Name: preparation of highly porous coordination polymer coatings on macroporous polymer monoliths for enhanced enrichment of phosphopeptides
Uploaded: 2015-07-14T13:30:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides at JoVE.com

This work has been performed at the Molecular Foundry, Lawrence Berkeley National Laboratory and supported by the Office of Science, Office of Basic Energy Sciences, Scientific User Facilities Division of the US Department of Energy, under Contract No. DE-AC02&#8211;05CH11231. The financial support of F.M. by a ME-Fulbright fellowship and A.S. by Higher Education Commission of Pakistan are gratefully acknowledged.

참고 : 시작하기 전에, 모든 관련 자료 데이터 시트 (MSDS (물질 안전 보건 자료))을 확인. 합성 절차 및 애플리케이션에 사용되는 화학 물질 중 일부는 독성. 모든 적절한 안전 관행에 따라 적절한 보호 장비 (실험실 코트, 전체 길이 바지, 폐쇄 발가락 신발, 보호 안경, 장갑)를 사용하십시오. 질소 흡착 측정을위한 액체 질소 (절연 장갑, 얼굴 가리개)를 처리 할 때 모든 극저온 개인 보호 장비를 사용하십시오. 벌크 및 모세관 컬럼 형식의 1 다공성 고분자 모노리스 준비 <ol><li> 특성에 대한 대량 폴리머 모노리스 <ol><li> 중합 억제제를 제거하기 위해, 염기성 알루미나 컬럼을 통해 스티렌, 디 비닐 벤젠 및 메타 크릴 산을 정제. 주사기 팁에 포장 유리 양모 섬유의 플러그가있는 25 ml의 일회용 플라스틱 주사기에 기본 알루미나 10g을 놓습니다. 칼럼을 통해 단량체의 약 10 ㎖을 관류. </li><li> 단량체 (50 mg의 스티렌, 디 비닐 벤젠 100 mg을 50 mg의 메타 크릴 산) 및 1 mL 유리 바이알에 기공 형성 제 (300 mg을 톨루엔 및 300 mg을 이소옥탄)을로드. 중합 개시제 2,2&#39;- 아조 비스 이소 부티로 니트릴 (AIBN, 단량체에 대하여 1 %) 4 mg을 추가. </li><li> 10 분 동안 초음파 처리하여 균질화. 10 분 동안 액체를 통해 질소를 버블 링하여 용존 산소를 제거합니다. 파라핀 필름으로 유리 병 마개를 밀봉하고, 혼합물을 중합하기 위해 6 시간 동안 60 ℃에서 수조에 배치. </li><li> 실온으로 냉각 조심스럽게 병을 깰. 셀룰로오스 추출 골무에 폴리머 기둥을 전송합니다. 속 슬렛 추출 챔버로 추출 골무를 놓고 추출 챔버의 적어도 세 번 볼륨 메탄올의 부피를 포함하는 둥근 바닥 플라스크를 조립한다. 추출 챔버의 상부에 콘덴서를 조립한다. 메탄올을 끓여 속 슬렛 추출을 수행16 시간 동안, 미 반응 단량체의 완전한 제거를 보장하고 기공 형성 제. </li><li> 60 ℃에서 진공 오븐에서 하룻밤 동안 건조. 푸리에에 의해 주치의 적외선 분광법 (FT-IR)를 변환 첨부 카르 복실 작용기의 존재를 확인합니다. 