Ngā mihi nui ki a koe mō tō toro mai ki Nature.com. Kei te whakamahi koe i tētahi putanga pūtirotiro me te iti o te tautoko CSS. Mō te wheako pai rawa atu, ka tūtohu mātou kia whakamahia e koe tētahi pūtirotiro kua whakahoutia (kia mono rānei i te Aratau Hototahi i roto i te Internet Explorer). Hei tāpiri, hei whakarite i te tautoko tonu, ka whakaatuhia e mātou te pae me te kore he momo kāhua me te JavaScript.
E whakaatu ana i tētahi carousel o ngā kiriata e toru i te wā kotahi. Whakamahia ngā pātene O mua me O muri hei neke i roto i ngā kiriata e toru i te wā kotahi, whakamahia rānei ngā pātene reti i te mutunga hei neke i roto i ngā kiriata e toru i te wā kotahi.
I whakaritea ngā matūriki silica porous mā te tikanga sol-gel me ētahi whakarerekētanga hei whiwhi i ngā matūriki whānui-porous. I whakatōngia ēnei matūriki ki te N-phenylmaleimide-methylvinyl isocyanate (PMI) me te styrene mā te whakapūtātanga whakawhiti mekameka whakamuri (RAFT) hei whakaputa i ngā polyamides kua whakauruhia ki roto i te N-phenylmaleimide. I whakakīia te Styrene (PMP) ki te wāhanga tūmau. I whakakīia ngā pou maitai kore waikura whaiti (100 × 1.8 mm te whānui o roto) ki te takai paru. I aromatawaihia te mahi chromatographic o te pou PMP hei wehe i te ranunga o ngā peptides hangai kei roto ko ngā peptides e rima (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) me te tryptic hydrolyzate o te albumin serum tangata (HAS). I raro i ngā tikanga elution tino pai, ko te maha o ngā pereti me te ranunga o ngā peptides i eke ki te 280,000 pereti/sq.m. I te whakatairite i te mahi wehenga o te pou i whakawhanakehia ki te pou Ascentis Express RP-Amide arumoni, i kitea he pai ake te whai huatanga wehenga o te pou PMP i te pou arumoni e pā ana ki te whai huatanga wehenga me te taumira.
Kua huri te umanga rongoā koiora hei mākete whānui o te ao, me te pikinga nui o te wahanga mākete i ngā tau tata nei. Nā te tipu tere o te umanga rongoā koiora1,2,3, he nui te hiahia mō te tātari peptide me te pūmua. Haunga te peptide ūnga, ka puta ngā poke maha i te wā o te hanganga peptide, nō reira me purenga chromatographic kia whiwhi ai i te parakore e hiahiatia ana o te peptide. 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 ine papatipu mō te raupapatanga o ngā peptide me ngā pūmua, ki te whakaurua tika aua tauira ki roto i te ine papatipu, kāore e pai te wehenga. Ka taea te whakatau i tēnei raruraru mā te mahi i te chromatography wai (LC) i mua i te tātari MS, ka whakaiti i te nui o ngā tātari e uru ana ki te ine papatipu i tētahi wā kua whakaritea4,5,6. Hei tāpiri, ka taea e ngā tātari te kukū ki tētahi rohe whaiti i te wā o te wehenga wāhanga wai, ā, ka kukū ēnei tātari me te whakanui ake i te aro o te kitenga MS. Kua tino whanake te chromatography wai (LC) i ngā tau tekau kua pahure ake nei, ā, kua noho hei tikanga whānuitia mō te tātari proteomic7,8,9,10.
