (100 × 1.8 mm) he 20,000 ± 100. Hōʻike ʻia nā waiwai N no nā kolamu PMP ʻekolu ma ka Papa 3 a hōʻike ʻia nā chromatograms ma Figure 5A. ʻO ka nānā wikiwikiʻana i ke kahe kahe kiʻekiʻe (700 µl / min) ma kahi kolamu PMP, ʻelima peptides i hoʻokahe ʻia i loko o hoʻokahi minuke, maikaʻi N waiwai o 13,500 ± 330 no kēlā me kēia kolamu (100 x 1.8 mm anawaena), e like me 135,000 mau papa / m (Fig. 5B). ʻEkolu kolamu o ka nui like (ka anawaena o loko 100 x 1.8 mm) i hoʻopiha ʻia me ʻekolu mau pūʻulu like ʻole o ka PMP stationary phase e hoʻāʻo ai i ka reproducibility. Ua hoʻopaʻa ʻia nā analytes no kēlā me kēia kolamu ma ka hoʻokaʻawale ʻana i ka hui hoʻāʻo like ma kēlā me kēia kolamu me ka hoʻohana ʻana i nā kūlana elution maikaʻi loa, ka helu o nā papa theoretical N, a me ka manawa paʻa. Hōʻike ʻia ka ʻikepili reproducibility no nā kolamu PMP ma ka Papa 4. Ua hoʻopili maikaʻi ʻia ka hana hou ʻana o ke kolamu PMP me nā koina %RSD haʻahaʻa loa e like me ka hōʻike ʻana ma ka Papa 3.
ʻO ka hoʻokaʻawale ʻana o nā hui peptide ma kahi kolamu PMP (B) a me kahi kolamu Ascentis Express RP-Amide (A), ka pae kelepona 60/40 ACN/H2O (TFA 0.1%), nā ana kolamu PMP (100 x 1.8 mm id) , ka nānā ʻana Elution kauoha o nā pūhui: 1 (Gly-Tyr-Tyr), 2- (Leu-Tyr), 2-(Gly-Tyr) (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) a me 5 (leucic acid enkephalin).
Ua loiloi ʻia kahi kolamu PMP (ka anawaena o loko 100 x 1.8 mm) no ka hoʻokaʻawale ʻana o ka tryptic hydrolyzate o ka albumin serum kanaka e HPLC. Hōʻike ka chromatogram ma ke Kiʻi 6 ua hoʻokaʻawale maikaʻi nā laʻana me ka hoʻonā maikaʻi loa. Hoʻopili ʻia nā haʻina HSA me ka hoʻohana ʻana i kahi kahe kahe o 100 μl / min, kahi pae kelepona o 70/30 acetonitrile / wai a me 0.1% TFA. Ua māhele ʻia ʻo Cleavage o HSA i 17 peaks, e like me ka mea i hōʻike ʻia ma ka chromatogram (Fig. 6), pili i 17 peptides. Ua helu ʻia ka hoʻokaʻawale ʻana o nā kiʻekiʻe o kēlā me kēia mai ka HSA hydrolyzate a hōʻike ʻia nā waiwai ma ka Papa 5.
Hoʻokaʻawale ʻia nā hydrolysates HSA tryptic ma kahi kolamu PMP (ka anawaena o loko 100 x 1.8 mm), kahe kahe (100 μl / min), ka pae kelepona 60/40 acetonitrile / wai, a me 0.1% TFA.
ʻo L ka lōʻihi o ke kolamu, ʻo η ka viscosity o ka māhele paʻa, ΔP ke kaomi hope o ke kolamu, a ʻo u ka wikiwiki laina o ka pae paʻa. ʻO ka permeability o ke kolamu PMP he 2.5 × 10-14 m2, he 25 µl / min ka kahe kahe, hoʻohana ʻia 60/40 v / v. ACN/wai. Ua like ka permeability o ke kolamu PMP (ID 100 × 1.8 mm) me kā mākou noiʻi Ref.34 mua. ʻO ka permeability o kahi kolamu i hoʻopiha ʻia me nā ʻāpana porous superficially he 1.7×10 .6 µm, 2.5×10-14 m2 no nā ʻāpana 5 µm43. No laila, ua like ka permeability o ka māhele PMP me ka permeability o nā ʻāpana core-shell me ka nui o 5 μm.
