Mun gode da ziyartar Nature.com. Sigar burauzar da kuke amfani da ita tana da ƙarancin tallafi ga CSS. Domin mafi kyawun ƙwarewa, muna ba da shawarar ku yi amfani da burauzar da aka sabunta (ko ku kashe yanayin daidaitawa a cikin Internet Explorer). A halin yanzu, don tabbatar da ci gaba da tallafi, za mu nuna shafin ba tare da salo da JavaScript ba.
An shirya barbashi masu ramuka ta hanyar hanyar sol-gel tare da wasu gyare-gyare don samun barbashi masu macroporous. An samo waɗannan barbashi ta hanyar polymerization na ƙarin ƙarin rarrabuwar sarkar (RAFT) tare da N-phenylmaleemide-methylvinylisocyanate (PMI) da styrene don shirya haɗin N-phenylmaleemide na polystyrene (PMP) na tsaye. An naɗe ginshiƙan bakin ƙarfe masu ƙunci (100 × 1.8 mm id) ta hanyar marufi. An kimanta rabuwar ginshiƙi na PMP na cakuda peptide wanda ya ƙunshi peptides biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) da narkewar trypsin na albumin na jini na ɗan adam (HAS). A ƙarƙashin yanayin fitarwa mafi kyau, adadin farantin ka'idar cakuda peptide yana da girma har zuwa faranti 280,000/m². Idan aka kwatanta aikin rabuwar ginshiƙin da aka haɓaka. Tare da ginshiƙin Ascentis Express RP-Amide na kasuwanci, an lura cewa aikin raba ginshiƙin PMP ya fi ginshiƙin kasuwanci kyau dangane da ingancin rabuwa da warwarewa.
A cikin 'yan shekarun nan, masana'antar biopharmaceutical ta zama kasuwar duniya mai faɗaɗa tare da ƙaruwa mai yawa a cikin kasuwar. Tare da ƙaruwar masana'antar biopharmaceutical1,2,3, ana matuƙar son nazarin peptides da sunadarai. Baya ga peptide da aka yi niyya, ana samar da ƙazanta da yawa yayin haɗa peptide, don haka yana buƙatar tsarkakewar chromatographic don samun peptides na tsarkin da ake so. 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 gano su a cikin samfuri ɗaya. Kodayake mass spectrometry kayan aiki ne mai tasiri don jerin peptide da furotin, idan aka allura irin waɗannan samfuran a cikin mass spectrometer a cikin wucewa ɗaya, rabuwar ba za ta yi kyau ba. Ana iya rage wannan matsalar ta hanyar aiwatar da rabuwar 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, yayin rabuwar lokaci na ruwa, ana iya mai da hankali kan masu nazarin a cikin yankuna masu kunkuntar, ta haka ne ake mai da hankali kan waɗannan masu nazarin da inganta gano MS. hankali. Tsarin chromatography na ruwa (LC) ya ci gaba sosai a cikin shekaru goma da suka gabata kuma ya zama sanannen dabarar nazarin proteomic7,8,9,10.