He whānuitia te whakamahinga o te chromatography wai whakamuri-wāhanga (RP-LC) hei pure me te wehewehe i ngā ranunga o ngā peptide mā te whakamahi i te silica kua whakarerekētia octadecyl (ODS) hei wāhanga tūmau11,12,13. Heoi, nā te hanganga uaua me te āhua amphoteric,14,15 kāore e taea e ngā wāhanga tūmau RP te whakarato i te wehenga pai o ngā peptide me ngā pūmua. Nō reira, ko te tātari i ngā peptide me ngā pūmua me ngā kongakonga polar me ngā kongakonga kore-polar me whai wāhanga tūmau motuhake hei taunekeneke me te pupuri i ēnei tātaritanga16. Ko te chromatography whakauru, e tuku ana i ngā taunekeneke maha-momo, ka taea te noho hei huarahi kē atu i te RP-LC mō te wehewehe i ngā peptide, ngā pūmua, me ētahi atu ranunga uaua. He maha ngā wāhanga tūmau momo-whakauru i whakaritea, ā, i whakamahia ngā pou kua whakakiia ki ēnei wāhanga tūmau hei wehewehe i ngā peptide me ngā pūmua17,18,19,20,21. Nā te noho mai o ngā rōpū porowhita me ngā rōpū kore-porowhita, he pai ngā āhua tūmau aratau whakauru (WAX/RPLC, HILIC/RPLC, intercalation porowhita/RPLC) mō te wehenga o ngā peptides me ngā pūmua22,23,24,25,26,27,28. , ko ngā āhua tūmau intercalated porowhita me ngā rōpū porowhita hono covalently e whakaatu ana i ngā āheinga wehenga pai me te whiriwhiri ahurei mō ngā analytes porowhita me ngā kore-porowhita nā te mea ka whakawhirinaki te wehenga ki te taunekeneke i waenga i te analyte me te āhua tūmau Ngā taunekeneke maha-momo 29,30,31,32. Nō nā tata nei, i whiwhi a Zhang et al. 30 i ngā āhua tūmau behenyl-terminated o ngā polyamines, ā, i wehe angitu i ngā warowaihā, ngā rongoā ārai pouri, ngā flavonoids, ngā nucleosides, ngā estrogens, me ētahi atu analytes. He rōpū porowhita me ngā rōpū kore-porowhita te rauemi tūmau kua whakauruhia ki te porowhita, nō reira ka taea te whakamahi hei wehe i ngā peptides me ngā pūmua ki ngā wāhanga hydrophobic me hydrophilic. Kei te wātea ngā pou whakarārangi-polar (hei tauira, ngā pou C18 me te amide whakarārangi-polar) i raro i te ingoa hokohoko Ascentis Express RP-Amide, engari ko ēnei pou kua whakamahia mō te tātaritanga o te amine 33 anake.
I roto i te rangahau o nāianei, i whakaritea he wāhanga tūmau whakauru pōro (N-phenylmaleimide, whakauru polystyrene) me te aromatawai mō te wehenga peptide me te wehenga tryptic HSA. I whakamahia te rautaki e whai ake nei hei whakarite i te wāhanga tūmau. I whakaritea ngā matūriki silica porous i runga i ngā tikanga kua whakaahuatia i roto i ā mātou pukapuka o mua, me ētahi huringa i roto i ngā kaupapa whakarite 31, 34, 35, 36, 37, 38, 39. I whakatikatikaina ngā ōwehenga o te urea, te polyethylene glycol (PEG), te TMOS me te waikawa wai-acetic kia whiwhi ai i ngā matūriki silica me ngā rahi pore nui. Tuarua, i hangaia he ligand phenylmaleimide-methylvinyl isocyanate hou, ā, i whakamahia ōna matūriki silica i ahu mai i te matūriki hei whakarite i ngā wāhanga tūmau whakauru pōro. I tākaihia te wāhanga tūmau i whiwhihia ki roto i tētahi pou kowiri tira (diameter o roto 100 × 1.8 mm) i runga anō i tētahi kaupapa tākai kua arotauhia. Ka āwhinahia te tākai o te pou e te wiri miihini hei whakarite i tētahi paparanga ōrite i roto i te pou. I arotakehia te pou kua tākaihia mō te wehenga o tētahi ranunga peptides kei roto ko ngā peptides e rima (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide), me ngā hydrolysates tryptic o te albumin serum tangata (HSA). I ​​kitea he pai te whakataunga me te whai huatanga o te wehenga o te ranunga peptide me te HSA tryptic digest. I whakaritea te whai huatanga wehenga o te pou PMP ki te pou Ascentis Express RP-Amide. I kitea he pai te whakataunga me te whai huatanga wehenga o ngā peptides me ngā pūmua i runga i te pou PMP, ā, he teitei ake te whai huatanga wehenga o te pou PMP i te pou Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, waikawa acetic, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, albumin toto tangata (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy-TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) mō te HPLC, methanol, 2-propanol me te acetone. Kamupene Sigma-Aldrich (St. Louis, Missouri, 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, ā, i tāpirihia he 24 ml o te TMOS ki roto i raro i te whakamatao hukapapa. 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 ki te 70°C mō te 12 hāora. I hurihia māeneene ngā poraka ngohengohe maroke, ā, i whakakāhia ki roto i te oumu i te 550°C mō te 12 hāora. E toru ngā puranga i whakaritea, ā, i whakaahuatia hei whakamātautau i te āheinga ki te whakaputa anō i ngā rahi matūriki, te rahi o ngā pore me te horahanga o te mata.
Rōpū pōro me te wāhanga tūmau mō ngā mekameka polystyrene. Kei raro nei te whakamārama i te tukanga whakarite.
