Mun gode da ziyartar Nature.com. Kuna amfani da sigar burauza mai ƙarancin tallafin CSS. Don mafi kyawun ƙwarewa, muna ba da shawarar ku yi amfani da burauzar da aka sabunta (ko ku kashe Yanayin Haɗin gwiwa a cikin Internet Explorer). Bugu da ƙari, don tabbatar da ci gaba da tallafi, muna nuna shafin ba tare da salo da JavaScript ba.
Yana nuna carousel na zamiya guda uku a lokaci guda. Yi amfani da maɓallan da suka gabata da na gaba don motsawa ta zamiya guda uku a lokaci guda, ko kuma yi amfani da maɓallan zamiya a ƙarshen don motsawa ta zamiya guda uku a lokaci guda.
An shirya barbashi masu ramuka ta hanyar hanyar sol-gel tare da wasu gyare-gyare don samun barbashi masu faɗi. An samo waɗannan barbashi da N-phenylmaleemide-methylvinyl isocyanate (PMI) da styrene ta hanyar polymerization na juyawar sarkar canja wuri (RAFT) don samar da polyamides masu haɗuwa da N-phenylmaleemide. Styrene (PMP) standalone phase. An cika ginshiƙan bakin ƙarfe mai ƙunci (diamita na ciki 100 × 1.8 mm) da marufi mai slurry. An kimanta aikin chromatographic na ginshiƙin PMP don raba cakuda peptides na roba waɗanda suka ƙunshi peptides biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) da tryptic hydrolyzate na albumin jini na ɗan adam (HAS). A ƙarƙashin yanayin fitar da iska mai kyau, adadin faranti na ka'idar da ke ɗauke da gaurayen peptides ya kai faranti 280,000 a kowace murabba'in mita. Idan aka kwatanta aikin raba ginshiƙin da aka haɓaka da ginshiƙin Ascentis Express RP-Amide na kasuwanci, an lura cewa ingancin raba ginshiƙin PMP ya fi ginshiƙin kasuwanci kyau dangane da ingancin rabuwa da warwarewa.
Masana'antar biopharmaceutical ta zama kasuwar duniya mai faɗaɗa tare da ƙaruwa mai yawa a kasuwa a cikin 'yan shekarun nan. Tare da ƙaruwar masana'antar biopharmaceutical1,2,3, akwai buƙatar yin nazarin peptide da furotin sosai. Baya ga peptide da aka yi niyya, ana samun ƙazanta iri-iri yayin haɗa peptide, don haka ana buƙatar tsarkake chromatographic don samun tsarkin da ake so na peptide. Bincike da siffanta sunadarai a cikin ruwan jiki, kyallen takarda, da ƙwayoyin halitta aiki ne mai matuƙar ƙalubale saboda yawan nau'ikan da za a iya ganowa a cikin samfuri guda ɗaya. Kodayake mass spectrometry kayan aiki ne mai tasiri don tsara peptides da sunadarai, idan an shigar da irin waɗannan samfuran kai tsaye cikin mass spectrometer, rabuwar ba za ta gamsar ba. Ana iya magance wannan matsalar ta hanyar yin liquid chromatography (LC) kafin nazarin MS, wanda zai rage adadin masu nazarin da ke shiga mass spectrometer a wani lokaci da aka ƙayyade4,5,6. Bugu da ƙari, masu nazarin na iya tattarawa a cikin yanki mai kunkuntar yayin rabuwar lokaci na ruwa, ta haka ne suke mai da hankali kan waɗannan masu nazarin kuma suna ƙara yawan fahimtar gano MS. Tsarin chromatography na ruwa (LC) ya ci gaba sosai a cikin shekaru goma da suka gabata kuma ya zama hanyar da ake amfani da ita sosai don nazarin proteomic7,8,9,10.
Ana amfani da chromatography na ruwa mai juyawa (RP-LC) sosai don tsarkakewa da raba gaurayen peptides ta amfani da silica mai canzawa ta octadecyl (ODS) a matsayin mataki na tsaye11,12,13. Duk da haka, saboda tsarinsu mai rikitarwa da yanayin amphoteric, matakai 14,15 na RP marasa motsi ba za su iya samar da raba peptides da sunadarai mai gamsarwa ba. Saboda haka, nazarin peptides da sunadarai tare da gutsuttsuran polar da waɗanda ba polar ba yana buƙatar matakai na tsaye na musamman don hulɗa da riƙe waɗannan masu nazarin16. Chromatography mai gauraya, wanda ke ba da hulɗar hanyoyin sadarwa da yawa, na iya zama madadin RP-LC don raba peptides, sunadarai, da sauran gaurayawan hadaddun abubuwa. An shirya matakai da yawa na tsaye na gaurayawa kuma an yi amfani da ginshiƙai cike da waɗannan matakai na tsaye don raba peptides da sunadarai17,18,19,20,21. Saboda kasancewar ƙungiyoyin polar da waɗanda ba polar ba, matakan gauraye na yanayin tsayawa (WAX/RPLC, HILIC/RPLC, haɗin polar/RPLC) sun dace da rabuwar peptides da sunadarai22,23,24,25,26,27,28., matakan tsayawa na polar da aka haɗa tare da ƙungiyoyin polar da aka haɗa tare suna nuna kyawawan iyawar rabuwa da zaɓi na musamman don masu nazarin polar da waɗanda ba polar ba saboda rabuwa ya dogara da hulɗar da ke tsakanin mai nazarin da matakin tsayawa hulɗar Multimodal 29,30,31,32. Kwanan nan, Zhang et al. 30 sun sami matakan tsayawa na behenyl da aka ƙare tare da raba hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, da wasu masu nazarin da suka yi nasara. Kayan da aka saka a polar yana da ƙungiyoyin polar da waɗanda ba polar ba, don haka ana iya amfani da shi don raba peptides da sunadarai zuwa sassan hydrophobic da hydrophilic. Gilashin layi na polar (misali, ginshiƙan C18 tare da ginshiƙan amide) suna samuwa a ƙarƙashin sunan kasuwanci Ascentis Express RP-Amide ginshiƙai, amma an yi amfani da waɗannan ginshiƙan ne kawai don nazarin amine 33.