질소 흡착 세공 측정법에 의해 비 표면적을 측정한다. </li></ol></li><li> 모 놀리 식 기둥의 준비를 위해 실리카 모세 혈관의 작용 <ol><li> 폴리이 미드 코팅 된 100 ㎛의 ID 용융 실리카 모세관의 2m를 잘라. 0.25-0.50 mL 유리 주사기에 연결하고 아세톤으로 모세관을 씻는다. 물과 모세 혈관을 세척하여 아세톤을 제거합니다. </li><li> 모세관 내부의 실리카 코팅을 활성화하기 위해, 30 분 / μL에서 0.25 분, 0.2 M 수성 NaOH 용액을 흐르게 주사기 펌프를 사용한다. 유출 물이 중성이 될 때까지 물로 씻어. </li><li> 폐수의 pH를 확인 산도 종이 스트립을 사용합니다. 모세관의 실란 올 그룹을 양성자 화하기 위해, 0.2 M aqueo 펌프30 분 동안 0.25 μL / 분 모세관을 통해 우리 HCl 용액. 유출 물이 중성이 될 때까지 물로 씻어. 에탄올로 씻어. </li><li> 1 시간 / μL로 0.25 분 (w / w) 20 % 3- (트리 메 톡시 실릴) 프로필 메타 크릴 레이트 (아세트산으로 pH를 조정 5)의 에탄올 용액 펌프. 이 단계에서, 실리카 모세관은 모세관 내면 중합체 모노리스를 장착하기 위해 비닐기로 작용 화한다. </li><li> 질소 기류, 아세톤으로 세척하여 건조 밤새 사용하기 전에 실온에서 떠난다. 길이 20cm의 짧은 조각으로 모세관을 잘라. </li></ol></li><li> 모 놀리 식 모세관 컬럼의 준비 <ol><li> 고무 격막과 1 mL 유리 병에 일괄 폴리머 모노리스 (1.1 절)에 대해서는 동일한 중합 혼합물을 준비합니다. 단량체에 대해 기자에게 1 %의 AIBN을 추가합니다. 10 분 동안 초음파 처리하여 균질화. </li><li> 비 - 관능 화 실리카 모세관을 커플 링시킴으로써 질소로 중합 혼합물 퍼지질소 스트림. <ol><li> 유리 병의 고무 격막을 통해 질소 기류 모세관을 삽입하고 질소는 액체를 통해 거품 있도록 중합 혼합물로 몰입. 과도한 압력을 피하기 위해 약간 느슨한 유리 병 뚜껑을 남겨주세요. 10 분 동안 퍼징. </li><li> 바이알의 헤드 스페이스에 중합 혼합물로부터 질소 기류 모세관을 들어 올리고 뚜껑을 꼭 닫습니다. 중합 혼합물에 격막을 통해 작용 모세관을 삽입합니다. 상부 공간에 주입 된 질소를 통해 모세관에 생성 된 압력의 초과는 모세관 작용을 통해 중합 반응 혼합물을 펌프. </li><li> 완전히 충전되어 있는지 확인하고 고무 격막을 닫습니다 모세관의 유출에서 중합 혼합물의 몇 방울을 수집합니다. 매우 신중하게 유리 병 밖으로 모세관을 가지고 고무 격막에 모세관의 입구를 닫습니다. </li></ol></li><li> 믹스를 중합6 시간 동안 60 ℃에서 수욕 모세관에 포함 스트럭처. 실온에서 냉각시키고, 모세관 양단의 몇 밀리미터를 잘랐다. 30 분 동안 3 μL / min으로 HPLC 펌프를 사용하여 아세토 니트릴로 열을 세척하여 미 반응 모노머 및 기공 형성 제를 제거합니다. 모세관 컬럼의 배압을 확인합니다. </li></ol></li></ol> 철 benzenetrycarboxylate 2. 성장 (FeBTC) PCP <ol><li> 특성에 대한 대량 폴리머 모노리스에 FeBTC MOPH의 성장 <ol><li> 박격포와 유 봉을 사용하여 이전에 건조 된 모노리스를 갈기. </li><li> 15 분 동안 2 mM의 에탄올의 FeCl3 · 6H 2 O 5 ml의 모노리스 분말 100mg을 담가. 나일론 필터 (0.22 μm의)와 에탄올로 가루를 씻어을 사용하여 진공 필터. 15 분 동안 에탄올에서 2 mM의 1,3,5- 벤젠 트리 카르 복실 산 (BTC) 5ml에 모노리스 분말을 담근다. 