ʻo Wx ka nuipaʻa o ke kolamu i hoʻopiha ʻia me ka chloroform, ʻo Wy ka nuipaʻa o ke kolamu i hoʻopiha ʻia me ka methanol, a ʻo ρ ka mānoanoa o ka mea hoʻoheheʻe. Ka mānoanoa o ka methanol (ρ = 0.7866) a me ka chloroform (ρ = 1.484). ʻO ka nui o ka porosity o ke kolamu silica-C18 particle (100 × 1.8 mm ID)34 a me kā mākou kolamu C18-urea31 i aʻo mua ʻia ʻo 0.63 a me 0.55. ʻO kēia ke ʻano o ka hiki ʻana o nā ligand urea e hōʻemi i ka permeability o ka pae paʻa. Ma kekahi ʻaoʻao, ʻo ka nui o ka porosity o ke kolamu PMP (ka anawaena o loko 100 × 1.8 mm) ʻo 0.60. ʻOi aku ka liʻiliʻi o nā kolamu PMP ma mua o nā kolamu i hoʻopaʻa ʻia me nā ʻāpana silica paʻa C18 no ka mea ma nā ʻano paʻa ʻano C18 ua hoʻopili ʻia nā ligands C18 i nā ʻāpana silica i nā kaulahao laina, ʻoiai ma nā ʻano paʻa polystyrene ua hana ʻia kahi polymer mānoanoa a puni nā ʻāpana. papa A. Ma kahi hoʻokolohua maʻamau, helu ʻia ka porosity kolamu penei:
Ma ka fig. 7A, B hōʻike i nā ʻāpana Van Deemter no kahi kolamu PMP (id 100 x 1.8 mm) a me kahi kolamu Ascentis Express RP-Amide (id 100 x 1.8 mm) ma lalo o nā kūlana elution like, 60/40 ACN/H2O a me 0 .1% TFA 20 µl/min a i 80. ʻO nā koina HETP haʻahaʻa loa ma ke kahe kahe maikaʻi loa (80 µl/min) ʻo 2.6 µm a me 3.9 µm no ke kolamu PMP a me ke kolamu Ascentis Express RP-Amide. Hōʻike nā helu HETP i ke kiʻekiʻe o ka hoʻokaʻawale ʻana o ke kolamu PMP (100 x 1.8 mm id) ma mua o ke kolamu Ascentis Express RP-Amide i loaʻa i ke kālepa (100 x 1.8 mm id). Hōʻike ka pakuhi van Deemter ma Fig. 7 (A) i ka emi ʻana o ka waiwai N ʻaʻole i ʻoi aku ka kiʻekiʻe me ka piʻi ʻana o ke kahe i hoʻohālikelike ʻia i kā mākou noiʻi mua. ʻOi aku ka maikaʻi o ka hoʻokaʻawale ʻana o ke kolamu PMP (id 100 × 1.8 mm) i hoʻohālikelike ʻia me ka kolamu Ascentis Express RP-Amide ma muli o ke ʻano a me ka nui i hoʻomaikaʻi ʻia a me ke kaʻina hana hoʻopili kolamu maʻalahi i hoʻohana ʻia i ka hana o kēia manawa34.
(A) Van Deemter plot (HETP vs. mobile phase linear velocity) i loaʻa ma ke kolamu PMP (id 100 x 1.8 mm) ma 60/40 ACN/H2O me 0.1% TFA. (B) Van Deemter plot (HETP versus mobile phase linear velocity) i loaʻa ma kahi kolamu Ascentis Express RP-Amide (id 100 x 1.8 mm) ma 60/40 ACN/H2O me 0.1% TFA.