A cikin wannan binciken, an shirya kuma an kimanta wani tsari mai motsi na polar embedding static phase (N-phenylmaleimide, embedding polystyrene) don rabuwar peptide da tryptic HSA cleavage. An yi amfani da dabarar da ke ƙasa don shirya matakin tsaye. An shirya barbashi masu ramuka bisa ga hanyoyin da aka bayyana a cikin wallafe-wallafenmu na baya, tare da wasu canje-canje a cikin shirye-shiryen 31, 34, 35, 36, 37, 38, 39. An daidaita rabon urea, polyethylene glycol (PEG), TMOS da ruwa-acetic acid don samun barbashi silica masu girman ramuka. Na biyu, an haɗa sabon phenylmaleimide-methylvinyl isocyanate ligand kuma an yi amfani da barbashi silica da aka samo don shirya matakan tsaye na polar embeded stighet. An saka matakin tsaye da aka samu a cikin ginshiƙin ƙarfe mai bakin ƙarfe (diamita na ciki 100 × 1.8 mm) bisa ga tsarin shiryawa da aka inganta. Ana taimakawa wajen shirya ginshiƙin ta hanyar girgiza na injiniya don tabbatar da cewa akwai layi ɗaya a cikin ginshiƙin. An kimanta ginshiƙin da aka cika don raba cakuda peptides waɗanda suka ƙunshi peptides guda biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). da kuma tryptic hydrolysates na albumin jini na ɗan adam (HSA). An lura cewa cakuda peptide da HSA tryptic digest sun rabu da kyakkyawan ƙuduri da inganci. An kwatanta ingancin rabuwar ginshiƙin PMP da na ginshiƙin Ascentis Express RP-Amide. An lura cewa peptides da sunadarai suna da kyakkyawan ƙuduri da babban ingancin rabuwar a ginshiƙin PMP, kuma ingancin rabuwar ginshiƙin PMP ya fi na ginshiƙin Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) don HPLC, methanol, 2-propanol da acetone. Kamfanin Sigma-Aldrich (St. Louis, Missouri, Amurka).
An gauraya cakuda urea (8 g), polyethylene glycol (8 g) da 8 ml na 0.01 N. acetic acid na tsawon mintuna 10, sannan aka ƙara 24 ml na TMOS a ciki a lokacin sanyaya kankara. An dumama cakudawar a zafin 40°C na tsawon awanni 6 sannan a zafin 120°C na tsawon awanni 8 a cikin wani injin ƙarfe mai bakin ƙarfe. An cire ruwan kuma an busar da ragowar a zafin 70°C na tsawon awanni 12. An niƙa busassun tubalan masu laushi cikin sauƙi kuma an narkar da su a cikin tanda a zafin 550°C na tsawon awanni 12. An shirya rukuni uku kuma an siffanta su don gwada iya sake haifuwar girman ƙwayoyin cuta, girman ramuka da kuma yankin saman.
Rukunin polar da matakin da ba ya tsayawa don sarƙoƙin polystyrene. An bayyana tsarin shiri a ƙasa.
An narkar da N-phenylmaleemide (200 mg) da methyl vinyl isocyanate (100 mg) a cikin toluene mai narkewa, sannan aka ƙara 0.1 ml na 2,2′-azoisobutyronitrile (AIBN) a cikin kwalbar amsawa don samun copolymer na phenylmaleemide da methyl vinyl isocyanate (PMCP). ) An dumama cakuda a zafin 60°C na tsawon awanni 3, an tace kuma an busar da shi a cikin tanda a zafin 40°C na tsawon awanni 3.
An watsa busassun ƙwayoyin silica (2 g) a cikin busassun toluene (100 ml), aka juya aka kuma sonicated na tsawon minti 10 a cikin kwalba mai zagaye na ƙasa mai nauyin 500 ml. An narkar da PMCP (10 mg) a cikin toluene sannan aka ƙara dropwise zuwa cikin kwalbar amsawa ta hanyar mazurari mai ƙara. An sake haɗa cakuda a 100°C na tsawon awanni 8, aka tace, aka wanke da acetone sannan aka busar da shi a 60°C na tsawon awanni 3. Sannan, an narkar da ƙwayoyin silica da ke da alaƙa da PMCP (100 g) a cikin toluene (200 ml), sannan aka ƙara 4-hydroxy-TEMPO (2 ml) a kai a gaban 100 μl na dibutyltin dilaurate a matsayin mai kara kuzari. An juya cakuda a 50°C na tsawon awanni 8, aka tace sannan aka busar da shi a 50°C na tsawon awanni 3.
