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I whakaritea ngā matūriki silica porous mā te tikanga sol-gel me ētahi whakarerekētanga hei whiwhi i ngā matūriki macroporous. I tangohia ēnei matūriki mā te polymerization reversible addition fragmentation chain transfer (RAFT) me te N-phenylmaleimide-methylvinylisocyanate (PMI) me te styrene hei whakarite i te intercalation N-phenylmaleimide o te wāhanga tūmau polystyrene (PMP). I tākaihia ngā pou maitai kore waikura whaiti (100 × 1.8 mm id) mā te tākai paru. I aromatawaihia te wehenga pou PMP o te ranunga peptide kei roto e rima ngā peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) (te mahi chromatographic) me te nakunaku trypsin o te albumin serum tangata (HAS). I raro i ngā tikanga elution tino pai, ko te tatau pereti ariā o te ranunga peptide he teitei ki te 280,000 pereti/m². Mā te whakataurite i te mahi wehenga o te pou kua whakawhanakehia ki te Ascentis arumoni. I roto i te pou RP-Amide tere, i kitea he pai ake te mahi wehenga o te pou PMP i te pou arumoni e pā ana ki te whai huatanga me te taumira wehenga.
I ngā tau tata nei, kua huri te umanga rongoā koiora hei mākete whānui o te ao me te pikinga nui o te wahanga mākete. Nā te tipu tere o te umanga rongoā koiora1,2,3, he mea tino hiahiatia te tātari i ngā peptides me ngā pūmua. Haunga te peptide ūnga, he maha ngā poke ka puta i te wā o te hanganga peptide, nō reira me purenga chromatography kia whiwhi ai i ngā peptides o te parakore e hiahiatia ana. He mahi tino uaua te tātari me te tautuhi i ngā pūmua i roto i ngā wai tinana, ngā kiko me ngā pūtau nā te nui o ngā momo ka kitea pea i roto i te tauira kotahi. Ahakoa he taputapu whai hua te spectrometry papatipu mō te raupapatanga peptide me te pūmua, ki te werohia aua tauira ki roto i te spectrometer papatipu i te kotahi haerenga, kāore e pai te wehenga. Ka taea te whakaiti i tēnei raruraru mā te whakatinana i ngā wehenga chromatography wai (LC) i mua i te tātari MS, ka whakaiti i te maha o ngā tātaritanga e uru ana ki te spectrometer papatipu i tētahi wā kua whakaritea4,5,6. Hei tāpiri, i te wā o te wehenga wāhanga wai, ka taea te arotahi i ngā tātaritanga ki ngā rohe whaiti, ā, ka whakaemihia ēnei tātaritanga me te whakapai ake i te aro ki te kitenga MS. Kua... kua tino whanake haere i roto i te tekau tau kua pahure ake nei, ā, kua noho hei tikanga rongonui i roto i te tātaritanga proteomic7,8,9,10.