Ua hoʻomākaukau ʻia a loiloi ʻia kahi ʻāpana polar o ka polystyrene intercalated no ka hoʻokaʻawale ʻana o ka hui ʻana o nā peptides synthetic a me ka tryptic hydrolyzate o ka albumin serum kanaka (HSA) i ka chromatography wai hana kiʻekiʻe. ʻO ka hana chromatographic o nā kolamu PMP no nā hui peptide maikaʻi loa i nā ʻōlelo o ka hoʻokaʻawale ʻana a me ka hoʻonā. ʻO ka maikaʻi o ka hoʻokaʻawale ʻana o nā kolamu PMP ma muli o nā kumu he nui e like me ka nui o ka silica particle a me ka nui o ka pore, ka hoʻopaʻa ʻia ʻana o nā hana kū, a me nā mea hoʻopili kolamu paʻakikī. Ma waho aʻe o ka maikaʻi o ka hoʻokaʻawale kiʻekiʻe, ʻo kekahi pōmaikaʻi ʻē aʻe o kēia pae paʻa ʻo ke kolamu haʻahaʻa haʻahaʻa hope i nā kahe kahe kiʻekiʻe. Hiki ke hana hou ʻia nā kolamu PMP a hiki ke hoʻohana ʻia e nānā i nā hui ʻana o nā peptides a me ka tryptic digestion o nā protein like ʻole. Manaʻo mākou e hoʻohana i kēia kolamu no ka hoʻokaʻawale ʻana i nā pūhui bioactive mai nā huahana kūlohelohe, nā ʻāpana o nā mea kanu lāʻau lapaʻau a me nā halo i ka chromatography wai. I ka wā e hiki mai ana, e loiloi pū ʻia nā kolamu PMP no ka hoʻokaʻawale ʻana o nā protein a me nā antibodies monoclonal.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Ke noiʻi ʻana i nā ʻōnaehana hoʻokaʻawale ʻana o ka chromatography peptide ʻāpana I: Hoʻomohala ʻana i kahi protocol no ka hōʻike ʻana i ke kolamu. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Ke noiʻi ʻana i nā ʻōnaehana hoʻokaʻawale ʻana o ka chromatography peptide ʻāpana I: Ka hoʻomohala ʻana i kahi protocol no ka hōʻike ʻana i ke kolamu.Field, JK, Owerby, MR, Lau, J., Togersen, H., and Petersson, P. Investigation of Peptide Separation Systems by Reverse-Phase Chromatography, Part I: Developing a Protocol for Column Characterization. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Ke noiʻi ʻana i nā ʻōnaehana hoʻokaʻawale ʻana o ka chromatography peptide ʻāpana I: Hoʻomohala ʻana i kahi protocol no nā ʻano kolamu. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Ke noiʻi ʻana i nā ʻōnaehana hoʻokaʻawale ʻana o ka chromatography peptide ʻāpana I: Hoʻomohala ʻana i kahi protocol no nā ʻano kolamu.Field, JK, Owerby, MR, Lau, J., Togersen, H., and Petersson, P. Investigation of Peptide Separation Systems by Reverse-Phase Chromatography, Part I: Developing a Protocol for Column Characterization.J.色谱法。 1603，113-129。 https://doi.org/10.1016/j.chroma.2019.05.038(2019).
Gomez, B. et al. Nā ala no ka hana ʻana i nā peptides ikaika i hoʻomaikaʻi ʻia no ka mālama ʻana i nā maʻi maʻi. Biotechnology. Nā hana 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: ʻEpekema a me ka mākeke. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeutic peptides: ʻEpekema a me ka mākeke.Vliege P, Lisowski V, Martinez J a me Chreschatyski M. Synthetic therapeutic peptides: ʻepekema a me ka mākeke.ʻO Vliege P, Lisowski V, Martinez J a me Khreschatsky M. Synthetic therapeutic peptides: ʻepekema a me ka mākeke. ʻike lāʻau. I kēia lā 15 (1–2), 40–56. https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Kiʻekiʻe proteomic wai chromatography. Xie, F., Smith, RD & Shen, Y. Kiʻekiʻe proteomic wai chromatography.E ʻike iā F., Smith RD a me Shen Yu. Kromatography wai proteomic kiʻekiʻe. Xie, F., Smith, RD & Shen, Y. 高级蛋白质组液相色谱。 Xie, F., Smith, RD & Shen, Y. Hoʻohui pūmua kiʻekiʻe 液相色谱。E ʻike iā F., Smith RD a me Shen Yu. Kromatography wai proteomic kiʻekiʻe.J. Kromatography. A 1261, 78–90 (2012).