An watsa Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) da ƙwayoyin silica da aka haɗa da TEMPO-PMCP (1.5 g) a cikin toluene kuma an tsarkake su da nitrogen. An yi polymerization na styrene a 100°C na tsawon awanni 12. An wanke samfurin da aka samo da methanol kuma an busar da shi dare ɗaya a 60°C. Tsarin amsawar gabaɗaya an nuna shi a cikin fig. ɗaya.
An cire samfuran daga gas ɗin a 393 K na tsawon awa 1 har sai an sami matsin lamba na ƙasa da 10-3 Torr. An yi amfani da adadin N2 da aka sha a matsin lamba na P/P0 = 0.99 don tantance jimlar girman ramin. An duba yanayin ƙwayoyin silica masu tsarki da waɗanda aka ɗaure da ligand ta amfani da na'urar duba lantarki ta lantarki (Hitachi High Technologies, Tokyo, Japan). An sanya samfuran busassun (ƙwayoyin silica masu tsarki da waɗanda aka ɗaure da ligand) akan sandunan aluminum ta amfani da tef ɗin carbon. An sanya zinare akan samfurin ta amfani da na'urar sputtering Q150T, kuma an sanya wani Layer na Au mai kauri 5 nm akan samfurin. Wannan yana inganta ingancin tsarin ƙarancin ƙarfin lantarki kuma yana ba da feshi mai sanyi. An gudanar da nazarin abubuwa ta amfani da na'urar nazarin abubuwan da ke cikin Thermo Electron (Waltham, MA, Amurka) Flash EA1112. An yi amfani da na'urar nazarin girman ƙwayoyin Malvern (Worcestershire, UK) Mastersizer 2000 don samun rarraba girman ƙwayoyin. An watsa barbashin silica da ba a shafa ba da barbashin silica da aka ɗaure da ligand (5 mg kowanne) a cikin 5 ml na isopropanol, an saka su da sonicated na minti 10, an motsa su na minti 5, sannan aka sanya su a kan benci na gani na Mastersizer. Ana gudanar da nazarin thermogravimetric a cikin saurin 5 °C a minti ɗaya a cikin zafin jiki daga 30 zuwa 800 °C.
An lulluɓe ginshiƙan bakin ƙarfe masu siffar kunkuntar da aka lulluɓe da zare mai siffar gilasar gilashi mai girma (ID 100 × 1.8 mm) ta hanyar cika slurry ta hanyar bin wannan tsari kamar yadda yake a cikin nassoshi na 31. An haɗa ginshiƙin bakin ƙarfe (layin gilashi, ID 100 × 1 .8 mm) da kuma hanyar fita da ke ɗauke da frit mai girman 1 µm zuwa injin marufi na slurry (Alltech Deerfield, IL, Amurka). Shirya dakatarwar matakin da ba a lulluɓe ta hanyar dakatar da 150 mg na matakin da ba a lulluɓe a cikin 1.2 ml na methanol sannan a ciyar da shi a cikin ginshiƙin tafki. An yi amfani da methanol azaman mai narkewa da kuma mai sarrafa slurry. An lulluɓe ginshiƙin ta hanyar amfani da jerin matsi na 100 MP na minti 10, 80 MP na minti 15, da 60 MP na minti 30. Tsarin marufi ya yi amfani da masu girgiza ginshiƙin gas guda biyu (Alltech, Deerfield, IL, Amurka) don girgizar injiniya don tabbatar da marufi iri ɗaya na ginshiƙin. Rufe mai narkewa da kuma sakin matsin lamba a hankali don hana lalacewar igiyar. An cire ginshiƙin daga bututun slurry kuma an haɗa wani kayan aiki zuwa mashigar kuma an haɗa shi da tsarin LC don gwada aikinsa.
An gina wani MLC na musamman ta amfani da famfon LC (10AD Shimadzu, Japan), samfurin samfuri mai madauri na allurar nL 50 (Valco (USA) C14 W.05), mai cire gass ɗin membrane (Shimadzu DGU-14A), da kuma taga mai kauri UV-VIS. Na'urar ganowa (UV-2075) da kuma ƙaramin ginshiƙi mai enamel. Yi amfani da bututun haɗawa masu kunkuntar da gajeru don rage tasirin ƙarin faɗaɗa ginshiƙi. Bayan cike ginshiƙin, shigar da babban ginshiƙi (50 µm id 365) a mashigar mahaɗin rage 1/16″ kuma shigar da babban ginshiƙi (50 µm) na mahaɗin ragewa. Ana gudanar da tattara bayanai da sarrafa chromatogram ta amfani da software na Multichro 2000. A 254 nm, an sa ido kan shan UV na masu nazarin abubuwan a 0. An yi nazarin bayanan Chromatographic ta amfani da OriginPro8 (Northampton, MA).