I whakarewaina te N-phenylmaleimide (200 mg) me te methyl vinyl isocyanate (100 mg) ki roto i te toluene anhydrous, kātahi ka tāpirihia te 0.1 ml o te 2,2′-azoisobutyronitrile (AIBN) ki te pounamu tauhohenga hei whiwhi i te copolymer o te phenylmaleimide me te methyl vinyl isocyanate (PMCP). ) I whakamahanahia te ranunga ki te 60°C mō ngā hāora e 3, i tātarihia, ā, i whakamarokehia 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 mō te 10 meneti ki roto i te pounamu raro porowhita 500 ml. I whakarewaina te PMCP (10 mg) ki roto i te toluene, ā, i tāpirihia kia maturuturu ki te pounamu tauhohenga mā te hāpara tāpiri. I whakahurihia te ranunga ki te 100°C mō te 8 hāora, i tātarihia, i horoia ki te acetone, ā, i whakamaroketia ki te 60°C mō te 3 hāora. Kātahi ka whakarewaina ngā matūriki silica e hono ana ki te PMCP (100 g) ki roto i te toluene (200 ml), ā, i tāpirihia te 4-hydroxy-TEMPO (2 ml) ki roto 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 i piri 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 mō te pō i te 60°C. Kei te whakaatuhia te kaupapa whānui o te tauhohenga i te pikitia tuatahi.
I tangohia te hau o ngā tauira i te 393 K mō te 1 hāora tae noa ki te whiwhi i te pēhanga toenga i raro i te 10–3 Torr. Ko te nui o te N2 i mimitihia i te pēhanga whanaunga P/P0 = 0.99 i whakamahia hei whakatau i te rōrahi katoa o ngā pore. I tirohia te āhua o ngā matūriki silica parakore me ngā matūriki silica e herea ana ki te ligand mā te whakamahi i te karu hiko matawai (Hitachi High Technologies, Tokyo, Hapani). I whakatakotoria ngā tauira maroke (ngā matūriki silica parakore me ngā matūriki silica e herea ana ki te ligand) ki runga i ngā tokotoko konumohe mā te whakamahi i te rīpene waro. I whakatakotoria te koura ki runga i te tauira mā te whakamahi i te taputapu pupuhi Q150T, ā, i whakatakotoria he paparanga Au matotoru 5 nm ki runga i te tauira. Mā tēnei ka whakapai ake i te whai huatanga o te tukanga ngaohiko iti, ā, ka whakaratohia he rehu matao pai. I mahia te tātari huānga mā te whakamahi i te tātari hanganga huānga Thermo Electron (Waltham, MA, USA) Flash EA1112. I whakamahia he tātari rahi matūriki Malvern (Worcestershire, UK) Mastersizer 2000 hei tiki i te tohatoha rahi matūriki. I whakamararatia ngā matūriki silica kāore i pania me ngā matūriki silica e herea ana ki te ligand (5 mg ia) ki roto i te 5 ml o te isopropanol, i whakakorikoria mō te 10 meneti, i whakaohokia mō te 5 meneti, ā, i whakatakotoria ki runga i te paepae whatu Mastersizer. Ka mahia te tātaritanga thermogravimetric i te tere o te 5 °C ia meneti i roto i te awhe pāmahana mai i te 30 ki te 800 °C.
I tākaihia ngā pou kowiri tira whaiti kua whakakikoruatia ki te muka karāhe, he whārite (ID 100 × 1.8 mm) mā te tikanga whakakī paru, e whai ana i te tukanga kotahi i roto i te tohutoro 31. I honoa te pou kowiri tira (kua whakakikoruatia ki te karāhe, ID 100 × 1 .8 mm) me te putanga kei roto he frit 1 µm ki tētahi mīhini tākai paru (Alltech Deerfield, IL, USA). Whakaritea he whakatārewatanga o te wāhanga tūmau mā te whakatārewa i te 150 mg o te wāhanga tūmau ki roto i te 1.2 ml o te methanol me te whāngai ki roto i tētahi pou puna. I whakamahia te methanol hei whakarewa paru me te whakarewa whakahaere. Tākaihia te pou mā te whakamahi i te raupapa pēhanga o te 100 MP mō te 10 meneti, 80 MP mō te 15 meneti, me te 60 MP mō te 30 meneti. I whakamahia e te tukanga tākai e rua ngā wiri pou chromatography hau (Alltech, Deerfield, IL, USA) mō te wiri miihini kia rite ai te tākai pou. Katia te tākai paru ka tukuna mārire te pēhanga kia kore ai e pakaru te aho. I momotuhia te pou i te pūwero para, ā, i tāpirihia tētahi atu hononga ki te pūwero tomo, ā, i honoa ki te pūnaha LC hei whakamātautau i tana mahi.