Ana amfani da chromatography na ruwa mai juyawa (RP-LC) sosai don tsarkakewa da rabuwa da gaurayen peptide ta amfani da silica mai canzawa ta octadecyl (ODS) a matsayin lokaci mai tsayawa11,12,13. Duk da haka, matakan RP masu tsayawa ba sa samar da isasshen rabuwa na peptides da sunadarai saboda tsarinsu mai rikitarwa da yanayin amphiphilic 14,15. Saboda haka, ana buƙatar matakai na tsaye na musamman don nazarin peptides da sunadarai tare da sassan polar da waɗanda ba polar ba don hulɗa da kuma riƙe waɗannan masu nazarin16. Chromatography na yanayin haɗuwa, wanda ke ba da hulɗa da yawa, na iya zama madadin RP-LC don rabuwa da peptides, sunadarai, da sauran gauraye masu rikitarwa. An shirya matakai da yawa na gauraye masu tsayawa, kuma an yi amfani da ginshiƙai masu cike da waɗannan matakai don rabuwa da peptide da furotin17,18,19,20,21. Matakan tsaye na yanayin haɗuwa (Wax/RPLC, HILIC/RPLC, haɗin polar/RPLC) sun dace da peptide da furotin rabuwa saboda kasancewar ƙungiyoyin polar da waɗanda ba polar ba22,23,24,25,26,27,28. Hakazalika, matakan da ke tsakanin polar da ƙungiyoyin polar da aka haɗa da juna suna nuna kyakkyawan ikon rabuwa da zaɓi na musamman ga masu nazarin polar da waɗanda ba polar ba, kamar yadda rabuwa ya dogara da hulɗar da ke tsakanin masu nazarin analyte da waɗanda ba polar ba. Hulɗar hanyoyin sadarwa da yawa 29, 30, 31, 32. Kwanan nan, Zhang et al. 30 ya shirya wani tsari na polyamine mai tsayayye wanda aka ƙare da dodecyl kuma ya sami nasarar raba hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, da sauran masu nazarin da yawa. Mai haɗa polar yana da ƙungiyoyin polar da waɗanda ba polar ba, don haka ana iya amfani da shi don raba peptides da sunadarai waɗanda ke da ƙwayoyin hydrophobic da hydrophilic. Gilashin da aka haɗa da polar (misali, ginshiƙan C18 da amide ya haɗa) suna samuwa a kasuwa a ƙarƙashin sunan kasuwanci Ascentis Express RP-Amide ginshiƙai, amma ana amfani da waɗannan ginshiƙan don nazarin amine 33 kawai.
A cikin wannan binciken, an shirya wani lokaci mai tsayi da aka haɗa da polar-embedded static phase (N-phenylmaleimide-embedded polystyrene) kuma an kimanta shi don raba peptides da trypsin na HSA. An shirya matakin mai tsayi ta amfani da dabarar da ke ƙasa. An shirya barbashi silica masu ramuka bisa ga hanyar da aka bayar a cikin littafinmu na baya tare da wasu gyare-gyare ga tsarin shiri. An daidaita rabon urea, polyethylene glycol (PEG), TMOS, da ruwa acetic acid don shirya barbashi silica tare da babban girman rami. Na biyu, an haɗa sabon ligand, phenylmaleimide-methyl vinyl isocyanate, kuma an yi amfani da shi don samo barbashi silica don shirya wani lokaci mai tsayi da aka haɗa da polar. An saka matakin mai tsayi da aka samu a cikin ginshiƙin bakin ƙarfe (100 × 1.8 mm id) ta amfani da tsarin shiryawa da aka inganta. Ana taimaka wa shirya ginshiƙin da girgiza na injiniya don tabbatar da cewa an samar da gado iri ɗaya a cikin ginshiƙin. Kimanta rabuwar ginshiƙin da aka haɗa da gaurayen peptide waɗanda suka ƙunshi peptides biyar; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) da kuma Trypsin digest na albumin na jini na ɗan adam (HAS). An lura da cewa cakuda peptide da trypsin digest na HSA sun rabu da kyakkyawan ƙuduri da inganci. An kwatanta aikin raba ginshiƙin PMP da na ginshiƙin Ascentis Express RP-Amide. An lura cewa duka peptides da sunadarai suna da kyau kuma suna da inganci akan ginshiƙin PMP, wanda ya fi inganci fiye da ginshiƙin Ascentis Express RP-Amide.
PEG (Polyethylene Glycol), Urea, Acetic Acid, 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, da Acetone An saya daga Sigma-Aldrich (St. Louis, MO, Amurka).
An juya 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 ƙarƙashin yanayin sanyin ƙanƙara. An dumama cakudawar amsawar a 40°C na tsawon awanni 6 sannan a 120°C na tsawon awanni 8 a cikin autoclave na bakin ƙarfe. An zuba ruwan kuma an busar da sauran kayan a 70°C na tsawon awanni 12. An niƙa busasshen laushin taro mai laushi a cikin tanda kuma an bar shi a 550°C na tsawon awanni 12. An shirya rukuni uku kuma an siffanta su don bincika iyawar sake haifuwa a girman barbashi, girman rami da kuma yankin saman.