He whānuitia te whakamahinga o te chromatography wai wāhanga-whakahuri (RP-LC) mō te purenga me te wehenga o ngā ranunga peptide mā te whakamahi i te silica whakarerekētia octadecyl (ODS) hei wāhanga tūmau11,12,13. Heoi, kāore ngā wāhanga tūmau RP e whakarato i te wehenga pai o ngā peptide me ngā pūmua nā te mea he hanganga uaua me te āhua amphiphilic 14,15. Nō reira, me hanga ngā wāhanga tūmau motuhake hei tātari i ngā peptide me ngā pūmua me ngā wāhanga polar me ngā wāhanga kore-polar hei taunekeneke me te pupuri i ēnei tātaritanga16. Ko te chromatography aratau-whakaranu, e whakarato ana i ngā taunekeneke maha-momo, ka taea te noho hei huarahi kē atu ki te RP-LC mō te wehenga o ngā peptide, ngā pūmua, me ētahi atu ranunga uaua. He maha ngā wāhanga tūmau aratau-whakaranu kua whakaritea, ā, kua whakamahia ngā pou kua whakakiia ki ēnei wāhanga mō ngā wehenga peptide me te pūmua17,18,19,20,21. He pai ngā wāhanga tūmau aratau-whakaranu (WAX/RPLC, HILIC/RPLC, intercalation polar/RPLC) mō ngā wehenga peptide me te pūmua nā te mea kei te noho ngā polar me ngā ngā rōpū kore-pōra22,23,24,25,26,27,28. Waihoki, ko ngā wāhanga tūmau honohono pōra me ngā rōpū pōra e hono tahi ana te hononga e whakaatu ana i te kaha wehewehe pai me te whiriwhiri ahurei mō ngā tātari pōra me ngā tātari kore-pōra, nā te mea ko te wehenga e whakawhirinaki ana ki te taunekeneke i waenga i te tātari me te wāhanga tūmau. Ngā taunekeneke maha-momo 29, 30, 31, 32. Nō nā tata nei, i whakarite a Zhang et al. 30 i tētahi wāhanga tūmau polyamine kua mutu te dodecyl, ā, i wehe angitu i ngā warowaihā, ngā rongoā ārai mate, ngā flavonoid, ngā nucleosides, ngā estrogens, me ētahi atu tātari. Kei te tātari pōra ngā rōpū pōra me ngā rōpū kore-pōra, nō reira ka taea te whakamahi hei wehe i ngā peptides me ngā pūmua he wāhanga hydrophobic me hydrophilic. Kei te wātea ngā pou whakauru-pōra (hei tauira, ngā pou C18 whakauru-amide) i raro i te ingoa hokohoko ko ngā pou Ascentis Express RP-Amide, engari ko ēnei pou e whakamahia ana mō te tātari i te amine 33 anake.
I roto i te rangahau o nāianei, i whakaritea he wāhanga tūmau kua whakauruhia ki te poro (N-phenylmaleimide-polystyrene kua whakauruhia) ā, i aromatawaihia hoki mō te wehenga o ngā peptide me ngā trypsin digests o te HSA. I whakaritea te wāhanga tūmau mā te whakamahi i te rautaki e whai ake nei. I whakaritea ngā matūriki silica porous i runga i te tikanga i hoatu i roto i tā mātou pukapuka o mua me ētahi whakarerekētanga ki te kawa whakarite. I whakatikatikaina te ōwehenga o te urea, te polyethylene glycol (PEG), te TMOS, te waikawa acetic wai hei whakarite i ngā matūriki silica me te rahi o ngā pore nui. Tuarua, i hangaia he ligand hou, te phenylmaleimide-methyl vinyl isocyanate, ā, i whakamahia hei tango i ngā matūriki silica hei whakarite i tētahi wāhanga tūmau kua whakauruhia ki te poro. I tākaihia te wāhanga tūmau hua ki roto i tētahi pou kowiri tira (100 × 1.8 mm id) mā te whakamahi i te kaupapa tākai kua arotauhia. Ka āwhinahia te tākai pou mā te wiri miihini hei whakarite kia hangaia he moenga ōrite i roto i te pou. Aromatawai i te wehenga pou kua tākaihia o ngā ranunga peptide e rima ngā peptide; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) me te Trypsin digest o te human serum albumin (HAS). I kitea te wehenga o te ranunga peptide me te trypsin digest o te HSA me te pai o te whakataunga me te whai huatanga. I whakaritea te mahi wehenga o te pou PMP ki te pou Ascentis Express RP-Amide. I kitea te rewa pai o ngā peptide me ngā pūmua me te whai huatanga i runga i te pou PMP, he pai ake te whai huatanga i te pou Ascentis Express RP-Amide.
PEG (Polyethylene Glycol), Urea, Waikawa Acetic, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, me te Acetone i hokona mai i Sigma-Aldrich (St. Louis, MO, USA).