Albamin jini na ɗan adam, foda mai laushi, ≥ 96% (electrophoresis na gel agarose) 3 mg a gauraya da trypsin (1.5 mg), urea 4.0 M (1 ml) da 0.2 M ammonium bicarbonate (1 ml). An juya maganin na tsawon minti 10 sannan a ajiye a cikin ruwan wanka a zafin jiki na 37°C na tsawon awanni 6, sannan a kashe shi da 1 ml na 0.1% TFA. A tace maganin sannan a adana a ƙasa da 4°C.
An tantance rabuwar cakuda peptides da tryptic digest HSA akan ginshiƙin PMP daban. Duba tryptic hydrolysis na cakuda peptides da HSA da aka raba ta ginshiƙin PMP sannan a kwatanta sakamakon da ginshiƙin Ascentis Express RP-Amide. An ƙididdige adadin faranti na ka'ida ta amfani da lissafi mai zuwa:
An nuna hotunan SEM na barbashi masu tsarki na silica da barbashi masu ɗaure da ligand a cikin Hoto na 2. Hotunan SEM na barbashi masu tsarki na silica (A, B) suna nuna siffar zagaye inda barbashi suke da tsayi ko kuma suna da daidaito mara tsari idan aka kwatanta da bincikenmu na baya. Fuskar barbashi masu ɗaure da ligand (C, D) ta fi santsi fiye da na barbashi masu tsarki na silica, wanda wataƙila ya faru ne saboda sarƙoƙin polystyrene da ke rufe saman barbashi masu ɗaure da ligand.
Ana duba ƙwayoyin lantarki na ƙwayoyin silica masu tsarki (A, B) da ƙwayoyin silica masu ɗaure da ligand (C, D).
An nuna rarrabawar girman barbashi na tsantsar barbashi da barbashi na silica da aka ɗaure da ligand a cikin Hoto na 2. 3(A). Lanƙwasa rarrabawar girman barbashi na volumetric ya nuna cewa girman barbashi na silica ya ƙaru bayan gyaran sinadarai (Hoto na 3A). An kwatanta bayanan rarrabawar girman barbashi na silica daga binciken da ake yi yanzu da kuma binciken da ya gabata a cikin Tebur na 1(A). Girman barbashi na volumetric d(0.5) na PMP shine 3.36 µm, idan aka kwatanta da ƙimar ad(0.5) na 3.05 µm a cikin bincikenmu na baya (barbashi na silica da aka ɗaure da polystyrene)34. Saboda canjin rabon PEG, urea, TMOS da acetic acid a cikin cakuda amsawa, rarrabawar girman barbashi na wannan rukunin ya fi kunkuntar idan aka kwatanta da bincikenmu na baya. Girman barbashi na matakin PMP ya ɗan fi girma fiye da na matakin barbashi na silica da aka ɗaure da polystyrene wanda muka yi nazari a baya. Wannan yana nufin cewa aikin saman barbashi na silica tare da styrene yana sanya Layer polystyrene kawai (0.97 µm) akan saman silica, yayin da a cikin matakin PMP kauri Layer shine 1.38 µm.
Rarraba girman barbashi (A) da rarraba girman rami (B) na barbashi masu tsarki na silica da barbashi masu ɗaure da ligand.
An nuna girman rami, girman rami, da kuma yankin saman barbashin silica da aka yi amfani da su a wannan binciken a cikin Jadawali na 1 (B). An nuna bayanan PSD na barbashin silica masu tsarki da barbashin silica masu ɗaure ligand a cikin Fig. 3(B). Sakamakon ya yi daidai da bincikenmu na baya34. Girman ramin barbashin silica masu tsarki da waɗanda aka ɗaure ligand sune 310 Å da 241 Å, bi da bi, yana nuna cewa bayan gyaran sinadarai, girman ramin ya ragu da 69 Å, kamar yadda aka nuna a Jadawali na 1 (B), kuma an nuna lanƙwasa na juyawa a cikin Hoto. Takamaiman yankin saman barbashin silica a cikin binciken na yanzu shine 116 m2/g, wanda yayi daidai da bincikenmu na baya (124 m2/g). Kamar yadda aka nuna a Jadawali na 1(B), yankin saman (m2/g) na barbashin silica bayan gyaran sinadarai shi ma ya ragu daga 116 m2/g zuwa 105 m2/g.