Ta hanyar gyaran saman barbashi na silica tare da ligand phenylmaleemide-methylvinylisocyanate (PCMP) da kuma radial polymerization tare da styrene, an shirya wani abu mai ɗauke da rukuni na polar. Matakin tsayawa don haɗakarwa da sarƙoƙi na polystyrene. An bayyana tsarin shiri a ƙasa.
An narkar da N-phenylmaleimide (200 mg) da methyl vinyl isocyanate (100 mg) a cikin busasshen toluene, kuma an ƙara 0.1 mL na 2,2′-azoisobutyronitrile (AIBN) a cikin kwalbar amsawa don shirya phenylmaleimide-methyl vinyl isocyanate copolymer (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 watsar da busassun ƙwayoyin silica (2 g) a cikin busasshen toluene (100 mL), aka juya aka saka su a cikin kwalba mai zagaye na ƙasa mai nauyin 500 mL na minti 10. An narkar da PMCP (10 mg) a cikin toluene sannan aka ƙara su a cikin kwalbar amsawa ta hanyar mazurari mai faɗuwa. An sake haɗar da cakuda a 100 °C na tsawon awanni 8, an tace kuma an wanke da acetone sannan aka busar da shi a 60 °C na tsawon awanni 3. Sannan, an narkar da ƙwayoyin silica masu ɗaure da PMCP (100 g) a cikin toluene (200 ml) kuma an ƙara 4-hydroxy-TEMPO (2 mL) a gaban 100 µL na dibutyltin dilaurate a matsayin mai haɓaka. An juya cakuda a 50 °C na tsawon awanni 8, an tace kuma an busar da shi a 50 °C na tsawon awanni 3.
An watsa Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), da kuma 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 samu da methanol kuma an busar da shi a 60 °C cikin dare. Tsarin amsawar gabaɗaya an nuna shi a Hoto na 1.
An cire samfuran daga gas a 393 K na tsawon awa 1 don samun 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 barbashi silica marasa siliki da aka haɗa da ligand tare da na'urar duba electron (Hitachi High Technologies, Tokyo, Japan). An sanya samfuran busassun (barbashi silica marasa siliki da barbashi masu ɗaure da ligand) akan ginshiƙin aluminum ta amfani da tef ɗin carbon mai manne. An yi wa samfuran ado da zinare ta amfani da murfin sputter na Q150T, kuma an sanya Layer na Au na 5 nm akan samfuran. Wannan yana inganta ingancin aiki ta amfani da ƙananan ƙarfin lantarki kuma yana samar da hatsi mai kyau, sputtering mai sanyi. An yi amfani da Thermo Electron (Waltham, MA, Amurka) Flash EA1112 elemental analyzer don nazarin elemental. An yi amfani da Malvern (Worcestershire, UK) Mastersizer 2000 mai nazarin girman barbashi don samun rarraba girman barbashi. Silica mara siliki An rarraba barbashi da barbashi masu haɗin ligand (5 mg kowanne) a cikin 5 mL na isopropanol, an yi masa sonicated na minti 10, an yi masa vortex na minti 5, sannan aka sanya shi a kan benci na gani na Mastersizer. An gudanar da nazarin thermogravimetric a cikin 5 °C a minti ɗaya a kan zafin jiki na 30 zuwa 800 °C.