I whakaohokia he ranunga o te urea (8 karamu), te polyethylene glycol (8 karamu), me te 8 mL o te waikawa acetic 0.01 N mō te 10 meneti, kātahi ka tāpirihia he 24 mL o te TMOS ki roto i raro i ngā āhuatanga makariri. I whakamahanahia te ranunga tauhohenga ki te 40°C mō te 6 hāora, kātahi ki te 120°C mō te 8 hāora i roto i te autoclave maitai kore waikura. I ringihia te wai, ā, i whakamaroketia te toenga rauemi ki te 70°C mō te 12 hāora. I hurihia te papatipu ngohengohe maroke ki roto i te oumu, ā, i whakawerahia ki te 550°C mō te 12 hāora. E toru ngā puranga i whakaritea, ā, i whakaahuatia hei tirotiro i te āheinga ki te whakaputa anō i te rahi o te matūriki, te rahi o ngā pore me te horahanga o te mata.
Mā te whakarerekētanga mata o ngā matūriki silica me te phenylmaleimide-methylvinylisocyanate (PCMP) ligand kua hangaia i mua, ā, whai muri ko te polymerization radial me te styrene, i whakaritea he matū kei roto he rōpū porowhita. Wāhanga tūmau mō ngā whakahiato me ngā mekameka polystyrene. Kei raro nei te whakaahuatanga o te tukanga whakarite.
I whakarewaina te N-phenylmaleimide (200 mg) me te methyl vinyl isocyanate (100 mg) ki roto i te toluene maroke, ā, i tāpirihia te 0.1 mL o te 2,2′-azoisobutyronitrile (AIBN) ki te pounamu tauhohenga hei whakarite i te phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP). I whakamahanahia te ranunga ki te 60°C mō ngā hāora e 3, i tātarihia, ā, i whakamaroketia ki roto i te oumu ki te 40°C mō ngā hāora e 3.
I whakamararatia ngā matūriki silica maroke (2 g) ki roto i te toluene maroke (100 mL), i whakaohokia, ā, i whakakorikoria ki roto i te pounamu raro porowhita 500 mL mō te 10 meneti. I whakarewaina te PMCP (10 mg) ki roto i te toluene, ā, i tāpirihia kia maturuturu ki roto i te pounamu tauhohenga mā te awaawa maturuturu. I whakahurihia te ranunga ki te 100°C mō te 8 hāora, i tātarihia, ā, i horoia ki te acetone, ā, i whakamaroketia ki te 60°C mō te 3 hāora. Kātahi ka whakarewaina ngā matūriki silica kua herea ki te PMCP (100 g) ki roto i te toluene (200 ml) ā, i tāpirihia te 4-hydroxy-TEMPO (2 mL) i te aroaro o te 100 µL o te dibutyltin dilaurate hei whakakorikori. I whakaohokia te ranunga ki te 50°C mō te 8 hāora, i tātarihia, ā, i whakamaroketia ki te 50°C mō te 3 hāora.
I whakamararatia te Styrene (1 mL), te benzoyl peroxide BPO (0.5 mL), me ngā matūriki silica e piri ana ki te TEMPO-PMCP (1.5 g) ki roto i te toluene, ā, i horoia ki te hauota. I whakahaerehia te polymerization o te styrene i te 100°C mō ngā haora 12. I horoia te hua hua ki te methanol, ā, i whakamaroketia i te 60°C i te pō. Kei te whakaatuhia te kaupapa tauhohenga whānui i te Pikitia 1.
I tangohia te hau o ngā tauira i te 393 K mō te 1 hāora kia whiwhi ai i te pēhanga toenga iti iho i te 10-3 Torr. Ko te nui o te N2 i mimitihia i te pēhanga whanaunga o te P/P0 = 0.99 i whakamahia hei whakatau i te rōrahi katoa o ngā pore. I tirotirohia te āhua o ngā matūriki silica tahanga me ngā matūriki silica e hono ana ki te ligand mā te karu hiko matawai (Hitachi High Technologies, Tokyo, Japan). I whakatakotoria ngā tauira maroke (silica tahanga me ngā matūriki silica e hono ana ki te ligand) ki runga i te pou konumohe mā te whakamahi i te rīpene waro piri. I whakakikoruatia te koura ki ngā tauira mā te whakamahi i te Q150T sputter coater, ā, i whakatakotoria he paparanga Au 5 nm ki runga i ngā tauira. Ka whakapai ake tēnei i te whai huatanga o te tukanga mā te whakamahi i ngā ngaohiko iti, ā, ka whakarato i te witi pai, te sputtering makariri. I whakamahia he tātari huānga Thermo Electron (Waltham, MA, USA) Flash EA1112 mō te tātari huānga. I whakamahia he tātari rahi matūriki Malvern (Worcestershire, UK) Mastersizer 2000 hei whiwhi i te tohatoha rahi matūriki. Ngā matūriki silica tahanga me te silica e hono ana ki te ligand I whakamararatia ngā matūriki (5 mg ia matūriki) ki roto i te 5 mL o te isopropanol, i whakakorikoria mō te 10 meneti, i hurihia mō te 5 meneti, ā, i whakatakotoria ki runga i te paepae whatu o te Mastersizer. I mahia te tātaritanga thermogravimetric i te tere o te 5 °C ia meneti i runga i te awhe pāmahana mai i te 30 ki te 800 °C.