An gabatar da sakamakon nazarin abubuwan da ke cikin matakin da ba ya canzawa a cikin Tebur. 2. Yawan sinadarin carbon a cikin matakin da ba ya canzawa a yanzu shine 6.35%, wanda ya yi ƙasa da na bincikenmu na baya (ƙwayoyin silica da ke da alaƙa da polystyrene, 7.93%35 da 10.21%, bi da bi) 42. Yawan sinadarin carbon a cikin matakin da ba ya canzawa a yanzu a ƙasa, tunda an yi amfani da wasu ligands na polar kamar phenylmaleemide methyl vinyl isocyanate (PCMP) da 4-hydroxy-TEMPO ban da styrene wajen shirya SP. Kashi na nauyin nitrogen a cikin matakin da ba ya canzawa a yanzu shine 2.21% idan aka kwatanta da 0.1735 da 0.85% a cikin binciken da ya gabata42. Wannan yana nufin cewa matakin da ba ya canzawa a yanzu yana da babban kaso na nitrogen saboda phenylmaleemide. Hakazalika, samfuran (4) da (5) suna da sinadarin carbon na 2.7% da 2.9%, bi da bi, yayin da samfurin ƙarshe (6) yana da sinadarin carbon na 6.35%, kamar yadda aka nuna a cikin Jadawali na 2. An yi amfani da nazarin thermogravimetric (TGA) a kan matakin PMP mai tsayawa don gwada asarar nauyi, kuma an nuna lanƙwasa TGA a cikin Hoto na 4. Lanƙwasa TGA yana nuna asarar nauyi na 8.6%, wanda ya yi daidai da sinadarin carbon (6.35%), tunda ligands ɗin ba su ƙunshi C kawai ba, har ma da N, O da H.
An zaɓi ligand phenylmaleemide-methylvinyl isocyanate don gyara saman barbashi na silica saboda rukunin phenylmaleemide da vinylisocyanate na polar. Rukunin isocyanate na Vinyl na iya ƙara amsawa da styrene ta hanyar polymerization mai rai. Dalili na biyu shine a saka ƙungiyar da ke da matsakaicin hulɗa da analyte kuma babu wata hulɗa mai ƙarfi ta electrostatic tsakanin analyte da matakin tsayawa, tunda ɓangaren phenylmaleemide ba shi da caji na kama-da-wane a pH na al'ada. Ana iya sarrafa polarity na matakin tsayawa ta hanyar mafi kyawun adadin styrene da lokacin amsawa na polymerization mai sassauci. Mataki na ƙarshe na amsawa (polymerization mai sassauci) yana da mahimmanci saboda yana canza polarity na matakin tsayawa. An gudanar da bincike na abubuwa don duba abubuwan da ke cikin carbon a cikin waɗannan matakan tsayawa. An lura cewa ƙara yawan styrene da lokacin amsawa yana ƙara yawan carbon na matakin tsayawa da akasin haka. SPs da aka shirya tare da yawan styrene daban-daban suna da nauyin carbon daban-daban. Hakazalika, an sanya waɗannan matakan da ba a tsayawa ba a kan ginshiƙan ƙarfe na bakin ƙarfe kuma an duba halayen chromatographic ɗinsu (zaɓi, ƙuduri, ƙimar N, da sauransu). Dangane da waɗannan gwaje-gwajen, an zaɓi ingantaccen tsari don shirya matakin PMP mai tsayawa don samar da madaidaicin polarity da kuma riƙe mai nazarin sosai.
An kuma kimanta ginshiƙin PMP don nazarin gaurayawan peptides guda biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) ta amfani da ƙarfin matakin motsi. 60/40 (v/v) ACN/ruwa (0.1% TFA) a ƙimar kwararar 80 µl/min. A ƙarƙashin yanayin fitarwa mafi kyau (faranti 200,000/m), adadin faranti na ka'ida (N) a kowane ginshiƙi (100 × 1.8 mm) shine 20,000 ± 100. An nuna ƙimar N don ginshiƙan PMP guda uku a cikin Jadawali na 3 kuma an nuna chromatograms a cikin Hoto na 5A. Bincike cikin sauri a babban gudu (700 µl/min) akan ginshiƙin PMP, an cire peptides guda biyar cikin minti ɗaya, kyakkyawan ƙimar N na 13,500 ± 330 a kowace ginshiƙi (diamita 100 x 1.8 mm), daidai da faranti 135,000/ m (Hoto na 5B). An cika ginshiƙai uku masu girman iri ɗaya (diamita na ciki 100 x 1.8 mm) da rukuni uku daban-daban na matakin PMP mai tsayawa don gwada sake haifuwa. An yi rikodin masu nazari ga kowane ginshiƙi ta hanyar raba cakuda gwaji ɗaya akan kowane ginshiƙi ta amfani da yanayin fitarwa mafi kyau, adadin faranti na ka'ida N, da lokacin riƙewa. An nuna bayanan sake haifuwa don ginshiƙan PMP a cikin Jadawali na 4. Kwaikwayon kwafin PMP ya yi daidai da ƙarancin ƙimar %RSD kamar yadda aka nuna a Jadawali na 3.
Raba gaurayen peptide akan ginshiƙin PMP (B) da ginshiƙin Ascentis Express RP-Amide (A), matakin wayar hannu 60/40 ACN/H2O (TFA 0.1%), girman ginshiƙin PMP (100 x 1.8 mm id), bincike Tsarin fitarwa na mahadi: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) da 5 (leucic acid enkephalin).