나일론 필터 (0.22 μm의)와 에탄올로 가루를 씻어을 사용하여 진공 필터.</li><li> 원하는대로 단계를 반복 번호 2. 최종 금속 - 유기 피막의 성장이 적용 사이클의 수에 의해 정의 될 뿐이다. 일반적으로 10 ~ 30 사이클이 수행됩니다. 질소 흡착 압 입법에 의한 새로운 구멍의 존재를 확인합니다. 열 중량 분석 (TGA)에 의해 추가 된 금속 부위의 양을 측정한다. </li></ol></li><li> 포스 포의 농축을위한 모세관 모 놀리 식 열에 FeBTC MOPH의 성장 <ol><li> 주사기 펌프를 이용. 2 μL / 분으로 15 분 동안 2 mM의 에탄올 중의 FeCl3 · 6H 2 O와 모세관 모노리스 플러시. 2 μL / 분에서 15 분 동안 에탄올로 씻으십시오. 2 μL / 분에서 15 분간 에탄올에서 2 mM의 BTC와 모세관 모노리스 플러시. 2 μL / 분에서 15 분 동안 에탄올로 씻으십시오. </li><li> 필요에 따라 1 단계를 반복합니다. 최종 금속 - 유기 피막의 성장이 수행 사이클의 수에 의해 정의 될 뿐이다. </li></ol></li></ol> 3. 단백질 소화와 E포스 포의 nrichment <ol><li> 단백질 소화 <ol><li> 물 1 ㎖에 무 지방 우유 0.5 ㎖에 용해시키고, 200 μL 분획으로 나눈다. </li><li> 단백질 소화 이황화 결합을 절단하기 위해, 각각의 분획을 160 ㎕의 1 M 중탄산 암모늄 및 50 ㎕의 45 mM의 디티 오 트레이 톨을 추가한다. 15 분 동안 써모에서 50 ° C에서 인큐베이션. </li><li> 용액 내려 실온으로 냉각하면서, 요오도 아세트 아미드 100 mM이 수용액 서서히 50 μl를 추가. 요오도 아세트 아미드는 새로운 이황화 결합의 형성을 방지한다. </li><li> 실온에서 15 분 동안 어두운 데에서 인큐베이션. 탈 이온수 1 ML을 추가합니다. 14 시간 동안 37 ℃에서 써모에서 단백질을 2 μg의 트립신을 추가하고 소화. </li><li> 1 % 트리 플루오로 아세트산 10 μL로 산성화하여 소화를 종료하고, 실온에서 5 분 동안 써모에 배치. 소화 단백질에 보관 -20 ° C. </li></ol></li><li> 모세관 MOPH 열을 사용하는 포스 포의 농축. <ol><li> 1 μL / 분의 유속에서 10 분 동안 0.1 % 트리 플루오로 아세트산 함유 아세토 니트릴의 1 : 1 혼합물 100 ㎕ 4와 칼럼 플러시. 30 분 / 2 μL에서 분 칼럼을 통해 단백질 소화 펌프. </li><li> 1 μL / 분의 유속에서 10 분 동안 0.1 % 트리 플루오로 아세트산 함유 아세토 니트릴의 혼합물을 1 : 4로 다시 인산화되지 않은 펩티드를 세척 할 것. 1 μL / 분의 유속으로 10 분 동안 물로 세척한다. </li><li> 15 분 동안 1 μL / 분으로 펌핑 된 250 mM의 pH가 7 인산염 완충액을 사용하여 용출 포스 포. 유리 병에 용리액을 수집하고 표준 프로토콜 19을 사용하여 솔루션을 탈염. 매트릭스 보조 레이저 탈착 / 이온화 비행 시간 형 질량 분석계 (MALDI-TOF-MS)에 대한 매트릭스로서 사용하는 2 ㎎ / ㎖ 2,5- 디 히드 록시 벤조산을 준비한다. phosphope을 용출하는 팁에 2,5- 디 히드 록시 벤조산의 2 μl를 그린ptides는 MALDI 판에 직접 자리합니다. </li><li> MALDI-TOF-MS에 의해 명소를 분석하고 물과 메탄올로 충분히 세척하여 열을 생성합니다. </li></ol></li></ol>