I hangaia he MLC ritenga mā te whakamahi i tētahi papu LC (10AD Shimadzu, Hapani), he tauira me te koropiko werohanga 50 nL (Valco (USA) C14 W.05), he tango hau kiriuhi (Shimadzu DGU-14A), me tētahi matapihi kapiti UV-VIS. Pūrere kimi (UV-2075) me tētahi pou iti enamel. Whakamahia he ngongo hononga tino whaiti, poto hoki hei whakaiti i te pānga o te whakawhānui atu o te pou. I muri i te whakakī i te pou, tāutahia he kapiti (50 µm id 365) ki te putanga o te hononga whakaiti 1/16″ ka tāutahia he kapiti (50 µm) o te hononga whakaiti. Ka mahia te kohikohinga raraunga me te tukatuka chromatogram mā te whakamahi i te pūmanawa Multichro 2000. I te 254 nm, i aroturukitia te mimiti UV o ngā tātaritanga kaupapa i te 0. I tātarihia ngā raraunga chromatographic mā te whakamahi i te OriginPro8 (Northampton, MA).
Pūmua toto tangata, paura lyophilized, ≥ 96% (hikohiko agarose gel) 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 te ranunga o ngā peptide me te tryptic digest HSA i runga i te pou PMP. Tirohia te hydrolysis tryptic o te ranunga o ngā peptide me te HSA i wehea e te pou PMP, ā, whakatairitehia ngā hua ki te pou Ascentis Express RP-Amide. Ka tatauhia te maha o ngā pereti ariā mā te whakamahi i te whārite e whai ake nei:
Kei te Pikitia 2 ngā whakaahua SEM o ngā matūriki silica parakore me ngā matūriki silica e herea ana ki te ligand. E whakaatu ana ngā whakaahua SEM o ngā matūriki silica parakore (A, B) i tētahi āhua porowhita e toro atu ana ngā matūriki, e ōrite ana rānei ki ā mātou rangahau o mua. He maeneene ake te mata o ngā matūriki silica e herea ana e te ligand (C, D) i ērā o ngā matūriki silica parakore, tērā pea nā ngā mekameka polystyrene e kapi ana i te mata o ngā matūriki silica.
Ngā whakaahua iti o ngā irahiko matawai o ngā matūriki silica parakore (A, B) me ngā matūriki silica e herea ana ki te ligand (C, D).
Ko te tohatoha rahi matūriki o ngā matūriki silica parakore me ngā matūriki silica e herea ana e te ligand e whakaaturia ana i te Pikitia 2. 3(A). I ​​whakaatuhia e ngā ānau tohatoha rahi matūriki rōrahi te pikinga o te rahi matūriki silica i muri i te whakarerekētanga matū (Pikitia 3A). Ko ngā raraunga tohatoha rahi matūriki silica mai i te rangahau o nāianei me te rangahau o mua ka whakaritea ki te Ripanga 1(A). Ko te rahi matūriki rōrahi d(0.5) o te PMP he 3.36 µm, ki te whakataurite ki te uara ad(0.5) o te 3.05 µm i roto i tā mātou rangahau o mua (ngā matūriki silica herea polystyrene)34. Nā te huringa o te ōwehenga o te PEG, te urea, te TMOS me te waikawa acetic i roto i te ranunga tauhohenga, he whaiti ake te tohatoha rahi matūriki o tēnei puranga ki tā mātou rangahau o mua. He paku nui ake te rahi matūriki o te wāhanga PMP i te wāhanga matūriki silica herea polystyrene i akohia e mātou i mua. Ko te tikanga o tēnei ko te mahi mata o ngā matūriki silica me te styrene i whakatakotoria he paparanga polystyrene anake (0.97 µm) ki runga i te mata silica, i te wā 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 parakore 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 i whakamahia i roto i tēnei rangahau e whakaaturia ana i te Ripanga 1 (B). Ko ngā kōtaha PSD o ngā matūriki silica parakore me ngā matūriki silica e herea ana ki te ligand e whakaaturia ana i roto i ngā Pikitia 3(B). He rite ngā hua ki tā mātou rangahau o mua34. Ko ngā rahi o ngā pūwero o ngā matūriki silica parakore me ngā matūriki silica e herea ana ki te ligand he 310 Å me te 241 Å, ia, e tohu ana i muri i te whakarerekētanga matū, i heke te rahi o ngā pūwero mā te 69 Å, e whakaaturia ana i roto i te Ripanga 1 (B), ā, ko te pihi nekehanga e whakaaturia ana i roto i te Pikitia. Ko te horahanga mata motuhake o ngā matūriki silica i roto i te rangahau o nāianei ko te 116 m2/g, he rite ki tā mātou rangahau o mua (124 m2/g). E whakaaturia ana i roto i te Ripanga 1(B), ko te horahanga mata (m2/g) o ngā matūriki silica i muri i te whakarerekētanga matū i heke anō mai i te 116 m2/g ki te 105 m2/g.