An kimanta ginshiƙin PMP (diamita na ciki 100 x 1.8 mm) don raba tryptic hydrolyzate na albumin na jini na ɗan adam ta HPLC. Chromatogram da ke cikin Hoto na 6 ya nuna cewa samfuran an raba su sosai tare da kyakkyawan ƙuduri. An yi nazarin mafita na HSA ta amfani da ƙimar kwarara na 100 μl/min, matakin motsi na 70/30 acetonitrile/ruwa da 0.1% TFA. An raba raba HSA zuwa kololuwa 17, kamar yadda aka nuna a cikin chromatogram (Hoto na 6), wanda ya dace da peptides 17. An ƙididdige ingancin rabuwar kololuwa ɗaya daga HSA hydrolyzate kuma an nuna ƙimar a cikin Tebur 5.
An raba HSA tryptic hydrolysates akan ginshiƙin PMP (diamita na ciki 100 x 1.8 mm), ƙimar kwarara (100 μl/min), matakin motsi na 60/40 acetonitrile/ruwa, da 0.1% TFA.
inda L shine tsawon ginshiƙi, η shine danko na matakin wayar hannu, ΔP shine matsin lamba na baya na ginshiƙi, kuma u shine saurin layi na matakin wayar hannu. Rarraba ginshiƙi na PMP shine 2.5 × 10–14 m2, ƙimar kwararar shine 25 µl/min, an yi amfani da 60/40 v/v. ACN/ruwa. Rarraba ginshiƙi na PMP (ID 100 × 1.8 mm) yayi kama da na bincikenmu na baya na Ref.34. Rarraba ginshiƙi cike da barbashi masu ramuka a saman shine 1.7 × 10 .6 µm, 2.5 × 10-14 m2 ga barbashi 5 µm43. Saboda haka, rarraba ginshiƙi na PMP yayi kama da rarraba ƙwayoyin harsashi na tsakiya tare da girman 5 μm.
inda Wx shine nauyin ginshiƙin da aka cika da chloroform, Wy shine nauyin ginshiƙin da aka cika da methanol, kuma ρ shine yawan sinadarin da ke narkewa. Yawan methanol (ρ = 0.7866) da chloroform (ρ = 1.484). Jimillar porosity na ginshiƙin silica-C18 (100 × 1.8 mm ID)34 da kuma ginshiƙin C18-urea31 da muka yi nazari a baya shine 0.63 da 0.55, bi da bi. Wannan yana nufin cewa kasancewar ligands na urea yana rage ƙarfin yanayin tsayawa. A gefe guda kuma, jimlar porosity na ginshiƙin PMP (diamita na ciki 100 × 1.8 mm) shine 0.60. Ginshiƙan PMP ba su da yawa fiye da ginshiƙan da aka cika da ƙwayoyin silica da aka ɗaure na C18 saboda a cikin matakan tsayawa na nau'in C18, ligands na C18 suna haɗe da ƙwayoyin silica a cikin sarƙoƙi masu layi, yayin da a cikin matakan tsayawa na nau'in polystyrene, ana samar da polymer mai kauri a kusa da ƙwayoyin. Layer A. A cikin wani gwaji na yau da kullun, ana ƙididdige porosity na ginshiƙi kamar haka:
A hoto na 7A, B ya nuna zane-zanen Van Deemter don ginshiƙin PMP (id 100 x 1.8 mm) da ginshiƙin Ascentis Express RP-Amide (id 100 x 1.8 mm) a ƙarƙashin yanayin fitarwa iri ɗaya, 60/40 ACN/H2O da 0 .1% TFA 20 µl/min zuwa 800 µl/min akan ginshiƙan biyu. Mafi ƙarancin ƙimar HETP a mafi kyawun ƙimar kwarara (80 µl/min) sune 2.6 µm da 3.9 µm don ginshiƙin PMP da ginshiƙin Ascentis Express RP-Amide, bi da bi. Ƙimar HETP sun nuna cewa ingancin rabuwar ginshiƙin PMP (id 100 x 1.8 mm) ya fi na ginshiƙin Ascentis Express RP-Amide da ake da shi a kasuwa (id 100 x 1.8 mm). Jadawalin van Deemter a cikin Hoto na 7(A) ya nuna cewa raguwar ƙimar N ba ta da girma sosai tare da ƙaruwar kwarara idan aka kwatanta da bincikenmu na baya. Ingantaccen ingancin rabuwa na ginshiƙin PMP (id 100 × 1.8 mm) idan aka kwatanta da ginshiƙin Ascentis Express RP-Amide ya dogara ne akan ingantaccen siffa da girman barbashi da kuma tsarin tattara ginshiƙi mai kyau da ake amfani da shi a cikin aikin yanzu34.
(A) Tsarin Van Deemter (HETP vs. saurin layi na matakin wayar hannu) da aka samu akan ginshiƙin PMP (id 100 x 1.8 mm) a cikin 60/40 ACN/H2O tare da 0.1% TFA. (B) Tsarin Van Deemter (gudun layi na HETP idan aka kwatanta da saurin layi na matakin wayar hannu) da aka samu akan ginshiƙin RP-Amide na Ascentis Express (id 100 x 1.8 mm) a cikin 60/40 ACN/H2O tare da 0.1% TFA.