<ol>
	<li>Li, H., Eddaoudi, M., O&#8217;Keeffe, M., Yaghi, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+an+exceptionally+stable+and+highly+porous+metal-organic+framework.">Design and synthesis of an exceptionally stable and highly porous metal-organic framework.</a> Nature. 402, 276-279 (1999).</li><li>Kitagawa, S., Kitaura, R., Noro, S. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+porous+coordination+polymers.">Functional porous coordination polymers.</a> Angew. Chem. Int. Ed. 43, 2334-2375 (2004).</li><li>Furukawa, H., Cordova, K. E., O&#8217;Keeffe, M., Yaghi, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+and+applications+of+metal-organic+frameworks.">The chemistry and applications of metal-organic frameworks.</a> Science. 341, 974 (2013).</li><li>Ma, S., Zhou, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+storage+in+porous+metal-organic+frameworks+for+clean+energy+applications.">Gas storage in porous metal-organic frameworks for clean energy applications.</a> Chem. Commun. 46, 44-53 (2010).</li><li>Li, J. R., Sculley, J., Zhou, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-organic+frameworks+for+separations.">Metal-organic frameworks for separations.</a> Chem. Rev. 112, 869-932 (2012).</li><li>Lee, J., Farha, O. K., Roberts, J., Scheidt, K. A., Nguyen, S. T., Hupp, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-organic+framework+materials+as+catalysts.">Metal-organic framework materials as catalysts.</a> Chem. Soc. Rev. 38, 1450-1459 (2009).</li><li>Gu, Z. Y., Yang, C. X., Chang, N., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-organic+frameworks+for+analytical+chemistry:+From+sample+collection+to+chromatographic+separation.">Metal-organic frameworks for analytical chemistry: From sample collection to chromatographic separation.</a> Acc. Chem. Res. 45, 734-745 (2012).</li><li>Ahmad, R., Wong-Foy, A. G., Matzger, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microporous+coordination+polymers+as+selective+sorbents+for+liquid+chromatography.">Microporous coordination polymers as selective sorbents for liquid chromatography.</a> Langmuir. 25, 11977-11979 (2009).</li><li>Yang, C. X., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-organic+framework+MIL-101(Cr)+for+high-performance+liquid+chromatographic+separation+of+substituted+aromatics.">Metal-organic framework MIL-101(Cr) for high-performance liquid chromatographic separation of substituted aromatics.</a> Anal. Chem. 83, 7144-7150 (2011).</li><li>Fu, Y. Y., Yang, C. X., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+coordination+status+of+the+open+metal+sites+in+metal-organic+frameworks+for+high+performance+separation+of+polar+compounds.">Control of the coordination status of the open metal sites in metal-organic frameworks for high performance separation of polar compounds.</a> Langmuir. 28, 6802-6810 (2012).</li><li>Gu, Z. Y., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-organic+framework+MIL-101+for+high-resolution+gas-chromatographic+separation+of+xylene+isomers+and+ethylbenzene.">Metal-organic framework MIL-101 for high-resolution gas-chromatographic separation of xylene isomers and ethylbenzene.</a> Angew. Chem. Int. Ed. 49, 1477-1480 (2010).</li><li>Chang, N., Gu, Z. Y., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zeolitic+imidazolate+framework-8+nanocrystal+coated+capillary+for+molecular+sieving+of+branched+alkanes+from+linear+alkanes+along+with+high-resolution+chromatographic+separation+of+linear+alkanes.">Zeolitic imidazolate framework-8 nanocrystal coated capillary for molecular sieving of branched alkanes from linear alkanes along with high-resolution chromatographic separation of linear alkanes.</a> J. Am. Chem. Soc. 132, 13645-13647 (2010).</li><li>Yu, L. Q., Xiong, C. X., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room+temperature+fabrication+of+post-modified+zeolitic+imidazolate-90+as+stationary+phase+for+open-tubular+capillary+electrochromatography.">Room temperature fabrication of post-modified zeolitic imidazolate-90 as stationary phase for open-tubular capillary electrochromatography.</a> J. Chromatogr. A. 1343, 188-194 (2014).