I tākaihia ngā pou whaiti-poka he mea whakakikorua ki te karāhe, he mea hanga ki te kowiri tira, he rahi (100 × 1.8 mm te id) mā te whakamahi i te tikanga tākai paru, me te whakamahi i te tukanga kotahi i whakamahia i roto i te Tohutoro. 31. I honoa he pou kowiri tira (he karāhe te rārangi, 100 × 1.8 mm te whānui) me te hononga putanga kei roto he frit 1 µm te matotoru ki tētahi pūtāhui paru (Alltech Deerfield, IL, USA). Whakapaia he pūtāhui āhua tūmau mā te whakatarewa i te 150 mg o te āhua tūmau ki roto i te 1.2 mL o te methanol ka tukuna ki te pou rokiroki. I whakamahia te methanol hei whakarewa paru me te whakarewa pana. Whakakīia te pou i ia wā mā te pēhi i te 100 MP mō te 10 meneti, 80 MP mō te 15 meneti, me te 60 MP mō te 30 meneti. I te wā e tākai ana, i whakamahia te wiri ā-ringa me ngā wiri pou GC e rua (Alltech, Deerfield, IL, USA) kia rite ai te tākai o te pou. Katia te pūtāhui paru ka tukuna mārire te pēhanga kia kore ai e kino i roto i te pou. Momotuhia te pou mai i te wae tākai paru ka honoa tētahi atu hononga ki te tomokanga me te pūnaha LC hei tirotiro i tana mahi.
I hangaia he papu LC (10AD Shimadzu, Hapani), he werowero (Valco (USA) C14 W.05) me te koropiko werowero 50nL, he tango hau kiriuhi (Shimadzu DGU-14A), he matapihi pūwero UV-VIS. I hangaia he taputapu kite µLC motuhake (UV-2075) me ngā pou iti kua whakakikoruatia ki te karāhe. Whakamahia he ngongo hononga tino whaiti me te poto hei whakaiti i te pānga o te whānui o te roopu pou tāpiri. I muri i te takai, i tāutahia ngā pūwero (50 μm id 365 me ngā pūwero whakakotahi whakaiti (50 μm) ki te putanga 1/16″ o te whakakotahi whakaiti. I mahia te kohikohinga raraunga me te tukatuka chromatography mā te whakamahi i te pūmanawa Multichro 2000. I whakamatautauria te aroturuki i te 254 nm Ngā tātaritanga mō te mimiti UV. I tātarihia ngā raraunga chromatographic e OriginPro8 (Northampton, MA).
He pūmua wai mai i te toto tangata, he paura kua whakakīia ki te wai, ≥ 96% (hiko whakapūkara agarose) 3 mg i konatunatua ki te trypsin (1.5 mg), 4.0 M urea (1 mL), me te 0.2 M ammonium bicarbonate (1 mL). I whakaohokia te otinga mō te 10 meneti, ā, i puritia ki roto i te kaukau wai i te 37°C mō te 6 hāora, kātahi ka tineia ki te 1 mL o te 0.1% TFA. Tātarihia te otinga, ā, rongoatia ki raro i te 4 °C.