An shirya kuma an kimanta wani lokaci mai tsayi na polystyrene mai hade da juna don raba cakuda peptides na roba da tryptic hydrolyzate na albumin na jini na ɗan adam (HSA) a cikin chromatography na ruwa mai aiki mai girma. Aikin chromatographic na ginshiƙan PMP don gaurayawan peptide yana da kyau kwarai da gaske dangane da ingancin rabuwa da ƙuduri. Ingantaccen ingancin rabuwa na ginshiƙan PMP ya faru ne saboda dalilai da yawa kamar girman barbashi na silica da girman rami, haɗakar matakai masu tsayi, da kayan tattara ginshiƙai masu rikitarwa. Baya ga babban ingancin rabuwa, wani fa'idar wannan matakin mai tsayi shine ƙarancin matsin lamba na baya na ginshiƙai a yawan kwarara mai yawa. Ginshiƙan PMP suna da matuƙar sake samarwa kuma ana iya amfani da su don nazarin gaurayawan peptides da narkewar tryptic na furotin daban-daban. Muna da niyyar amfani da wannan ginshiƙi don raba mahaɗan bioactive daga samfuran halitta, cirewar tsire-tsire masu magani da namomin kaza a cikin chromatography na ruwa. A nan gaba, za a kuma kimanta ginshiƙan PMP don raba sunadarai da ƙwayoyin rigakafi na monoclonal.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Bincike kan tsarin rabuwar peptide na chromatography na lokaci mai juyawa sashi na I: Haɓaka yarjejeniya don halayyar ginshiƙi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Bincike kan tsarin rabuwar peptide na chromatography na lokaci mai juyawa sashi na I: Haɓaka yarjejeniya don siffanta ginshiƙi.Field, JK, Owerby, MR, Lau, J., Togersen, H., da Petersson, P. Binciken Tsarin Raba Peptide ta hanyar Tsarin Chromatography na Juyawa, Kashi na I: Kirkirar Yarjejeniya don Halayen Ginshiƙi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Bincike kan tsarin rabuwar peptide na chromatography na lokaci mai juyawa sashi na I: Haɓaka yarjejeniya don halayen ginshiƙi. Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Bincike kan tsarin rabuwar peptide na chromatography na lokaci mai juyawa sashi na I: Haɓaka yarjejeniya don halayen ginshiƙi.Field, JK, Owerby, MR, Lau, J., Togersen, H., da Petersson, P. Binciken Tsarin Raba Peptide ta hanyar Tsarin Chromatography na Juyawa, Kashi na I: Kirkirar Yarjejeniya don Halayen Ginshiƙi.J.色谱法。 1603, 113-129. https://doi.org/10.1016/j.chroma.2019.05.038(2019).
Gomez, B. da sauransu. Hanyoyin ƙirƙirar ingantattun peptides masu aiki don maganin cututtuka masu yaɗuwa. Fasahar kere-kere. Nasarorin 36(2), 415–429. https://doi.org/10.1016/j.biotechadv.2018.01.004 (2018).
Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Peptides na roba masu warkarwa: Kimiyya da kasuwa. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Peptides na roba masu warkarwa: Kimiyya da kasuwa.Vliege P, Lisowski V, Martinez J da Chreschatyski M. Peptides na magani na roba: kimiyya da kasuwa.Vliege P, Lisowski V, Martinez J da Khreschatsky M. Peptides na roba masu warkarwa: kimiyya da kasuwa. gano magunguna. Yau 15 (1–2), 40–56. https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Tsarin gwajin ruwa mai suna proteomic chromatography mai ci gaba. Xie, F., Smith, RD & Shen, Y. Tsarin gwajin ruwa mai suna proteomic chromatography mai ci gaba.Duba F., Smith RD da Shen Yu. Tsarin chromatography na ruwa mai zurfi na proteomic. Xie, F., Smith, RD & Shen, Y. 高级蛋白质组液相色谱。 Xie, F., Smith, RD & Shen, Y. Advanced protein abun da ke ciki 液相色谱。Duba F., Smith RD da Shen Yu. Tsarin chromatography na ruwa mai zurfi na proteomic.J. Tsarin Tsari. A 1261, 78–90 (2012).
Liu, W. da sauransu. Advanced liquid chromatography-mass spectrometry yana iya haɗar da faffadan tushen metabolomics da proteomics. dubura. Chim. Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Matsayin UHPLC a cikin haɓaka magunguna. Chesnut, SM & Salisbury, JJ Matsayin UHPLC a cikin haɓaka magunguna.Chesnut, SM da Salisbury, JJ Matsayin UHPLC a cikin haɓaka magunguna.Chesnut, SM da Salisbury, JJ Matsayin UHPLC a fannin haɓaka magunguna. J. Satumba Science. 30(8), 1183–1190 (2007).