Kei te Ripanga 2 ngā hua o te tātari huānga o te wāhanga tūmau. Ko te ihirangi waro o te wāhanga tūmau o nāianei he 6.35%, he iti iho i tā mātou rangahau o mua (ngā matūriki silica e hono ana ki te polystyrene, 7.93%35 me te 10.21%, ia) 42. Kei raro nei te ihirangi waro o te wāhanga tūmau o nāianei, nā te mea kua whakamahia ētahi ligands polar pērā i te phenylmaleimide methyl vinyl isocyanate (PCMP) me te 4-hydroxy-TEMPO hei tāpiri atu ki te styrene i roto i te whakarite SP. Ko te ōrau taumaha o te hauota i roto i te wāhanga tūmau o nāianei he 2.21% ki te whakarite ki te 0.1735 me te 0.85% i roto i ngā rangahau o mua42. Ko te tikanga he nui te ōrau taumaha o te hauota o te wāhanga tūmau o nāianei nā te phenylmaleimide. Waihoki, ko ngā hua (4) me (5) he 2.7% me te 2.9% te nui o te waro, ia, ko te hua whakamutunga (6) he 6.35% te nui o te waro, e whakaaturia ana i te Ripanga 2. I whakamahia te tātaritanga thermogravimetric (TGA) i runga i te wāhanga tūmau o te PMP hei whakamātautau mō te whakahekenga taumaha, 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%, e rite ana ki te nui o te 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-methylvinyl isocyanate hei whakarerekē i te mata o ngā matūriki silica nā ōna rōpū phenylmaleimide me vinylisocyanate polar. 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 tauhohenga ngāwari ki te analyte, ā, kāore he tauhohenga 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) nā te mea ka whakarerekē i te polarity o te wāhanga tūmau. I whakahaerehia te tātari huānga hei tirotiro i te ihirangi waro i roto i ēnei wāhanga tūmau. Kua kitea ko te whakanui ake i te nui o te styrene me te wā tauhohenga ka whakanui ake i te ihirangi waro o te wāhanga tūmau, ā, ko te ritenga kē. Ko ngā SP i whakaritea me ngā kukū rerekē o te styrene he rerekē ngā kawenga waro. Waihoki, i whakanohoia ēnei wāhanga tūmau ki runga i ngā pou kowiri tira, ā, i tirohia ō rātou āhuatanga chromatography (te whiriwhiri, te taumira, te uara N, me ētahi atu). I runga i ēnei whakamātautau, i whiriwhiria he hanganga kua arotauhia mō te whakarite i te wāhanga tūmau PMP hei whakarato i te polarity whakahaeretia me te pupuri pai o te analyte.
I arotakehia hoki te pou PMP mō te tātari i 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 kaha o te wāhanga neke. 60/40 (v/v) ACN/wai (0.1% TFA) i te tere rere o te 80 µl/min. I raro i ngā tikanga elution tino pai (200,000 pereti/m), ko te maha o ngā pereti ariā (N) mō ia pou (100 × 1.8 mm) he 20,000 ± 100. Ko ngā uara N mō ngā pou PMP e toru e whakaaturia ana i te Ripanga 3, ā, ko ngā chromatograms e whakaaturia ana i te Pikitia 5A. He tātari tere i te tere rere teitei (700 µl/min) i runga i te pou PMP, e rima ngā peptide i whakawāteatia i roto i te meneti kotahi, he uara N pai rawa atu o te 13,500 ± 330 mō ia pou (100 x 1.8 mm te whānui), he rite ki te 135,000 pereti/m (Pikitia 5B). E toru ngā pou o te rahi kotahi (te whānui o roto 100 x 1.8 mm) i whakakīia ki ngā puranga rerekē e toru o te wāhanga tūmau PMP hei whakamātautau i te āheinga tāruarua. I tuhia ngā tātaritanga mō ia pou mā te wehewehe i te ranunga whakamātautau kotahi i runga i ia pou mā te whakamahi i ngā tikanga tātaritanga pai rawa atu, te maha o ngā pereti ariā N, me te wā pupuri. Kei te Ripanga 4 ngā raraunga āheinga tāruarua mō ngā pou PMP. He pai te hononga o te āheinga tāruarua o te pou PMP ki ngā uara %RSD tino iti e whakaaturia ana i te Ripanga 3.
Te wehenga o ngā 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 x 1.8 mm id), te tātaritanga 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 (enkephalin waikawa leucic).
I arotakehia he pou PMP (diameter o roto 100 x 1.8 mm) mō te wehenga o te hydrolyzate tryptic o te albumin serum tangata mā te HPLC. E whakaatu ana te chromatogram i te Pikitia 6 he pai te wehenga o ngā tauira me te tino pai o te taumira. I tātarihia ngā otinga HSA mā te whakamahi i te tere rere o te 100 μl/min, he wāhanga neke o te 70/30 acetonitrile/wai ​​me te 0.1% TFA. I wehea te wehenga o te HSA kia 17 ngā tihi, e whakaaturia ana i te chromatogram (Pikitia 6), e rite ana ki te 17 peptides. I tatauhia te whai huatanga o te wehenga o ngā tihi takitahi mai i te hydrolyzate HSA, ā, kei te Ripanga 5 ngā uara.