An naɗe ginshiƙan bakin ƙarfe masu siffar gilashi masu siffar kunkuntar (100 × 1.8 mm id) ta amfani da hanyar tattara slurry, ana amfani da irin wannan hanyar da aka yi amfani da ita a cikin Ref. 31. An haɗa ginshiƙin bakin ƙarfe (mai layi da gilashi, 100 × 1.8 mm id) tare da matsewar fita wanda ke ɗauke da frit 1 µm zuwa matsewar slurry (Alltech Deerfield, IL, Amurka). Shirya matsewar lokaci mai tsayawa ta hanyar dakatar da 150 mg na lokaci mai tsayawa a cikin 1.2 mL na methanol kuma aika shi zuwa ginshiƙin ajiya. An yi amfani da Methanol azaman matsewar slurry da kuma matsewar propelling. Cika ginshiƙin a jere ta hanyar sanya matsin lamba na 100 MP na minti 10, 80 MP na minti 15, da 60 MP na minti 30. A lokacin matsewa, an yi amfani da girgizar injiniya tare da masu girgiza ginshiƙin GC guda biyu (Alltech, Deerfield, IL, Amurka) don tabbatar da matse ginshiƙin iri ɗaya. Rufe matsewar slurry kuma saki matsin lamba a hankali don hana duk wani lalacewa a cikin ginshiƙin. Cire ginshiƙin daga na'urar matse slurry kuma haɗa wani matsewa zuwa shigarwa da tsarin LC don duba aikin sa.
An gina famfon LC (10AD Shimadzu, Japan), injin allura (Valco (Amurka) C14 W.05) tare da madauri na allurar 50nL, mai cire gass na membrane (Shimadzu DGU-14A), taga mai kauri UV-VIS. An yi amfani da na'urar gano na'urar µLC ta musamman (UV-2075) da ƙananan ginshiƙai masu layi da gilashi. Yi amfani da bututun haɗawa mai kunkuntar da gajeru don rage tasirin faɗaɗa band ɗin ginshiƙi. Bayan marufi, an sanya capillaries (50 μm id 365 da ƙananan capillaries masu ragewa (50 μm) a matsewar 1/16″ na haɗin ragewa. An yi amfani da software na Multichro 2000 don tattara bayanai da sarrafa chromatographic. An gwada sa ido a 254 nm Analytes don sha UV. OriginPro8 (Northampton, MA) ta yi nazarin bayanan chromatographic.
Albumin daga jinin ɗan adam, foda mai laushi, ≥ 96% (electrophoresis na agarose gel) 3 mg a gauraya da trypsin (1.5 mg), 4.0 M urea (1 mL), da 0.2 M ammonium bicarbonate (1 mL). An juya maganin na tsawon mintuna 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 gaurayen peptide da narkar da HSA trypsin daban-daban akan ginshiƙan PMP. Duba rabuwar gaurayen peptide da narkar da trypsin na HSA ta hanyar ginshiƙin PMP kuma kwatanta sakamakon da ginshiƙin Ascentis Express RP-Amide. An ƙididdige lambar faranti na ka'ida kamar haka:
An nuna hotunan SEM na barbashin silica marasa siliki da barbashin silica masu haɗin ligand a cikin Hoto na 2. Hotunan SEM na barbashin silica marasa siliki (A, B) sun nuna cewa, sabanin bincikenmu na baya, waɗannan barbashin suna da siffar zagaye inda barbashin suke da tsayi ko kuma suna da daidaito mara tsari. Fuskar barbashin silica masu haɗin ligand (C, D) ta fi santsi fiye da na barbashin silica marasa siliki, wanda wataƙila ya faru ne saboda rufin sarƙoƙin polystyrene akan saman barbashin silica.
Ana duba hotunan ƙwayoyin silica marasa siliki (A, B) da ƙwayoyin silica masu haɗin ligand (C, D).
An nuna rarrabawar girman barbashi na barbashi silica marasa sirara da barbashi silica masu haɗin ligand a cikin Hoto na 3(A). Lanƙwasa rarrabawar girman barbashi bisa ga girma ya nuna cewa girman barbashi silica ya ƙaru bayan gyaran sinadarai (Hoto na 3A). An kwatanta bayanan rarrabawar girman barbashi na barbashi silica daga binciken da ake yi yanzu da kuma binciken da ya gabata a cikin Tebur 1(A). Girman barbashi bisa ga girma, d(0.5), na PMP shine 3.36 μm, idan aka kwatanta da bincikenmu na baya tare da ƙimar ad(0.5) na 3.05 μm (barbashi silica da aka haɗa da polystyrene)34. Wannan rukunin yana da rarrabawar girman barbashi mafi ƙanƙanta idan aka kwatanta da bincikenmu na baya saboda bambancin rabo na PEG, urea, TMOS, da acetic acid a cikin cakuda amsawa. Girman barbashi na matakin PMP ya ɗan fi girma fiye da na matakin barbashi silica da aka haɗa da polystyrene da muka yi nazari a baya. Wannan yana nufin cewa aikin saman barbashi silica tare da styrene kawai ya sanya layin polystyrene (0.97 µm) akan saman silica, yayin da a cikin Matakin PMP kauri na Layer shine 1.38 µm.