Liu, W. et al. Hiki i ka chromatography-mass spectrometry kiʻekiʻe ke hoʻohui i nā metabolomics ākea a me nā proteomics. anus. ʻO Chim. Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ ʻO ke kuleana o UHPLC i ka hoʻomohala lāʻau. Chesnut, SM & Salisbury, JJ ʻO ke kuleana o UHPLC i ka hoʻomohala lāʻau.Chesnut, SM a me Salisbury, JJ ʻO ke kuleana o UHPLC i ka hoʻomohala lāʻau.Chesnut, SM a me Salisbury, JJ ʻO ke kuleana o UHPLC i ka hoʻomohala lāʻau. J. Sept Science. 30(8), 1183–1190 (2007).
Wu, N. & Clausen, AM Nā hiʻohiʻona kumu a me ka hana o ka ultrahigh pressure wai chromatography no ka hoʻokaʻawale wikiwiki. Wu, N. & Clausen, AM Nā hiʻohiʻona kumu a me ka hana o ka ultrahigh pressure wai chromatography no ka hoʻokaʻawale wikiwiki.Wu, N. a me Clausen, AM Nā hiʻohiʻona kumu a me ka hana o ka chromatography wai kaomi kiʻekiʻe no ka hoʻokaʻawale wikiwiki. 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 Nā hiʻohiʻona kumu a me ka hana o ka chromatography wai kaomi ultra-kiʻekiʻe no ka hoʻokaʻawale wikiwiki.Wu, N. a me Clausen, AM Nā hiʻohiʻona kumu a me ka hana o ka chromatography wai kaomi kiʻekiʻe no ka hoʻokaʻawale wikiwiki.J. Sept. Naauao. 30(8), 1167–1182. https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Hoʻohana i ka ultra-performance liquid chromatography i ka hoʻomohala lāʻau. Wren, SA & Tchelitcheff, P. Hoʻohana i ka ultra-performance liquid chromatography i ka hoʻomohala lāʻau.Ren, SA a me Chelischeff, P. Ka hoʻohana 'ana i ka ultra high performance liquid chromatography i ka hoʻomohala lāʻau. Wren, SA & Tchelitcheff, P. 超高效液相色谱在药物开发中的应用。 Wren, SA & Tchelitcheff, P.Ren, SA a me Chelischeff, P. Hoʻohana i ka ultra-performance liquid chromatography i ka hoʻomohala ʻana i ka lāʻau.J. Kromatography. 1119(1-2), 140-146. https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al. He monolithic macroporous hydrogel i loaʻa mai kahi emulsion aila-i-wai me kahi pae kūloko kiʻekiʻe no ka hoʻomaʻemaʻe pono ʻana o enterovirus 71. Kemika. papahana. Nūpepa 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA ʻO ke kuleana o ka chromatography wai i nā proteomics. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA ʻO ke kuleana o ka chromatography wai i nā proteomics.Shi, Y., Xiang, R., Horvath, C. a me Wilkins, JA ʻO ke kuleana o ka chromatography wai i nā proteomics. 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. a me Wilkins, JA ʻO ke kuleana o ka chromatography wai i nā proteomics.J. Kromatography. A 1053 (1-2), 27-36 (2004).