</li><li>Fu, Y. Y., Yang, C. X., Yan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+metal-organic+framework+UiO-66+into+porous+polymer+monoliths+to+enhance+the+liquid+chromatographic+separation+of+small+molecules.">Incorporation of metal-organic framework UiO-66 into porous polymer monoliths to enhance the liquid chromatographic separation of small molecules.</a> Chem. Commun. 49, 7162-7164 (2013).</li><li>Lin, C. L., Lirio, S., Chen, Y. T., Lin, C. H., Huang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+hybrid+metal-organic+framework-polymeric+monolith+for+solid-phase+extraction.">A novel hybrid metal-organic framework-polymeric monolith for solid-phase extraction.</a> Chem. Eur. J. 20, 3317-3321 (2014).</li><li>Svec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+polymer+monoliths:+Amazingly+wide+variety+of+techniques+enabling+their+preparation.">Porous polymer monoliths: Amazingly wide variety of techniques enabling their preparation.</a> J. Chromatogr. A. 1217, 902-924 (2010).</li><li>Shekhah, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+route+for+the+synthesis+of+metal-organic+frameworks.">Step-by-step route for the synthesis of metal-organic frameworks.</a> J. Am. Chem. Soc. 129, 15118-15119 (2007).</li><li>Shekhah, O., Fu, L., Belmabkhout, Y., Cairns, A. J., Giannelis, E. P., Eddaoudi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+implementation+of+the+stepwise+layer-by-layer+growth+of+MOF+thin+films+on+confined+surfaces:+mesoporous+silica+foam+as+a+first+case+study.">Successful implementation of the stepwise layer-by-layer growth of MOF thin films on confined surfaces: mesoporous silica foam as a first case study.</a> Chem. Commun. 48, 11434-11436 (2012).</li><li>Saeed, A., Maya, F., Xiao, D. J., Naham-ul-Haq, M., Svec, F., Britt, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+highly+porous+coordination+polymer+on+a+macroporous+polymer+monolith+support+for+enhanced+immobilized+metal+ion+affinity+chromatographic+enrichment+of+phosphopeptides.">Growth of a highly porous coordination polymer on a macroporous polymer monolith support for enhanced immobilized metal ion affinity chromatographic enrichment of phosphopeptides.</a> Adv. Funct. Mater. 24, 5797-5710 (2014).</li><li>Krenkova, J., Lacher, N. A., Svec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+selectivity+via+nanochemistry:+Monolithic+capillary+column+containing+hydroxyapatite+nanoparticles+for+separation+of+proteins+and+enrichment+of+phosphopeptides.">Control of selectivity via nanochemistry: Monolithic capillary column containing hydroxyapatite nanoparticles for separation of proteins and enrichment of phosphopeptides.</a> Anal. Chem. 82, 8335-8341 (2010).</li><li>Jabeen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica-lanthanum+oxide:+Pioneer+composite+of+rare-earth+metal+oxide+in+selective+phosphopeptides+enrichment.">Silica-lanthanum oxide: Pioneer composite of rare-earth metal oxide in selective phosphopeptides enrichment.</a> Anal. Chem. 84, 10180-10185 (2012).</li><li>Hussain, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+diamond+nanopowder+for+phosphopeptides+enrichment+from+complex+biological+fluids.">Functionalized diamond nanopowder for phosphopeptides enrichment from complex biological fluids.</a> Anal. Chim. Acta. 775, 75-84 (2013).</li><li>Aprilita, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(glycidyl+methacrylate/divinylbenzene)-IDA-FeIII+in+phosphoproteomics.">Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.</a> J. Proteom. Res. 4, 2312-2319 (2005).</li><li>Lo, C. Y., Chen, W. Y., Chen, C. T., Chen, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enrichment+of+phosphopeptides+from+tryptic+digests+of+proteins+using+iron+oxide+nanocomposites+of+magnetic+particles+coated+with+zirconia+as+the+concentrating+probes.">Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes.</a> J. Proteom. Res. 6, 887-893 (2007).</li><li>Aryal, U. K., Ross, A. R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+and+analysis+of+phosphopeptides+under+different+experimental+conditions+using+titanium+dioxide+affinity+chromatography+and+mass+spectrometry.">Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 24, 219-231 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Select Iron Affinity Gel Technical Bulletin. , (2015).</li></ol>

