We gratefully acknowledge Dr. Markus Zweckstetter for providing the pdb coordinate file for the 'native' tau ensemble predicted from his NMR work, with contributed analysis tools provided by Dr. Adnan Sljoka. Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC) ENGAGE Grant program.

참고 : 사용하기 전에 모든 관련 물질 안전 보건 자료 (MSDS)를 참조하십시오. 폴리 (메틸 메타 크릴 레이트)의 레이저 어블 레이션 (PMMA)에 의해 생성 된 증기는 독성이있을 수있다. 레이저 조각사가 작동 환기 시스템에 연결되어 있는지 확인합니다. (, 보안경, 얼굴 마스크, 장갑, 실험실 코트, 전체 길이 바지 폐쇄 발가락 신발) 공학적 관리 (흄 후드, 예리한 컨테이너) 및 개인 보호 장비의 사용을 포함하여 마이크로 유체 장치를 구축 할 때 모든 적절한 안전 방법을 사용합니다. 그것은 가능한 분석 과정을 방해 오염 물질을 감소 ACS 등급 이상의 모든 존재와 함께, 고성능 액체 크로마토 그래피 (HPLC) 등급의 시약을 사용하는 것이 가장 중요합니다. 미세 유동 장치의 제조 1 <ol><li> PMMA 마이크로 유체 플랫폼의 건설 <ol><li> 표준 PMMA 블록 (8.9 X 3.8 X 0.6 cm) 및 구 시약을 도입하기위한 proteo을 입력 채널을 레이저 어블 레이션-용해 챔버 및 레이저 조각기 (17), (18)를 이용하여 출력 채널. 주의 : 연장 된 타원형 (30 × 5 mm X 0.05)의 단백질 분해 챔버 에칭. 입력 및 출력 채널은 PMMA 블록의 단부까지 연장하고, GA (30) 모세관을 수용하기 위해 두 폭과 깊이보다 더 큰 75 μm의 없어야한다. </li><li> . 1/64 &quot;두께 차단 디스크와 회전 공구를 사용하여 약 10cm 각의 두 부분으로 30 GA 스테인레스 금속 모세관 컷 모세관의 단부 (이것은 아래 보면서 용이해진다 부드럽게 사포를 사용하여 광학 현미경). </li><li> 납땜 인두를 사용하여 에칭 된 폴리 (메틸 메타 크릴 레이트) 블록으로 모세관 녹아. 입력 채널은 자동 주사기 펌프에 연결되며, 상기 출력 채널은 MS에 결합을 위해 사용될 것이다. </li></ol></li><li> 연속 흐름 시간 해결 운동 믹서의 건설 <ol><li> 융합을 구합니다석영 유리 모세관 (ID : 75 μm의, OD 150 μm의) 약 40cm의 약 15cm의 28 GA 스테인레스 금속 모세관 삽입. 필요한 반응 시간에 따라, 더 큰 ID와 장기간 외부 금속 또는 모세관을 사용한다. 주 : 금속 모세관의 &#39;참&#39;내경을 결정하는 정확한 반응 시간을 얻기 위해 중요하다. 이것은 비록 용매 모세관 유동 및 후방 압력, 레코딩 HPLC에 금속 모세관을 부착시킴으로써 이루어질 수있다. 내경 계산에 배압이 이루어질 수 있으며, 금속 모세관의 실제의 내경이 결정. 이것은 (윈도우 버전 6.49의 경우) 분자량 계산기 소프트웨어를 사용하여 계산 될 수있다. </li><li> 내측 유리 모세관의 일단에 저출력의 레이저 조각기를 이용하여 설정 2mm 노치 생산 내측 유리 모세관 (도 2)의 단부를 밀봉. 또한, 세라믹 유리 capillar를 사용하여 노치을Y 커터 공구 나 미세 절단 날과 회전 분쇄기. </li><li> 라인업 금속 모세관의 끝 부분과 상기 내부 유리 모세관 (이는 광 현미경에 의해 용이하게 볼 수있다). </li><li> 믹싱 티의 일단 (도 2)이 부착 운동 믹서. </li><li> 믹싱 티 운동 혼합기의 반대쪽에 약 40cm의 (가 150㎛ : 75 μm의, OD ID) 융합 된 석영 유리 모세관을 첨부. 이러한 반응을 종결 산 (5 % 아세트산의 pH 2.4)을 제공하기 위해 사용된다. 주의 : 반응물 자동 주입 펌프를 사용하여 테트라 플루오로 에틸렌 (PTFE) 튜브를 통해 기밀 주사기로 장치에 공급된다. </li></ol></li><li> 펩신의 활성화 <ol><li> 돼지의 위 점막에서 20mg의 펩신을 달아 결합 완충액 (0.1 M 인산 나트륨, 0.15 M 염화나트륨, pH를 6.0) 1 ㎖에 중단. </li><li> N 개의 -hydroxysuccinimide (NHS) 부활 아가 로스 비드 50 mg의 체중을 재들에 추가uspended 프로테아제 부드럽게 밤새 4 ℃로 회전한다. </li><li> 수지를 수집하고 실온에서 2 분 동안 1,000 XG에서 스핀 다운. </li><li> 언 바운드 