I wehea ngā waikura HSA tryptic i runga i te pou PMP (diameter o roto 100 x 1.8 mm), 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, i whakamahia he 60/40 v/v. ACN/wai. Ko te urunga o te pou PMP (ID 100 × 1.8 mm) he rite ki tā mātou rangahau Ref.34 o mua. Ko te urunga o te pou kua whakakiia ki ngā matūriki porous papa ko 1.7×10 .6 µm, 2.5×10-14 m2 mō ngā matūriki 5 µm43. Nō reira, ko te urunga o te wāhanga PMP he rite ki te urunga o ngā matūriki anga-matua me te rahi o te 5 μm.
ko Wx te papatipu o te pou kua whakakīia ki te chloroform, ko Wy te papatipu o te pou kua whakakīia ki te methanol, ā, ko ρ te mātotoru o te whakarewa. Te mātotoru o te methanol (ρ = 0.7866) me te chloroform (ρ = 1.484). Ko te porosity katoa o te pou matūriki silica-C18 (100 × 1.8 mm ID)34 me tā mātou pou C18-urea31 i akohia i mua ko 0.63 me 0.55, ia. Ko te tikanga o tēnei ko te noho mai 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 (diameter o roto 100 × 1.8 mm) ko 0.60. He iti ake te urunga o ngā pou PMP i ngā pou kua whakakīia 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 i roto i ngā mekameka raina, i roto i ngā wāhanga tūmau momo polystyrene ka hangaia he polymer matotoru huri noa i ngā matūriki. paparanga A. I roto i tētahi whakamātautau noa, ka tatauhia te pūngao o te pou penei:
I te pikitia 7A, B, e whakaatu ana i ngā kauwhata Van Deemter mō te pou PMP (id 100 x 1.8 mm) me te pou Ascentis Express RP-Amide (id 100 x 1.8 mm) i raro i ngā tikanga whakaputanga ōrite, 60/40 ACN/H2O me te 0 .1% TFA 20 µl/min ki te 800 µl/min i ngā pou e rua. Ko ngā uara HETP iti rawa i te tere rere pai (80 µl/min) he 2.6 µm me te 3.9 µm mō te pou PMP me te pou Ascentis Express RP-Amide. E whakaatu ana ngā uara HETP he nui ake te whai huatanga wehenga o te pou PMP (100 x 1.8 mm id) i tērā o te pou Ascentis Express RP-Amide e wātea ana i te hokohoko (100 x 1.8 mm id). E whakaatu ana te kauwhata van Deemter i te Pikitia 7(A) kāore te hekenga o te uara N i tino teitei ake me te pikinga o te rere ki tā mātou rangahau o mua. Ko te pai ake o te wehenga o te pou PMP (id 100 × 1.8 mm) ki te whakarite ki te pou Ascentis Express RP-Amide e hangai ana ki te āhua me te rahi o te matūriki kua whakapaitia me te tikanga tākai pou matatau e whakamahia ana i roto i ngā mahi o nāianei34.
(A) Kauwhata Van Deemter (HETP vs. tere raina ā-wāhanga nekeneke) i whiwhihia i runga i te pou PMP (id 100 x 1.8 mm) i roto i te 60/40 ACN/H2O me te 0.1% TFA. (B) Kauwhata Van Deemter (HETP vs tere raina ā-wāhanga nekeneke) i whiwhihia i runga i te pou Ascentis Express RP-Amide (id 100 x 1.8 mm) i roto i te 60/40 ACN/H2O me te 0.1% TFA.