I arotakehia motuhaketia te wehenga o ngā ranunga peptide me ngā keri trypsin HSA i runga i ngā pou PMP. Tirohia te wehenga o te ranunga peptide me te keri trypsin o HSA e te pou PMP, ā, whakatairitehia ngā hua ki te pou Ascentis Express RP-Amide. Ka tatauhia te tau pereti ariā penei:
Kei te whakaatuhia ngā whakaahua SEM o ngā matūriki silica tahanga me ngā matūriki silica e herea ana e te ligand i te PIK. 2. E whakaatu ana ngā whakaahua SEM o ngā matūriki silica tahanga (A, B), he rerekē ki ā mātou rangahau o mua, he porowhita ēnei matūriki, ā, he roa, he ōrite koretake rānei ngā matūriki. He maeneene ake te mata o ngā matūriki silica e herea ana e te ligand (C, D) i ērā o ngā matūriki silica tahanga, tērā pea nā te paninga o ngā mekameka polystyrene i runga i te mata o ngā matūriki silica.
Ngā whakaahua o te karu hiko matawai o ngā matūriki silica tahanga (A, B) me ngā matūriki silica herea-ligan (C, D).
Ko ngā tohatoha rahi matūriki o ngā matūriki silica tahanga me ngā matūriki silica here-ligan e whakaaturia ana i te Pikitia 3(A). I ​​whakaatuhia e ngā ānau tohatoha rahi matūriki e hangai ana ki te rōrahi te pikinga o te rahi o ngā matūriki silica i muri i te whakarerekētanga matū (Pikitia 3A). Ko ngā raraunga tohatoha rahi matūriki o ngā matūriki silica mai i te rangahau o nāianei me te rangahau o mua ka whakaritea ki te Ripanga 1(A). Ko te rahi matūriki e hangai ana ki te rōrahi, d(0.5), o te PMP he 3.36 μm, ki te whakataurite ki tā mātou rangahau o mua me te uara ad(0.5) o te 3.05 μm (ngā matūriki silica here-polystyrene)34. He whaiti ake te tohatoha rahi matūriki o tēnei puranga ki tā mātou rangahau o mua nā te rerekētanga o ngā ōwehenga o te PEG, te urea, te TMOS, me te waikawa acetic i roto i te ranunga tauhohenga. He paku nui ake te rahi matūriki o te wāhanga PMP i tērā o te wāhanga matūriki silica here-polystyrene i akohia e mātou i mua. Ko te tikanga o tēnei ko te mahi mata o ngā matūriki silica me te styrene i whakatakoto noa i te paparanga polystyrene (0.97 µm) ki runga i te mata silica, engari i te wāhanga PMP ko te matotoru o te paparanga he 1.38 µm.
Te tohatoha rahi o te matūriki (A) me te tohatoha rahi o ngā pūwero (B) o ngā matūriki silica tahanga me ngā matūriki silica e herea ana ki te ligand.
Ko te rahi o ngā pūwero, te rōrahi o ngā pūwero me te horahanga o te mata o ngā matūriki silica o tēnei rangahau kei roto i te Ripanga 1(B). Ko ngā kōtaha PSD o ngā matūriki silica tahanga me ngā matūriki silica here-ligan e whakaaturia ana i te Pikitia 3(B). He rite ngā hua ki tā mātou rangahau o mua. Ko ngā rahi o ngā pūwero o ngā matūriki silica tahanga me ngā matūriki silica here-ligan he 310 me te 241, ia, e tohu ana ka heke te rahi o ngā pūwero mā te 69 i muri i te whakarerekētanga matū, e whakaaturia ana i te Ripanga 1(B), ā, ko te huringa o te pihi e whakaaturia ana i te Pikitia 3(B). Waihoki, i heke te rōrahi o ngā pūwero o ngā matūriki silica mai i te 0.67 ki te 0.58 cm3/g i muri i te whakarerekētanga matū. Ko te horahanga mata motuhake o ngā matūriki silica e akohia ana i tēnei wā ko te 116 m2/g, he rite ki tā mātou rangahau o mua (124 m2/g). E whakaaturia ana i te Ripanga 1(B), i heke anō hoki te horahanga mata (m2/g) o ngā matūriki silica mai i te 116 m2/g ki te 105 m2/g i muri i te whakarerekētanga matū. whakarerekētanga