Fekete, S., Vutey, J.-L. & Guillarme. & Guillarme. & Guillarme, D. Новые тенденции в разделении терапевтических пептидов и белков с помощью жидкостной хроматонграфира хроматографира теория и приложения. & Guillarme, D. Nā ʻano hou i ka hoʻokaʻawale ʻana o nā peptides therapeutic a me nā protein e ka reverse phase liquid chromatography: theory and applications. & 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。 & Guillarme, D.a me Guillarmé, D. Nā hana hou i ka hoʻokaʻawale ʻana o nā peptides therapeutic a me nā protein e ka reverse phase liquid chromatography: theory and applications.J. Pharm. ʻEpekema Biomedical. anus. 69, 9–27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Hoʻokaʻawale ʻelua-dimensional o nā peptides me ka hoʻohana ʻana i ka ʻōnaehana RP-RP-HPLC me ka pH like ʻole i nā ana hoʻokaʻawale mua a me ka lua. Gilar, M., Olivova, P., Daly, AE & Gebler, JC Hoʻokaʻawale ʻelua-dimensional o nā peptides me ka hoʻohana ʻana i ka ʻōnaehana RP-RP-HPLC me ka pH like ʻole i nā ana hoʻokaʻawale mua a me ka lua.ʻO Gilar M., Olivova P., Dali AE a me Gebler JK ʻO ka hoʻokaʻawale ʻana i ʻelua mau ʻāpana o nā peptides me ka hoʻohana ʻana i ka ʻōnaehana RP-RP-HPLC me nā pH like ʻole i ka nui o ka hoʻokaʻawale mua a me ka lua.ʻO Gilar M., Olivova P., Dali AE a me Gebler JK Hoʻokaʻawale ʻelua ʻāpana o nā peptides me ka hoʻohana ʻana i nā waiwai pH like ʻole i ka nui o ka hoʻokaʻawale mua a me ka lua me ka hoʻohana ʻana i ka ʻōnaehana RP-RP-HPLC. J. Sept Science. 28 (14), 1694–1703 (2005).
Fellitti, S. et al. Ke noiʻi ʻana i ka hoʻololi nui a me nā hiʻohiʻona kinetic o nā kolamu chromatography hana kiʻekiʻe i hoʻopiha ʻia me nā ʻāpana C18 porous piha a me ka porous ma lalo o 2 µm. J. Sept Science. 43 (9–10), 1737–1745 (2020).
Piovesana, S. et al. ʻO nā ʻano hou a me nā pilikia loiloi i ka hoʻokaʻawale ʻana, ka ʻike, a me ka hōʻoia ʻana o nā peptides bioactive mea kanu. anus. Anal kino. Kemika. 410(15), 3425-3444. https://doi.org/10.1007/s00216-018-0852-x (2018).
Muller, JB et al. ʻO ka ʻāina proteomic o ke aupuni o ke ola. Nature 582 (7813), 592–596. https://doi.org/10.1038/s41586-020-2402-x (2020).
De Luca, K. et al. Ma hope o ka mālama ʻana i nā peptides therapeutic e ka preparative liquid chromatography. Nā Molekula (Basel, Kuikilana) 26(15), 4688 (2021).
ʻO Yang, Y. & Geng, X. ʻO ka chromatography mode Mixed-mode a me kāna mau noi i nā biopolymers. ʻO Yang, Y. & Geng, X. ʻO ka chromatography mode Mixed-mode a me kāna mau noi i nā biopolymers.Yang, Yu. a me Geng, X. ʻO ka chromatography mode hui pū ʻia a me kona hoʻohana ʻana i nā biopolymers. Yang, Y. & Geng, X. 混合模式色谱及其在生物聚合物中的应用。 ʻO Yang, Y. & Geng, X. ʻO ka chromatography mode hui ʻia a me kāna hoʻohana ʻana i nā biopolymers.Yang, Yu. a me Gene, X. ʻO ka chromatography mode hui pū ʻia a me kona hoʻohana ʻana i nā biopolymers.J. Kromatography. A 1218(49), 8813–8825 (2011).