단백질 분해 효소를 대기음. </li><li> 버퍼 (1 M 트리스 -HCl, pH를 6.0) 차단를 1ml 아가 로스를 인큐베이션하고 1 시간 동안 실온에서 부드럽게 회전한다. </li><li> 수지를 수집하고 실온에서 2 분 동안 1,000 XG에서 스핀 다운. </li><li> 블로킹 버퍼 대기음. </li><li> 1 mL의 5 % 아세트산, 5 분 동안 pH를 2.4 펩신 아가 로스 부화. </li><li> 수지를 수집하기 위해 2 분 동안 1,000 XG에서 스핀 다운. </li><li> 뜨는을 대기음. </li><li> 세 세척의 총 1.3.10 - 반복 1.3.8 단계를 반복합니다. </li><li> 장시간 사용을 위해 4 ℃에서 1 mL의 아세트산 비드를 저장한다. </li></ol></li><li> 장치 조립 <ol><li> 멸균 주걱을 사용하여 5 % 아세트산 펩신 활성 아가 로스 비드 슬러리와 단백질 분해 챔버를 채운다. </li><li> PMMA 마이크로 유체 platfor 배치장치를 밀봉하는 덮개와 같은 두 개의 빈 PMMA 블록 사이에서 m은 액밀 시일을 만드는 실리콘 고무 늘어서. </li><li> 압력 실에 장치를 금속 클램핑 플레이트를 사용한다. 주 : 클램프 맞춤 미세 유체 플랫폼을 맞추기 위해 제출되었다 10.1 cm X 7.4 cm X 1.2 cm 측정 개의 플레이트로 구성되어있다. </li><li> 10 μL / 분의 속도로 5 % 아세트산, pH가 2.4를 흐른다. 1 μL / 분의 속도로 단백질 라인 비록의 50 mM 아세트산 암모늄 완충액 (pH 6.9)을 흐른다. 주 : 아세트산 연속적 실험 전체에 걸쳐 디바이스를 통해 흐르는 것이 매우 중요하다. 거기에는 누설이없는 그 유체 ESI 소스 역할을한다 출력 채널에서만 종료되었는지 확인. </li><li> 몇 일렉트로 최적 조건을 달성하기 위해 조절 가능한 절연 스테이지를 사용하여 개질 중극 비행 시간 형 (Q-TOF) 질량 분광기의 선단부에 장치. 참고 : 바이 패스 스위치상업 ESI 소스의 존재를 시뮬레이션하기 위해 도입된다. (17) </li></ol></li></ol> 2. 시간 해결 전기 분무 이온화 수소 - 중수소 교환 <ol><li> 펩신의 수집 및 단백질 만 스펙트럼 주 : ESI-MS 취득 60 V 디 클러스터링 전위에 5,000 4,500의 전압 양이온 모드에서 수행되며, 250 V 전위 집중. 스펙트럼은 (1 개) (S)의 주사 속도 350-1,500 m / z 범위에 걸쳐 취득 -1. <ol><li> 펩신 유일한 스펙트럼을 획득. 단백질이 추가되면이 스펙트럼에서 나타나는 모든 피크가 감산된다. </li><li> 50 mM의 아세트산 암모늄 완충액 이전 흘렀다 1 μL / 분의 속도로 50 ~ 100 μM 타우 / 인산화 타우 단백질 (19 정화 앞서 설명한 제조 13) 소개. </li><li> 단백질 만 스펙트럼을 획득. </li></ol></li><li> 취득 오F 시간 포인트 <ol><li> 100 μM 타우 / 인산화 타우 단백질은 1 μL / 분으로 흐르는 동안, 티 커넥터를 통해 3 μL / 분의 속도로 D O 2를 도입하고 운동 믹서에서 반응 할 수있다. 스펙트럼의 취득 전에 적어도 10 분 동안 평형을 허용 시스템. 음표 : 교환 후, 라벨링 반응 표지 단백질의 10 μL / 분으로 아세트산의 pH 2.4의 흐름과 절단에 의해 급냉된다 단백질 분해 챔버 내에서 발생한다. </li><li> 수동 레이블 시간을 증가 8의 42 밀리 초의 시간 혼합 달성 내측 유리 모세관의 위치를 ​​위로 끌어하기 위하여. 각 풀 - 백 사이에 적어도 10 분 동안 평형을 허용 시스템. </li></ol></li></ol> 3. 데이터 및 통계 분석 <ol><li> 펩티드를 확인하고 중수소 교환의 비율을 계산 <ol><li> , MS 스펙트럼 mMass 소프트웨어를 사용하여 분석 버전 5.5.0 (20)를 수행 </suP&gt;. </li><li> ExPASy FindPept 단백체 서버를 사용하여 펩티드를 확인하고 (21)을 필요한 경우 충돌 - 유도 해리 (CID)에 의해 확인한다. 참고 : 여기, 중수소 흡수 통합은 동위 원소 분포 분석 (22), (23)에 대한 자체 개발 FORTRAN 소프트웨어를 사용하여 측정 하였다. </li><li> 구체 웹 도구 (22, 24)를 사용하여 기본 시퀀스에 기초하여 이론적 극한 요금을 계산한다. </li><li> 단일 지수 비선형 회귀를 사용하여 데이터를 장착하고, 그래프와 통계 소프트웨어 (예 SigmaPlot)를 이용하여 정규화. 주 : 비율 K INT / K OBS 보호 인자 (PF)의 특정 영역은 상기 입체 앙상블 내의 구조화 정도 반 정량적 측정 값을 산출한다. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+protein+isoforms,+phosphorylation+and+role+in+neurodegenerative+disorders1.">Tau protein isoforms, phosphorylation and role in neurodegenerative disorders1.</a> Brain Res. Rev. 33, 95-130 (2000).</li><li>Zhang, H. M., 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=Enhanced+Digestion+Efficiency,+Peptide+Ionization+Efficiency,+and+Sequence+Resolution+for+Protein+Hydrogen/Deuterium+Exchange+Monitored+by+FT-ICR+Mass+Spectrometry.">Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by FT-ICR Mass Spectrometry.</a> Anal. Chem. 80 (23), 9034-9041 (2008).</li><li>Deng, B., Lento, C., Wilson, D. 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We have nothing to disclose.

그들이 단백질의 매우 상세한 구조를 제공하기 때문에 이러한 X 선 결정학 및 NMR 구조 생물학 등의 방법이 유리하지만, 이러한 이미지들은 정적이다. 과도 종과 약하게 구조화 된 도메인의 특성은 이러한 기존의 방법으로 공부하면 어려운되고 있습니다. 따라서, 이러한 유형의 시스템에 동적 통찰력을 얻기 위해이 빠른 시간 규모에서 일을하는 것이 중요합니다. 우리는 성공적으로 지역화 된 펩?...

지난 몇 년 동안 상당한 발전 단백질 구조와 역학 1, 2, 3, 4를 측정하기위한 분석 기술의 개발되었습니다. X 선 결정학은 단백질 구조를 결정하기위한 원리 공구 유지하는 동안, 단백질의 고농도가 필요하다 광범위한 최적화 회절 품질의 결정을 제조 할 필요가있다. 이러한 막과 본질적으로 연관된 장애 단백질로서 결정화하기 어려운 단백질, 고전 수소 - 중수소 교환 (HDX) NMR (5)에 의해 연구되었다. 그러나 최근 수십 년간, HDX에 전기 분무 이온화 질량 분석 (ESI-MS)의 커플 링은 빠르게 인기 6, 7 얻고있다. 질량 분석은 솔루션을 제공합니다X 선 결정학 및 NMR로 인한 제약 사항에. 특히, MS는 매우 민감한 (필수 μM 농도로 ㎚)이고, 단백질의 크기에는 제한이 거의 없다. 또한, MS 분석의 높은 듀티 사이클은 효소 회전율, 미스 폴딩, 복합체 및 기타 중요한 생물학적 과정을 겪는다 같은 단백질 연구의 가능성을 허용한다. 이러한 프로세스는 종종 두 번째 규모로 밀리 초에서 발생 및 분석에 앞서 시약의 신속한 혼합이 필요합니다. 2003 허용 반응에서 윌슨과 Konermann 소요 시간 - 해결 된 전기 분무 이온화 (TRESI)의 개발은 ESI-MS에 의한 의사 실시간으로 모니터링 할 수 있습니다. 이들의 설정은 계속 조정 가능한 반응 실 용적 8 모세관 믹서로 통합된다. 장치는 좁은 상호 capillar 내 혼합을 허용하도록 밀봉 내부 모세관의 측면으로 잘라 노치 두 동심 모세관 이루어져내부 모세관 (일반적으로 2mm)의 단부에 절 결부의 공간 Y. HDX 실험에 적용 할 때, 상기 내부 모세관 관심 단백질을 운반 외측 모세관 표지 D를 다음 ESI로 직접 전송하기 전에 HDX 라벨링 있도록 조절 반응 챔버로 들어가기 전에 단백질과 혼합 겪는다 2 O 용액을 운반 출처. 간단히, HDX 용액 9, 10 중수소 원자 교환을 진행 백본 아미드 수소에 의존한다. 교환은 염기 - 촉매 생리적 pH에서, 산 촉매는 약 2.6 이하의 pH에서 함께 널리 사용되고있다. 산도, 온도, 용매 접근성과 분자간 수소 결합 : 교환 율은 네 가지 요인에 기초한다. 이전의 두 가지 요인이 실험 특히 펩티드 백본 아미드 위치에서의 환율에 걸쳐 일정하게 유지되기 때문에, 우선적 인 dependen단백질 구조 (11)에 t. 밀접 루프와 무질서 영역 (때때로 전혀) (12)에 비해 실질적으로 느린 속도로 중수소 소요될 것이다 α 나선과 β-시트 광범위한 안정된 수소 결합 네트워크 영역으로 접혀. 이 글로벌 단백질 분석을 가능 여기서 구조 섭동 (예., 응집 또는 기질 결합시) 중수소 흡수 (도 1)에 다른 리드. 