Ko ngā hua o te tātari huānga o te wāhanga tūmau e whakaaturia ana i te Ripanga 2. Ko te utaina waro o te wāhanga tūmau o nāianei he 6.35%, he iti iho i te utaina waro o tā mātou rangahau o mua (ngā matūriki silica herea polystyrene, 7.93%35 me te 10.21%, ia). 42. He iti te utaina waro o te wāhanga tūmau o nāianei, Nā te mea i roto i te whakarite o te SP o nāianei, hei tāpiri atu ki te styrene, i whakamahia ētahi ligands polar pērā i te phenylmaleimide-methylvinylisocyanate (PCMP) me te 4-hydroxy-TEMPO. Ko te ōrau taumaha hauota o te wāhanga tūmau o nāianei he 2.21%, ki te whakaritea ki te 0.1735 me te 0.85% mā te taumaha o te hauota i ngā rangahau o mua. Ko te tikanga o tēnei he teitei ake te ōrau taumaha o te hauota i roto i te wāhanga tūmau o nāianei nā te phenylmaleimide. Waihoki, ko ngā utaina waro o ngā hua (4) me te (5) he 2.7% me te 2.9%, ia, ko te utaina waro o te whakamutunga Ko te hua (6) he 6.35%, e whakaaturia ana i te Ripanga 2. I tirohia te whakahekenga taumaha mā te PMP stationary phase, ā, e whakaaturia ana te pihi TGA i te Pikitia 4. E whakaatu ana te pihi TGA i te whakahekenga taumaha o te 8.6%, he mea e rite ana ki te uta waro (6.35%) nā te mea ehara i te mea ko te C anake kei roto i ngā ligands engari ko te N, O, me te H anō hoki.
I whiriwhiria te ligand phenylmaleimide-methylvinylisocyanate mō te whakarerekētanga mata o ngā matūriki silica nā te mea he rōpū phenylmaleimide polar me ngā rōpū vinylisocyanate. Ka taea e ngā rōpū vinyl isocyanate te tauhohe atu ki te styrene mā te polymerization radical ora. Ko te take tuarua ko te whakauru i tētahi rōpū he taunekeneke ngāwari ki te analyte, ā, kāore he taunekeneke hiko kaha i waenga i te analyte me te wāhanga tūmau, nā te mea kāore he utu mariko o te wāhanga phenylmaleimide i te pH noa. Ka taea te whakahaere i te polarity o te wāhanga tūmau mā te nui o te styrene pai rawa atu me te wā tauhohenga o te polymerization radical kore utu. He mea nui te taahiraa whakamutunga o te tauhohenga (polymerization radical kore utu) ā, ka taea te whakarerekē i te polarity o te wāhanga tūmau. I mahia te tātari huānga hei tirotiro i te uta waro o ēnei wāhanga tūmau. I kitea ko te whakanui ake i te nui o te styrene me te wā tauhohenga i whakanui ake i te uta waro o te wāhanga tūmau, me te ritenga kē. Ko ngā SP i whakaritea me ngā kukū rerekē o te styrene he uta waro rerekē. Anō, utaina ēnei wāhanga tūmau ki roto i ngā pou kowiri tira, ā, tirohia ō rātou mahi chromatographic. (te whiriwhiringa, te taumira, te uara N, me ētahi atu). I runga i ēnei whakamātautau, i tīpakohia he whakatakotoranga kua arotauhia hei whakarite i te wāhanga tūmau PMP kia mau ai te polarity whakahaeretia me te pupuri pai o te analyte.