The authors have nothing to disclose.

원래 고분자 모노리스는 금속에 결합 할 수 카르 복실 작용기를 포함하고 있습니다. 원래 소재에 초기 금속 자리 코디 우리 미다 네트워크 정형 추가적인 금속 자리의 숫자를 통합 PCP 코팅 (도 1a)을 성장할 수있다. 이것은 고정 된 금속 이온 친 화성 크로마토 그래피 (IMAC) 기술과 같은 금속의 종 관련된 추출 또는 정제 과정, 매력적인 제시된 MOPH 재료를 만든다. 포스 포의 농축 용 캐 ?...

다공성 배위 고분자 (의 PCP)는 비정질 또는 결정질 일 수있다 1-3 1, 2 또는 3 차원으로 연장되는 조정 엔티티 반복 유기 리간드에 의해 연결된 금속 중심에 기초한 배위 화합물이다. 최근에, 다공성 물질이 클래스는 그들의 높은 다공성, 넓은 화학 가변성, 및 안정성으로 널리 주목 받고있다. 의 PCP는 가스 저장, 가스 분리 및 촉매 3-6, 매우 최근의 PCP의 분석 애플리케이션 제 7 기술되었다을 포함한 다양한 애플리케이션에 대해 탐구되어왔다. 때문에 강화 화학 기능과 높은 기공율의 PCP 정제 공정 및 크로마토 그래피 분리의 개선을위한 그들의 엄청난 잠재적 대상으로되어 있고,이 항목에 관한 다수의 보고서가 7-13 발표되었다. 그러나,의 PCP의 성능 equivale에서 현재 사용하지 않는그들의 입자 또는 결정의 그들의 일반적으로 불규칙한 모양의 형태학에 의한 이러한 고체의 충전 층에서 큰 입자 간 공극을 통해 빠르게 확산 가능성이 기존의 크로마토 그래피 재료와 NT 수준. 이 불규칙하게 분산 포장 낮은 예상보다 성능뿐만 아니라 높은 열 배압 및 바람직하지 않은 피크 모양 형태학 14, 15로 연결됩니다. 입자 간 공극을 통해 빠르게 확산의 문제를 해결하고 부수적 분석 애플리케이션의 PCP의 성능을 향상시키기 위하여, 거대 기공의 표면에 PCP를 포함하는 거대 다공성 중합체 모노리스 (16)에 기초하여 복합 재료의 개발하고자 것이 바람직하다. 고분자 모노리스 패킹 비드 성공적 여러 C 상용화되어 가장 효율적인 대안들 중 하나를들을 수있는, 그들의 구멍을 통해 대류 흐름을 유지할 수 단일 피스 재료 자체 포함 된<html> ompanies (17, 18). 다공성 중합체는 일반적으로 모노리스 단량체의 중합, 전형적으로 유기 용매의 혼합물이다 이진 포로 겐의 존재 하에서 가교제에 기초한다. 얻어진 재료는 모 놀리 microglobular 구조 및 높은 다공성 및 유동 투과도. 간단한 방법은 PCP를 함유하는 중합체 단일체 모노리스의 중합 혼합물의 PCP 합성 된 상태의 직접 첨가에 기초 제조 이들 재료를 통합한다. 결과의 PCP이 방식은 주로 최종 14,15 재료의 상기 어플리케이션에 대해 활성 인 고분자 지지체 내에 매립되는 것은 아니다. 다른 합성 방법이 명확하게, 예를 들면, 결정에 포함 된 기공의 대부분이 중합체 모노리스의 거대에서 액세스의 PCP, 또는 결정 성 금속 - 유기 프레임 워크 (MOFs는)의​​ 균일 한 필름을 개발하기 위해 필요하다. t &quot;&gt; 여기서 우리는 쉽게 구현 될 수의 PCP의 부착에 적합한 작용기를 가진 거대 다공성 고분자 지지체에 기초한 유기 금속 중합체 복합 재료 (MOPH)의 제조를위한 간단한 프로토콜보고 급식 단일 관류 애플리케이션을위한 최적의 특성을 갖는 열 형식 -piece 중합체 모놀리스. 중합체 합성 절차가 간단 실온 용액 기반 뒤에 &#160; 방법 19-20 모노리스의 공극의 내부 표면 상에 코팅 PCP 성장. 첫 번째 예로서, 우리는 거대 다공성 폴리 (스티렌 - 디 비닐 벤젠 - 메타 크릴 산) 모노리스 내의 철 (III) 벤젠 (FeBTC) 배위 고분자 필름의 제조를 기술한다. 이 방법은 벌크 분말의 제조뿐만 아니라, 캐 필러 리 컬럼을위한 효과적이고 설명은 다른 프로토콜의 PCP에게 용이하게 구현 가능하다. 유동 throu위한 기능성 재료로서 MOPHs의 전위의 예로서GH 응용 프로그램, 우리는 센터가 철에 포스 포의 결합 친화력을 악용 소화 단백질 혼합물에서 포스 포 농축 (III) 철의 고밀도 코팅을 포함하는 개발 FeBTC MOPH을 적용 (III). 개발 된 프로토콜 (21)는 세 가지 주요 부분 : 상기 거대 다공성 유기 고분자 모노리스 지원 준비; 모노리스의 기공의 표면에 코팅 PCP의 성장; 포스 포의 농축을위한 응용 프로그램입니다.