Wu, N. & Clausen, AM Abubuwa na asali da amfani na chromatography na ruwa mai matsin lamba mai yawa don rabuwa cikin sauri. Wu, N. & Clausen, AM Abubuwa na asali da amfani na chromatography na ruwa mai matsin lamba mai yawa don rabuwa cikin sauri.Wu, N. da Clausen, AM Abubuwa na asali da na aiki na chromatography na ruwa mai matsin lamba don rabuwa cikin sauri. 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 Abubuwa na asali da na aiki na chromatography na ruwa mai matsin lamba mai yawa don rabuwa cikin sauri.Wu, N. da Clausen, AM Abubuwa na asali da na aiki na chromatography na ruwa mai matsin lamba don rabuwa cikin sauri.J. Satumba. Kimiyya. 30(8), 1167–1182. https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Amfani da chromatography na ruwa mai aiki sosai a cikin haɓaka magunguna. Wren, SA & Tchelitcheff, P. Amfani da chromatography na ruwa mai aiki sosai a cikin haɓaka magunguna.Ren, SA da Chelischeff, P. Amfani da sinadarin chromatography na ruwa mai matuƙar aiki a cikin haɓaka magunguna. 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 da Chelischeff, P. Amfani da chromatography na ruwa mai aiki sosai a cikin haɓaka magunguna.J. Tsarin Chromatography. 1119(1-2), 140-146. https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al. Hydrogel mai kama da macroporous wanda aka samo daga wani sinadarin mai a cikin ruwa tare da babban matakin ciki don tsarkakewa mai inganci na enterovirus 71. Chemical. aikin. Journal 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Matsayin sinadarin chromatography na ruwa a cikin proteomics. Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Matsayin sinadarin chromatography na ruwa a cikin proteomics.Shi, Y., Xiang, R., Horvath, C. da Wilkins, JA Matsayin sinadarin chromatography na ruwa a cikin 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. da Wilkins, JA Matsayin sinadarin chromatography na ruwa a cikin proteomics.J. Tsarin Chromatography. A 1053 (1-2), 27-36 (2004).
Fekete, S., Vutey, J.-L. & Guillarme, D. Sabbin halaye a cikin rabuwar chromatographic na ruwa mai juyawa na peptides na warkewa da sunadarai: Ka'ida da aikace-aikace. & Guillarme, D. Sabbin halaye a cikin rabuwar chromatographic na ruwa mai juyawa na peptides na warkewa da sunadarai: Ka'ida da aikace-aikace. & Guillarme, D. Новые тенденции в разделении обращенной фазой: теория и приложения. & Guillarme, D. Sabbin salo a cikin rabuwar peptides na warkewa da sunadarai ta hanyar chromatography na ruwa na baya: ka'ida da aikace-aikace. & 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.da kuma Guillarmé, D. Sabbin hanyoyin rabuwa da peptides na warkewa da sunadarai ta hanyar amfani da chromatography na ruwa na baya: ka'ida da aikace-aikace.J. Pharm. Kimiyyar Halittu. dubura. 69, 9–27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Raba peptides masu girma biyu ta amfani da tsarin RP-RP-HPLC tare da pH daban-daban a cikin girman rabuwa na farko da na biyu. Gilar, M., Olivova, P., Daly, AE & Gebler, JC Raba peptides masu girma biyu ta amfani da tsarin RP-RP-HPLC tare da pH daban-daban a cikin girman rabuwa na farko da na biyu.Gilar M., Olivova P., Dali AE da Gebler JK Raba peptides masu girma biyu ta amfani da tsarin RP-RP-HPLC tare da pH daban-daban a cikin girman rabuwa na farko da na biyu.Gilar M., Olivova P., Dali AE da Gebler JK Raba peptides masu girma biyu ta amfani da ƙimar pH daban-daban a cikin girman rabuwa ta farko da ta biyu ta amfani da tsarin RP-RP-HPLC. J. Satumba Science. 28 (14), 1694–1703 (2005).
Fellitti, S. da sauransu. Bincike kan canja wurin taro da halayen motsi na ginshiƙan chromatography masu aiki mai girma cike da ƙwayoyin C18 masu ramuka da ramuka a saman ƙasa da 2 µm. J. Satumba Science. 43 (9–10), 1737–1745 (2020).
Piovesana, S. da sauransu. Sabbin abubuwan da suka faru da ƙalubalen nazari a cikin ware, ganowa, da kuma tabbatar da peptides masu aiki a cikin tsirrai. dubura. Ƙwayar halitta. Sinadarai. 410(15), 3425-3444. https://doi.org/10.1007/s00216-018-0852-x (2018).
Muller, JB da sauransu. Tsarin Proteomic na masarautar rayuwa. Yanayi 582 (7813), 592–596. https://doi.org/10.1038/s41586-020-2402-x (2020).
De Luca, K. da sauransu. Bayan maganin peptides na warkewa ta hanyar shirya chromatography na ruwa. Molecules (Basel, Switzerland) 26(15), 4688 (2021).
Yang, Y. & Geng, X. Tsarin chromatography na yanayi iri-iri da aikace-aikacensa ga biopolymers. Yang, Y. & Geng, X. Tsarin chromatography na yanayi iri-iri da aikace-aikacensa ga biopolymers.Yang, Yu. da Geng, X. Haɗaɗɗen yanayin chromatography da aikace-aikacensa ga 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. Haɗaɗɗen yanayin chromatography da aikace-aikacensa a cikin biopolymers.Yang, Yu. da Gene, X. Haɗaɗɗen yanayin chromatography da aikace-aikacensa ga biopolymers.J. Tsarin Chromatography. A 1218(49), 8813–8825 (2011).