I arotakehia hoki ngā ranunga peptide e rima (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) mā te whakamahi i te pou PMP mā te whakamahi i te wāhanga nekeneke; 60/40 (v/v) acetonitrile/wai ​​(0.1% TFA) i te tere rere o te 80 μL/min. I raro i ngā tikanga elution tino pai, ko te tau pereti ariā (N) mō ia pou (100 × 1.8 mm id) he 20,000 ± 100 (200,000 pereti/m²). Kei te Ripanga 3 ngā uara N mō ngā pou PMP e toru, ā, kei te whakaatuhia ngā chromatogram i te Pikitia 5A. He tātari tere i runga i te pou PMP i te tere rere teitei (700 μL/min), e rima ngā peptide i eluhia i roto i te meneti kotahi, he tino pai ngā uara N, 13,500 ± 330 mō ia pou (100 × 1.8 mm id), e rite ana ki te 135,000 pereti/m (Pikitia 5B). E toru ngā pou rite te rahi (100 × 1.8 mm id) i whakakīia ki ngā rota rerekē e toru o te wāhanga tūmau PMP hei tirotiro i te taea te whakaputa anō. Ko te I tuhia te kukū o te tātaritanga mō ia pou mā te whakamahi i ngā tikanga whakapūtanga pai rawa atu me te maha o ngā pereti ariā N me te wā pupuri hei wehe i te ranunga whakamātautau kotahi i runga i ia pou. Kei te Ripanga 4 ngā raraunga tāruarua mō ngā pou PMP. He pai te hononga o te tāruarua o te pou PMP ki ngā uara %RSD tino iti, e whakaaturia ana i te Ripanga 3.
Te wehenga o te ranunga peptide i runga i te pou PMP (B) me te pou Ascentis Express RP-Amide (A); te wāhanga neke 60/40 ACN/H2O (TFA 0.1%), ngā rahi o te pou PMP (100 × 1.8 mm id); te tātaritanga Ko te raupapa elution o ngā pūhui: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) me te 5 (leucine) waikawa enkephalin)).
I arotakehia he pou PMP (100 × 1.8 mm id) mō te wehenga o ngā keri tryptic o te albumin toto tangata i roto i te chromatography wai mahi teitei. E whakaatu ana te chromatogram i te Pikitia 6 he pai te wehenga o te tauira, ā, he tino pai te taumira. I tātarihia ngā keri HSA mā te whakamahi i te tere rere o te 100 µL/min, te wāhanga neke 70/30 acetonitrile/wai ​​me te 0.1% TFA. E ai ki te chromatogram (Pikitia 6), kua wehea te keri HSA kia 17 ngā tihi e rite ana ki te 17 peptides. I tatauhia te whai huatanga wehenga o ia tihi i roto i te keri HSA, ā, kua hoatu ngā uara ki te Ripanga 5.
I wehea he whakarāpopototanga tryptic o te HSA (100 × 1.8 mm id) i runga i te pou PMP; te tere rere (100 µL/min), te wāhanga neke 60/40 acetonitrile/wai ​​me te 0.1% TFA.
ko L te roa o te pou, ko η te matotoru o te wāhanga neke, ko ΔP te pēhanga whakamuri o te pou, ā, ko u te tere raina o te wāhanga neke. Ko te urunga o te pou PMP he 2.5 × 10-14 m2, ko te tere rere he 25 μL/min, ā, i whakamahia he 60/40 v/v ACN/wai. Ko te urunga o te pou PMP (100 × 1.8 mm id) he rite ki tā mātou rangahau o mua Ref.34. Ko te urunga o te pou kua kī tonu i ngā matūriki porous papaku ko: 1.7 × 10-15 mō ngā matūriki 1.3 μm, 3.1 × 10-15 mō ngā matūriki 1.7 μm, 5.2 × 10-15 me te 2.5 × 10-14 m2 mō ngā matūriki 2.6 μm Mō ngā matūriki 5 μm 43. Nō reira, ko te urunga o te wāhanga PMP he rite ki ngā matūriki anga-matua 5 μm.