<table><tbody><tr><td>Polyimide-coated capillaries</td><td>Polymicro Technologies</td><td>TSP100375</td><td>100 &amp;mu;m i.d.</td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate, 98%</td><td>Sigma-Aldrich</td><td>440159</td><td></td></tr><tr><td>Styrene, 99%</td><td>Sigma-Aldrich</td><td>W323306</td><td>Technical grade</td></tr><tr><td>Divinylbenzene, 80%</td><td>Sigma-Aldrich</td><td>414565</td><td></td></tr><tr><td>Methacrylic acid, 98%</td><td>Mallinckrodt</td><td>MK150659</td><td></td></tr><tr><td>Toluene, &amp;ge;99.5%</td><td>EMD chemicals</td><td>MTX0735-6</td><td></td></tr><tr><td>Isooctane, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>650439</td><td></td></tr><tr><td>2,2&#039;-azobisisobutyronitrile, 98%</td><td>Sigma-Aldrich</td><td>441090</td><td></td></tr><tr><td>Aluminium oxide (basic alumina)</td><td>Sigma-Aldrich</td><td>199443</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate, 97%</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>1,3,5-benzenetrycarboxylic acid, 95%</td><td>Sigma-Aldrich</td><td>482749</td><td></td></tr><tr><td>Acetonitrile, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>360457</td><td></td></tr><tr><td>Ammonium bicarbonate, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>Trifluoroacetic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>302031</td><td></td></tr><tr><td>Ethanol, &amp;ge;99.8%</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Iodoacetamide, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Dithiothreitol, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>Monobasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71505</td><td></td></tr><tr><td>Dibasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Phosphoric acid, &amp;ge;85%</td><td>Sigma-Aldrich</td><td>438081</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>85707</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8003</td><td>Bovine pancreas</td></tr><tr><td>&amp;beta;-casein</td><td>Sigma-Aldrich</td><td>C6905</td><td>Bovine milk</td></tr><tr><td>ZipTip pipette tips</td><td>Merck Millipore</td><td>ZTC18S096</td><td>C18 resin</td></tr></tbody></table>

preparation of highly porous coordination polymer coatings on macroporous polymer monoliths for enhanced enrichment of phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

유기 고분자 모노리스의 기공 표면에 PCP 성장의 개략도는도 1에 도시된다.이 도면에서, 우리는 예시 초기의 Fe (III) 원자 복실 작용기에 배위 원래 중합체 모노리스의 기공 표면에 보유 . 프로토콜을 사용하는 것은 본원에 추가로 유기 리간드와 설명의 Fe (III) 이온이 중합체 모노리스 내의 다공질 코디 네트워크를 성형 표면에 첨가된다. 또한 개략적위한 관류 지지체로서 준비한 모세?...

Watch this Scientific Journal Video about Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides at JoVE.com

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

포스 포의 향상된 농축을위한 거대 다공성 고분자 모노리스에 다공성 조정 폴리머 코팅의 제조

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

preparation-highly-porous-coordination-polymer-coatings-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

10.0K 조회수.  E. O. Lawrence Berkeley National Laboratory. A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

비디오: Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.

We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.

플라즈마 Polymerized 빈 입자의 캡슐화 및 투과율 특성

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma ...

연구

공학

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

시퀀스 및 선택적 인식 하 여 단일 가닥 RNAs에 더블-좌초 RNAs의 화학적 펩 티 드 핵 산 수정

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

유전학

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

폴리스타이렌을 사용하여<em&gt; 블록</em&gt; 폴리 (아크릴산) 및 이들의 단독 중합체는 공중합 용 단량체로 피복 된 금속 나노 입자

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

화학

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

전립선 암에 있는 Phosphoproteome를 조사 하기 위해 양적 라벨 무료 질량 분석으로 결합 된 Phosphopeptide 농축

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

암 연구학

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine 기반 펩타이드 Amphiphiles (PPAs) 및 관련된 생체 자기 조립의 합성에 대 한 손쉬운 프로토콜

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

단일 분자 힘 분광학을 위한 OaAEP1 중재된 효소 합성 및 중합단백질의 고정화

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

생화학

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

모 세관 지역 전기 이동 법 탠덤 질량 분석을 사용 하 여 대규모 하향식 단백질

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

복잡한 생물 학적 샘플에서 선택적으로 - glycosylated 펩티드을 풍요롭게하는 렉틴 HPLC 방법

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

생물학

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

단백질 정화에 대 한 Poly(pentafluorophenyl acrylate) 기능성 SiO2 구슬의 준비

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

정보 베어링 펩토이드의 합성과 시퀀스 지향 동적 공유자 자체 조립

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

포스 포의 향상된 농축을위한 거대 다공성 고분자 모노리스에 다공성 조정 폴리머 코팅의 제조

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