운동 모세관 믹서 중수소 흡수 제이션을위한 단백질 분해 챔버를 포함하는 미세 유체 플랫폼으로 통합 될 수있다. 이러한 단백질 분해 챔버 효과적으로 교환 반응을 급냉시키기 위해 낮은 pH로 유지하고, 지역화 된 펩티드 (도 2)로 단백질을 소화하기 위해 고정화 산 프로테아제를 필요로한다. 두 번째 시간 규모에 밀리 초에서 모니터링 백본 교환은 특히 중요하다어려운 내 구조적 변화 특성 루프 영역, 용융 구체와 본질적으로 무질서 단백질 (실향민) (13) (14)을 특성화한다. 대안 적으로, TRESI-HDX 또한 COREX 알고리즘 (DX-COREX) 방법 (15, 16)에 결합 된 중수소 교환을 이용하여, 현재 X 선 결정학 및 NMR의 방법을 통해 해결할 원자 구조를 갖고 있지 않은 단백질을 특성화하는데 사용될 수있다. 이 상세한 프로토콜은 병원성 hyperphosphorylated 상태입니다으로 모두가 기본 형태뿐만 아니라의에, 타우, 실향민을 연구하기 위해 TRESI - HDX을 적용합니다. 기본 타우가 가장 잘 연구 실향민 중 하나이지만, 약간은 아밀로이드 대응 (13)에 대한 알려져있다.

<table><tbody><tr><td>Poly(methyl methacrylate) or PMMA</td><td>Professional Plastics</td><td>SACR.250CCP</td><td>8.9 cm x 3.8 cm x 0.6 cm</td></tr><tr><td>Fused Silica Glass Capillary</td><td>Polymicro Technologies</td><td>106815-0018</td><td>ID: 75 &amp;micro;m, OD: 150 &amp;micro;m</td></tr><tr><td>Metal Capillaries</td><td>McMaster-Carr</td><td>28 ga &amp;ndash; 89875K97&lt;br/&gt; 30 ga&amp;nbsp; - 89875K99</td><td></td></tr><tr><td>Fluorinated Ethylene Propylene (FEP) Tubing</td><td>IDEX</td><td>1477&lt;br/&gt; 1548</td><td>ID: 0.007&amp;rdquo;, OD: 1/16&amp;rdquo;&lt;br/&gt; ID: 0.020&amp;rdquo;, OD: 1/16&amp;rdquo;</td></tr><tr><td>Standard Polymer Tubing Cutter</td><td>IDEX</td><td>A-327</td><td>for 1/16&amp;rdquo; and 1/8&amp;rdquo; OD tubing</td></tr><tr><td>Micro Static Mixing Tee</td><td>IDEX</td><td>M-540</td><td>for 1/16&amp;rdquo; OD tubing</td></tr><tr><td>or</td><td></td><td></td><td></td></tr><tr><td>Stainless Steel Tee, 0.25&amp;nbsp;mm Bore</td><td>Valco Instruments Co., Inc. (VICI)</td><td>ZT1C</td><td>for 1/16&amp;rdquo; OD tubing</td></tr><tr><td>PEEK Tee for 1/16&amp;rdquo; OD Tubing</td><td>IDEX</td><td>P-727</td><td></td></tr><tr><td>10-32 Female to Female Luer</td><td>IDEX</td><td>P-659</td><td></td></tr><tr><td>10-32 PEEK Double-Winged Nut</td><td>IDEX</td><td>F-300</td><td></td></tr><tr><td>Ferrule for 1/16&amp;rdquo; OD Tubing</td><td>IDEX</td><td>F-142</td><td></td></tr><tr><td>100 Series Rotary Tool</td><td>Dremel</td><td>F013010001</td><td></td></tr><tr><td>Cut-Off Discs</td><td>Jobmate</td><td></td><td>1/64&amp;rdquo; thickness</td></tr><tr><td>Stereomaster Digital Zoom Microscope</td><td>Fisher Scientific</td><td>12-563-411</td><td></td></tr><tr><td>Soldering Iron</td><td>Mastercraft</td><td>58-6301-2</td><td></td></tr><tr><td>VersaLaser</td><td>Universal