I whakaritea, ā, i aromatawaitia hoki tētahi wāhanga tūmau porowhita o te polystyrene kua whakauruhia, hei wehewehe i te ranunga o ngā peptide hangai me te hydrolyzate tryptic o te albumin serum tangata (HSA) i roto i te chromatography wai mahi teitei. He tino pai te mahi chromatography o ngā pou PMP mō ngā ranunga peptide e pā ana ki te pai o te wehenga me te taumira. Ko te pai ake o te pai o te wehenga o ngā pou PMP nā ētahi take pēnei i te rahi o te matūriki silica me te rahi o ngā pore, te hanganga whakahaere o ngā wāhanga tūmau, me ngā rauemi tākai pou uaua. Haunga te pai o te wehenga teitei, ko tētahi atu painga o tēnei wāhanga tūmau ko te iti o te pēhanga whakamuri o te pou i ngā tere rere teitei. He tino taea te whakaputa anō i ngā pou PMP, ā, ka taea te whakamahi hei tātari i ngā ranunga o ngā peptide me te nakunaku tryptic o ngā momo pūmua. Kei te whakaaro mātou ki te whakamahi i tēnei pou hei wehewehe i ngā matū koiora mai i ngā hua taiao, ngā tangohanga o ngā tipu rongoā me ngā 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.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Te Rangahau mō ngā pūnaha wehenga peptide chromatography wāhanga whakahurihia wāhanga I: Te whakawhanaketanga o tētahi kawa mō te whakaahuatanga pou. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Te Rangahau mō ngā pūnaha wehenga peptide chromatography wāhanga whakahurihia wāhanga I: Te whakawhanaketanga o tētahi kawa mō te tautuhi i ngā pou.Field, JK, Owerby, MR, Lau, J., Togersen, H., me Petersson, P. Te Rangahau i ngā Pūnaha Wehewehe Peptide mā te Chromatography Āhua-Whakamuri, Wāhanga I: Te Whakawhanake i tētahi Tikanga mō te Whakaahuatanga Pou. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Te Rangahau mō ngā pūnaha wehenga peptide chromatography wāhanga whakahurihia wāhanga I: Te whakawhanaketanga o tētahi kawa mō ngā āhuatanga o te pou. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Te Rangahau mō ngā pūnaha wehenga peptide chromatography wāhanga whakahurihia wāhanga I: Te whakawhanaketanga o tētahi kawa mō ngā āhuatanga o te pou.Field, JK, Owerby, MR, Lau, J., Togersen, H., me Petersson, P. Te Rangahau i ngā Pūnaha Wehewehe Peptide mā te Chromatography Āhua-Whakamuri, Wāhanga I: Te Whakawhanake i tētahi Tikanga mō te Whakaahuatanga Pou.J.色谱法。 1603，113-129。 https://doi.org/10.1016/j.chroma.2019.05.038(2019).
Gomez, B. et al. Ngā tikanga mō te hanga i ngā peptide hohe kua whakapaitia hei rongoā i ngā mate pāwera. Hangarau Koiora. Ngā Whakatutukitanga 36(2), 415–429. https://doi.org/10.1016/j.biotechadv.2018.01.004 (2018).
Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Ngā peptide rongoā hangai: Pūtaiao me te mākete. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Ngā peptide rongoā hangai: Pūtaiao me te mākete.Vliege P, Lisowski V, Martinez J me Chreschatyski M. Ngā peptide rongoā hangai: te pūtaiao me te mākete.Vliege P, Lisowski V, Martinez J me Khreschatsky M. Ngā peptide rongoā hangai: te pūtaiao me te mākete. te kitenga rongoā. Today 15 (1–2), 40–56. https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Te chromatography wai proteomic matatau. Xie, F., Smith, RD & Shen, Y. Te chromatography wai proteomic matatau.Tirohia a F., Smith RD me Shen Yu. Te chromatography wai proteomic matatau. Xie, F., Smith, RD & Shen, Y. 高级蛋白质组液相色谱。 Xie, F., Smith, RD & Shen, Y. Hanganga pūmua matatau 液相色谱。Tirohia a F., Smith RD me Shen Yu. Te chromatography wai proteomic matatau.J. Te Whakamātautau Irahiko. A 1261, 78–90 (2012).
Liu, W. et al. Ka taea e te chromatography wai matatau-spectrometry papatipu te whakakotahi i te metabolomics whānui me te proteomics. anus. Chim. Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Te tūranga o te UHPLC i roto i te whanaketanga rongoā. Chesnut, SM & Salisbury, JJ Te tūranga o te UHPLC i roto i te whanaketanga rongoā.Chesnut, SM me Salisbury, JJ Te tūranga o te UHPLC i roto i te whanaketanga rongoā.Chesnut, SM me Salisbury, JJ Te tūranga o te UHPLC i roto i te whanaketanga rongoā. J. Sept Science. 30(8), 1183–1190 (2007).
Wu, N. & Clausen, AM Ngā āhuatanga taketake me ngā āhuatanga mahi o te chromatography wai pēhanga tino teitei mō ngā wehenga tere. Wu, N. & Clausen, AM Ngā āhuatanga taketake me ngā āhuatanga mahi o te chromatography wai pēhanga tino teitei mō ngā wehenga tere.Wu, N. me Clausen, AM Ngā āhuatanga matua me ngā āhuatanga mahi o te chromatography wai pēhanga teitei mō te wehenga tere. 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 Ngā āhuatanga taketake me ngā āhuatanga mahi o te chromatography wai pēhanga tino teitei mō te wehenga tere.Wu, N. me Clausen, AM Ngā āhuatanga matua me ngā āhuatanga mahi o te chromatography wai pēhanga teitei mō te wehenga tere.J. Sept. Pūtaiao. 30(8), 1167–1182. https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Te whakamahinga o te chromatography wai tino mahi i roto i te whanaketanga rongoā. Wren, SA & Tchelitcheff, P. Te whakamahinga o te chromatography wai tino mahi i roto i te whanaketanga rongoā.Ren, SA me Chelischeff, P. Te whakamahinga o te chromatography wai tino mahi teitei i roto i te whanaketanga rongoā. 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 me Chelischeff, P. Te whakamahinga o te chromatography wai tino mahi i roto i te whanaketanga rongoā.J. Te Whakamātautau Irahiko. 1119(1-2), 140-146. https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al. He hauwaiwai macroporous monolithic i ahu mai i te emulsion hinu-i-roto-i-te-wai me te wāhanga ā-roto teitei mō te purenga whai hua o te enterovirus 71. Kaupapa matū. Journal 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Te tūranga o te chromatography wai i roto i te proteomics. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Te tūranga o te chromatography wai i roto i te proteomics.Shi, Y., Xiang, R., Horvath, C. me Wilkins, JA Te tūranga o te chromatography wai i roto i te 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. me Wilkins, JA Te tūranga o te chromatography wai i roto i te proteomics.J. Te Whakamātautau Irahiko. A 1053 (1-2), 27-36 (2004).