Rarraba girman barbashi (A) da rarraba girman rami (B) na barbashi silica marasa siliki da barbashi silica masu ɗaure da ligand.
An bayar da girman rami, girman rami da kuma yankin saman barbashin silica na binciken da ake yi a yanzu a cikin Jadawali na 1(B). Bayanan PSD na barbashin silica marasa siliki da barbashin silica masu haɗin ligand an nuna su a Hoto na 3(B). Sakamakon ya yi daidai da binciken da muka yi a baya. Girman ramin barbashin silica marasa siliki da masu haɗin ligand sune 310 da 241, bi da bi, wanda ke nuna cewa girman ramin ya ragu da 69 bayan gyaran sinadarai, kamar yadda aka nuna a Jadawali na 1(B), kuma an nuna canjin lanƙwasa a Hoto na 3(B). Hakazalika, girman ramin barbashin silica ya ragu daga 0.67 zuwa 0.58 cm3/g bayan gyaran sinadarai. Takamaiman yankin saman barbashin silica da aka yi nazari a kai a yanzu shine 116 m2/g, wanda yayi daidai da binciken da muka yi a baya (124 m2/g). Kamar yadda aka nuna a Jadawali na 1(B), yankin saman (m2/g) na barbashin silica shi ma ya ragu daga 116 m2/g zuwa 105 m2/g bayan gyaran sinadarai.
An nuna sakamakon nazarin abubuwan da ke cikin matakin tsayawa a cikin Jadawali na 2. Loda carbon na matakin tsayawa a yanzu shine 6.35%, wanda ya yi ƙasa da nauyin carbon na bincikenmu na baya (ƙwayoyin silica masu haɗin polystyrene, 7.93%35 da 10.21%, bi da bi) 42. Loda carbon na matakin tsayawa a yanzu yana da ƙasa, Domin a cikin shirya SP na yanzu, ban da styrene, an yi amfani da wasu ligands na polar kamar phenylmaleemide-methylvinylisocyanate (PCMP) da 4-hydroxy-TEMPO. Kashi na nauyin nitrogen na matakin tsayawa a yanzu shine 2.21%, idan aka kwatanta da 0.1735 da 0.85% ta nauyin nitrogen a cikin binciken da ya gabata, bi da bi. Wannan yana nufin cewa wt% na nitrogen ya fi girma a matakin tsayawa a yanzu saboda phenylmaleemide. Hakazalika, loda carbon na samfura (4) da (5) sun kasance 2.7% da 2.9%, bi da bi, yayin da loda carbon na ƙarshe samfurin (6) ya kasance 6.35%, kamar yadda aka nuna a Jadawali na 2. An duba asarar nauyi tare da matakin PMP mai tsayawa, kuma an nuna lanƙwasa TGA a Hoto na 4. Lanƙwasa TGA yana nuna asarar nauyi na 8.6%, wanda ya yi daidai da nauyin carbon (6.35%) saboda ligands ɗin ba wai kawai sun ƙunshi C ba har ma da N, O, da H.
An zaɓi ligand ɗin phenylmaleemide-methylvinylisocyanate don gyaran saman barbashi na silica saboda yana da ƙungiyoyin phenylmaleemide na polar da ƙungiyoyin vinylisocyanate. Ƙungiyoyin isocyanate na Vinyl za su 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 kuma yana iya canza polarity na matakin tsayawa. An gudanar da bincike na abubuwa don duba nauyin carbon na waɗannan matakai tsayawa. An lura cewa ƙara yawan styrene da lokacin amsawa ya ƙara nauyin carbon na matakin tsayawa da akasin haka. SPs da aka shirya tare da yawan styrene daban-daban suna da nauyin carbon daban-daban. Kuma, ɗora waɗannan matakan tsayawa cikin ginshiƙan bakin ƙarfe kuma duba su Aikin chromatographic (zaɓi, ƙuduri, ƙimar N, da sauransu). Dangane da waɗannan gwaje-gwajen, an zaɓi ingantaccen tsari don shirya matakin PMP mai tsayawa don tabbatar da daidaiton polarity da riƙe mai nazari mai kyau.