ko Wx te taumaha o te pou kua whakakiia ki te chloroform, ko Wy te taumaha o te pou kua whakakiia ki te methanol, ā, ko ρ te kiato o te whakarewa. Ko ngā kiato o te methanol (ρ = 0.7866) me te chloroform (ρ = 1.484). Ko te porosity katoa o ngā pou SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 me ngā pou C18-Urea 31 i akohia e mātou i mua ko 0.63 me 0.55, ia. Ko te tikanga o tēnei ko te aroaro o ngā ligands urea ka whakaiti i te urunga o te wāhanga tūmau. I tētahi atu taha, ko te porosity katoa o te pou PMP (100 × 1.8 mm id) ko 0.60. He iti ake te urunga o ngā pou PMP i tērā o ngā pou kua whakakiia ki ngā matūriki silica kua herea ki te C18 nā te mea i roto i ngā wāhanga tūmau momo C18 ka piri ngā ligands C18 ki ngā matūriki silica hei mekameka raina, i roto i ngā wāhanga tūmau momo polystyrene, ka hangaia te paparanga polymer matotoru A huri noa i te I roto i tētahi whakamātautau noa, ka tatauhia te porosity o te pou penei:
E whakaatu ana te Pikitia 7A,B i te pou PMP (100 × 1.8 mm id) me te pou Ascentis Express RP-Amide (100 × 1.8 mm id) mā te whakamahi i ngā tikanga elution ōrite (arā, 60/40 ACN/H2O me te 0.1% TFA). ) o te kauwhata van Deemter. I whakaritea ngā ranunga peptide kua tīpakohia (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) i roto i te 20 µL/. Ko te tere rere iti rawa mō ngā pou e rua ko te 800 µL/min. Ko ngā uara HETP iti rawa i te tere rere tino pai (80 µL/min) mō te pou PMP me te pou Ascentis Express RP-Amide ko te 2.6 µm me te 3.9 µm, ia. E whakaatu ana ngā uara HETP he pai ake te whai huatanga wehenga o te pou PMP (100 × 1.8 mm id) i te pou Ascentis Express RP-Amide e wātea ana i te hokohoko (100 × 1.8 mm id). E whakaatu ana te kauwhata van Deemter i te Pikitia 7(A) kāore te hekenga o te uara N me te piki haere o te rere i te mea nui ki tā mātou rangahau o mua. Ko te whai huatanga wehenga teitei ake o te pou PMP (100 × 1.8 mm id) ki te whakarite ki te pou Ascentis Express RP-Amide e hangai ana ki ngā whakapainga o te āhua matūriki, te rahi, me ngā tikanga tākai pou matatini e whakamahia ana i roto i te mahi o nāianei34.
(A) kauwhata van Deemter (HETP versus tere raina ā-wāhanga nekeneke) i whiwhi mā te whakamahi i te pou PMP (100 × 1.8 mm id) i roto i te 60/40 ACN/H2O me te 0.1% TFA.(B) kauwhata van Deemter (HETP versus tere raina ā-wāhanga nekeneke) i whiwhi mā te whakamahi i te pou Ascentis Express RP-Amide (100 × 1.8 mm id) i roto i te 60/40 ACN/H2O me te 0.1% TFA.
I whakaritea, ā, i aromatawaitia he wāhanga tūmau polystyrene kua whakauruhia ki te porowhita mō te wehenga o ngā ranunga peptide hangai me ngā nakunaku trypsin o te albumin toto tangata (HAS) i roto i te chromatography wai mahi teitei. He tino pai te mahi chromatography o ngā pou PMP mō ngā ranunga peptide i roto i te pai o te wehenga me te taumira. Ko te pai ake o te mahi wehenga o ngā pou PMP nā te maha o ngā take, pērā i te rahi o te matūriki me te rahi o ngā pore o ngā matūriki silica, te hanganga whakahaere o te wāhanga tūmau, me te tākai pou matatini. Haunga te pai o te wehenga, ko te iti o te pēhanga whakamuri o te pou i ngā tere rere teitei tētahi atu painga o tēnei wāhanga tūmau. He pai te whakaputa uri o ngā pou PMP, ā, ka taea te whakamahi mō te tātari i ngā ranunga peptide me te nakunaku trypsin o ngā momo pūmua. Kei te whakaaro mātou ki te whakamahi i tēnei pou mō te wehenga o ngā hua taiao, ngā matū koiora mai i ngā tipu rongoā me ngā tango harore i roto i te chromatography wai. I te heke mai, ka aromatawaitia hoki ngā pou PMP mō te wehenga o ngā pūmua me ngā paturopi monoclonal.
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