Fekete, S., Vutey, J.-L. & Guillarme, D. Ngā au hou i roto i ngā wehenga chromatography wai-wāhanga-hurihuri o ngā peptide me ngā pūmua rongoā: Ariā me ngā tono. & Guillarme, D. Ngā au hou i roto i ngā wehenga chromatography wai-wāhanga-hurihuri o ngā peptide me ngā pūmua rongoā: Ariā me ngā tono. & Guillarme, D. Новые тенденции в разделении терапевтических пептидов и белков с помощью жидкостной хроматографира хроматографира: теория и приложения. & Guillarme, D. Ngā au hou i roto i te wehenga o ngā peptide me ngā pūmua rongoā mā te chromatography wai āhua whakamuri: te ariā me ngā tono. & 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.me Guillarmé, D. Ngā au hou i roto i te wehenga o ngā peptide me ngā pūmua rongoā mā te chromatography wai āhua whakamuri: te ariā me ngā tono.J. Pharm. Pūtaiao Hauora Koiora. anus. 69, 9–27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Te wehenga ā-rua o ngā peptide mā te whakamahi i te pūnaha RP-RP-HPLC me ngā pH rerekē i ngā ā-tuatahi me te ā-tuarua o te wehenga. Gilar, M., Olivova, P., Daly, AE & Gebler, JC Te wehenga ā-rua o ngā peptide mā te whakamahi i te pūnaha RP-RP-HPLC me ngā pH rerekē i ngā ā-tuatahi me te ā-tuarua o te wehenga.Gilar M., Olivova P., Dali AE me Gebler JK Wehenga ā-rua o ngā peptide mā te whakamahi i te pūnaha RP-RP-HPLC me ngā pH rerekē i ngā ā-wehenga tuatahi me te tuarua.Gilar M., Olivova P., Dali AE me Gebler JK Te wehenga ā-rua o ngā peptide mā te whakamahi i ngā uara pH rerekē i ngā ā-tuatahi me te tuarua o ngā ā-wāhanga mā te whakamahi i te pūnaha RP-RP-HPLC. J. Sept Science. 28 (14), 1694–1703 (2005).
Fellitti, S. et al. Te rangahau i te whakawhiti papatipu me ngā āhuatanga nekeneke o ngā pou chromatography mahi-teitei kua whakakiia ki ngā matūriki C18 tino pūwero me te papa pūwero iti iho i te 2 µm. J. Sept Science. 43 (9–10), 1737–1745 (2020).
Piovesana, S. et al. Ngā au hou me ngā wero tātari i roto i te wehenga, te tautuhi, me te whakamana i ngā peptide bioactive tipu. anus. Creature anal. Chemical. 410(15), 3425-3444. https://doi.org/10.1007/s00216-018-0852-x (2018).
Muller, JB me ētahi atu. Te whenua proteomic o te rangatiratanga o te ora. Nature 582 (7813), 592–596. https://doi.org/10.1038/s41586-020-2402-x (2020).
De Luca, K. et al. Te maimoatanga i muri i ngā peptide rongoā mā te chromatography wai whakarite. Molecules (Basel, Switzerland) 26(15), 4688 (2021).
Yang, Y. & Geng, X. Te chromatography aratau-whakaranu me ōna tono ki ngā biopolymers. Yang, Y. & Geng, X. Te chromatography aratau-whakaranu me ōna tono ki ngā biopolymers.Yang, Yu. me Geng, X. Te chromatography aratau whakauru me tōna tono ki ngā 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. Te chromatography aratau whakauru me tōna whakamahinga i roto i ngā biopolymers.Yang, Yu. me Gene, X. Te chromatography aratau whakauru me tōna tono ki ngā biopolymers.J. Te Whakamātautau Irahiko. A 1218(49), 8813–8825 (2011).