An kuma tantance gaurayen peptide guda biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) ta amfani da ginshiƙin PMP ta amfani da matakin motsi; 60/40 (v/v) acetonitrile/ruwa (0.1% TFA) a ƙimar kwararar 80 μL/min. A ƙarƙashin yanayin fitarwa mafi kyau, lambar farantin ka'ida (N) a kowace ginshiƙi (100 × 1.8 mm id) shine 20,000 ± 100 (faranti 200,000/m²). Tebur na 3 yana ba da ƙimar N don ginshiƙan PMP guda uku kuma an nuna chromatograms a cikin Hoto na 5A. Binciken sauri akan ginshiƙin PMP a ƙimar kwararar mai yawa (700 μL/min), an fitar da peptides guda biyar cikin minti ɗaya, ƙimar N sun kasance masu kyau sosai, 13,500 ± 330 a kowane ginshiƙi (100 × 1.8 mm id), Ya yi daidai da faranti 135,000/m (Hoto na 5B). An cika ginshiƙai uku masu girman iri ɗaya (100 × 1.8 mm id) tare da wurare uku daban-daban na matakin PMP mai tsayawa don dubawa sake haifuwa. An rubuta yawan nazarin kowane ginshiƙi ta amfani da yanayin fitarwa mafi kyau da adadin faranti na ka'ida N da lokacin riƙewa don raba cakuda gwaji ɗaya akan kowane ginshiƙi. Bayanan sake haifuwa don ginshiƙan PMP an nuna su a cikin Jadawali na 4. Sauƙin sake haifuwa na ginshiƙin PMP yana da kyau tare da ƙimar %RSD mai ƙarancin gaske, kamar yadda aka nuna a Jadawali na 3.
Rabawar cakuda 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 × 1.8 mm id); nazari Tsarin fitarwa na mahaɗan: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) da 5 (leucine) acid enkephalin)).
An kimanta ginshiƙin PMP (100 × 1.8 mm id) don raba tryptic narkar da albumin na jini na ɗan adam a cikin babban aikin chromatography na ruwa. Chromatogram a cikin Hoto na 6 ya nuna cewa samfurin ya rabu sosai kuma ƙudurin yana da kyau sosai. An yi nazarin narkar da HSA ta amfani da ƙimar kwarara na 100 µL/min, matakin motsi na 70/30 acetonitrile/ruwa da 0.1% TFA. Kamar yadda aka nuna a cikin chromatogram (Hoto na 6), an raba narkar da HSA zuwa kololuwa 17 daidai da peptides 17. An ƙididdige ingancin rabuwa na kowane kololuwa a cikin narkar da HSA kuma an ba da ƙimar a cikin Tebur na 5.
An raba wani tryptic digest na HSA (100 × 1.8 mm id) a kan ginshiƙin PMP; ƙimar kwarara (100 µL/min), matakin motsi na 60/40 acetonitrile/ruwa tare 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, kuma an yi amfani da 60/40 v/v ACN/ruwa. Rarraba ginshiƙi na PMP (100 × 1.8 mm id) yayi kama da na bincikenmu na baya Ref.34. Rarraba ginshiƙi da aka cika da barbashi masu ramuka a saman shine: 1.7 × 10-15 don barbashi 1.3 μm, 3.1 × 10-15 don barbashi 1.7 μm, 5.2 × 10-15 da 2.5 × 10-14 m2 don barbashi 2.6 μm Ga barbashi 5 μm 43. Saboda haka, rarraba ginshiƙi na PMP yayi kama da na barbashi 5 μm na tsakiya.
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 ginshiƙan SILICA-C18 (100 × 1.8 mm id) 34 da C18-Urea ginshiƙai 31 da muka yi nazari a baya sune 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 (100 × 1.8 mm id) shine 0.60. Fuskar ginshiƙan PMP ya yi ƙasa da na ginshiƙan da aka cika da ƙwayoyin silica masu haɗin C18 saboda a cikin matakan tsayawa na nau'in C18, ligands na C18 suna haɗe da ƙwayoyin silica a matsayin sarƙoƙi masu layi, yayin da a cikin polystyrene-type static matakai, an samar da Layer ɗin polymer mai kauri A a kusa da shi. A cikin wani gwaji na yau da kullun, ana ƙididdige porosity na ginshiƙi kamar haka:
Siffa ta 7A,B ta nuna ginshiƙin PMP (100 × 1.8 mm id) da ginshiƙin Ascentis Express RP-Amide (100 × 1.8 mm id) ta amfani da yanayin fitarwa iri ɗaya (watau 60/40 ACN/H2O da 0.1% TFA). ) na filin van Deemter. An shirya cakuda peptide da aka zaɓa (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) a cikin 20 µL/ Mafi ƙarancin ƙimar kwarara ga ginshiƙai biyu shine 800 µL/min. Mafi ƙarancin ƙimar HETP a mafi kyawun ƙimar kwarara (80 µL/min) don ginshiƙin PMP da ginshiƙin Ascentis Express RP-Amide sune 2.6 µm da 3.9 µm, bi da bi. Ƙimar HETP tana nuna cewa ingancin rabuwa na ginshiƙin PMP (100 × 1.8 mm id) ya fi kyau fiye da ginshiƙin Ascentis Express RP-Amide da ake da shi a kasuwa (100 × 1.8 mm id). Tsarin van Deemter a cikin Hoto na 7(A) ya nuna cewa raguwar ƙimar N tare da ƙaruwar kwarara ba ta da mahimmanci idan aka kwatanta da bincikenmu na baya. Ingantaccen ingancin rabuwa na ginshiƙin PMP (100 × 1.8 mm id) idan aka kwatanta da ginshiƙin Ascentis Express RP-Amide ya dogara ne akan haɓakawa a cikin siffar barbashi, girma, da hanyoyin tattara ginshiƙai masu rikitarwa da ake amfani da su a cikin aikin yanzu34.
(A) taswirar van Deemter (gudun HETP idan aka kwatanta da saurin motsi na motsi) da aka samu ta amfani da ginshiƙin PMP (100 × 1.8 mm id) a cikin 60/40 ACN/H2O tare da 0.1% TFA.(B) taswirar van Deemter (gudun HETP idan aka kwatanta da saurin motsi na motsi) da aka samu ta amfani da ginshiƙin Ascentis Express RP-Amide (100 × 1.8 mm id) a cikin 60/40 ACN/H2O tare da 0.1% TFA.
An shirya kuma an kimanta wani tsari na polystyrene mai tsayi da aka haɗa da polar don raba gaurayen peptide na roba da kuma narkewar trypsin na albumin na jini na ɗan adam (HAS) a cikin babban aikin chromatography na ruwa. Aikin chromatographic na ginshiƙan PMP don gaurayen peptide yana da kyau a cikin ingancin rabuwa da ƙuduri. Ingantaccen aikin rabuwa na ginshiƙan PMP ya faru ne saboda dalilai daban-daban, kamar girman barbashi da girman ramin barbashi na silica, haɗakar lokaci mai tsayi, da kuma tattara ginshiƙai masu rikitarwa. Baya ga ingantaccen rabuwa, ƙarancin matsin lamba na baya na ginshiƙi a yawan kwararar ruwa wani fa'ida ne na wannan lokaci mai tsayi. Ginshiƙan PMP suna nuna kyakkyawan sake haifuwa kuma ana iya amfani da su don nazarin gaurayen peptide da narkewar trypsin na furotin daban-daban. Muna da niyyar amfani da wannan ginshiƙi don raba samfuran halitta, mahaɗan bioactive daga tsire-tsire masu magani da kuma abubuwan da aka cire na fungal a cikin chromatography na ruwa. A nan gaba, za a kuma kimanta ginshiƙan PMP don rabuwar sunadarai da ƙwayoyin rigakafi na monoclonal.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Bincike kan Tsarin Raba Peptide ta hanyar Juyawa Tsarin Chromatography Kashi na I: Ci gaban Tsarin Halayyar Ginshiƙi.J. Chromatography.1603, 113–129.https://doi.org/10.1016/j.chroma.2019.05.038 (2019).
Gomez, B. da sauransu. Ingantaccen peptides masu aiki waɗanda aka tsara don maganin cututtuka masu yaɗuwa. Biotechnology.Advanced.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. gano magunguna.15 (1-2) a yau, 40-56.https://doi.org/10.1016/j.drudis.2009.10.009 (2010).
Xie, F., Smith, RD & Shen, Y. Advanced Proteomic Liquid Chromatography.J. Chromatography.A 1261, 78–90 (2012).
Liu, W. da sauransu. Advanced liquid chromatography-mass spectrometry yana ba da damar haɗa metabolomics da aka yi niyya sosai da proteomics.anus.Chim.Acta 1069, 89–97 (2019).
Chesnut, SM & Salisbury, JJ Matsayin UHPLC a cikin haɓaka magunguna.J. Satumba. Sci.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. J. Satumba. Sci.30(8), 1167-1182.https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Amfani da sinadarin chromatography na ruwa mai matuƙar aiki a cikin haɓaka magunguna. J. Chromatography.1119(1-2), 140-146.https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al. Hydrogels masu yawan ƙwayoyin cuta masu kama da monolithic da aka shirya daga emulsions na ciki mai zurfi a cikin mai don tsarkake ƙwayoyin cuta masu inganci.Chemical.Britain.J. 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA Matsayin chromatography na ruwa a cikin proteomics. J. Chromatography.A 1053(1-2), 27-36 (2004).
Fekete, S., Veuthey, J.-L. & Guillarme, D. Sabbin abubuwan da ke faruwa a cikin rabuwar chromatography na ruwa mai juye-juye na peptides na warkewa da sunadarai: ka'ida da aikace-aikace. J. Pharmacy.Biomedical Science.anus.69, 9-27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Raba peptides masu girma biyu ta amfani da tsarin RP-RP-HPLC ta amfani da ƙimar pH daban-daban a cikin girman rabuwa ta farko da ta biyu. J. Satumba. Sci.28(14), 1694-1703 (2005).
Feletti, S. da sauransu. An binciki halayen canja wurin taro da aikin motsi na ginshiƙan chromatographic masu inganci waɗanda aka cika da ƙwayoyin C18 sub-2 μm gaba ɗaya da kuma waɗanda ba su da ramuka a saman. J. Satumba. Sci.43 (9-10), 1737-1745 (2020).
Piovesana, S. da sauransu. Sabbin abubuwan da suka faru da ƙalubalen nazari a cikin warewa, ganowa da tabbatar da peptides masu aiki da tsirrai.anus.biological anus.Chemical.410(15), 3425–3444.https://doi.org/10.1007/s00216-018-0852-x (2018).
Mueller, JB da sauransu. Yanayin proteomic na masarautar rayuwa.Nature 582(7813), 592-596.https://doi.org/10.1038/s41586-020-2402-x (2020).
DeLuca, C. da sauransu. Tsarin sarrafa peptides na warkewa ta hanyar shirya chromatography na ruwa. Molecule (Basel, Switzerland) 26(15), 4688(2021).
Yang, Y. & Geng, X. Tsarin chromatography na yanayi iri-iri da aikace-aikacensa ga biopolymers. J. Tsarin Chromatography.A 1218(49), 8813–8825 (2011).
Zhao, G., Dong, X.-Y.& Sun, Y. Ligands don tsarin furotin mai gauraye: ƙa'ida, halaye, da ƙira. J. Biotechnology.144(1), 3-11 (2009).