Laser</td><td></td><td></td></tr><tr><td>Syringes</td><td>Hamilton</td><td>81220</td><td>500&amp;nbsp;&amp;micro;L capacity</td></tr><tr><td>Syringe Pumps</td><td>Harvard Apparatus</td><td>70-4501</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NHS-Activated Agarose</td><td>Fisher Scientific</td><td>26196</td><td></td></tr><tr><td>Pepsin from Porcine Gastric Mucosa</td><td>Sigma-Aldrich</td><td>P6887-250MG</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Sigma-Aldrich</td><td>151882-10X0.6ML</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma-Aldrich</td><td>695092-100ML</td><td></td></tr><tr><td>HPLC Grade Water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>A7330-500G</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S671-3</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software/Online Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CorelDraw X3</td><td>Corel</td><td></td><td></td></tr><tr><td>Molecular Weight Calculator</td><td></td><td>Version 6.49</td><td>Open Source MS Tool</td></tr><tr><td>mMass</td><td></td><td>Version 5.5.0</td><td>Open Source MS Tool</td></tr><tr><td>ExPASy FindPept</td><td>Swiss Institute of Bioinformatics</td><td></td><td></td></tr><tr><td>SigmaPlot</td><td>Systat Software</td><td>Version 11.0</td><td></td></tr><tr><td>PyMOL</td><td>Schr&amp;ouml;dinger</td><td>Version 1.5.0.4</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>QStar Elite Hybrid Q-TOF Mass Spectrometer</td><td>AB SCIEX</td><td></td><td></td></tr></tbody></table>

time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics

본질적으로 무질서 단백질 (실향민) 길이 때문에 안정적인 이차 구조 요소의 부족 구조적 생물 학자에 도전하고있다. 수소 - 중수소 빠른 시간 척도로 측정 교환 (HDX)는 네이티브 앙상블 일시적인 이형태 체의 특성을 허용 간단히 채워진 구조와 수소 결합 네트워크를 검출하는 고유 적합하다. 질량 분석에 HDX의 커플 링은 높은 감도, 낮은 샘플 소비와 단백질의 크기에 제한을 포함하여 몇 가지 중요한 이점을 제공합니다. 이 기술은 밀리 초 시간 규모 HDX 라벨 시간을 모니터링 할 수있는 기능을 포함하여, 지난 몇 년간 크게 발전했다. 또한, 산성 프로테아제 마이크로 리액터 하우징 미세 유체 플랫폼 상 HDX 흐름을 도입함으로써, 우리는 펩타이드 레벨에서의 동적 속성을 지역화 할 수있다. 본 연구에서는 시간 - 해결 된 전기 분무 이온화 질량 분석법 (TRESI-MS)는 HDX의 WA 결합S는 타우 단백질에 잔여 구조의 상세한 그림뿐만 아니라 과인산에 유도 된 형태 적 변화를 제공하기 위해 사용된다.

네이티브 및 인산화 타우 분해 프로파일이 각각 77.1과 71.7 %의 서열 커버리지를 수득 유사 하였다. 펩타이드 중수소 흡수 값은 사내 개발 FORTRAN 소프트웨어를 사용하여 생성 된 이론적 인 분포로 관찰 동위 원소 분포를 피팅하여 구 하였다. 최상의 결합 분포 연관된 중수소 흡수 값과 함께 (도 3a)을 나타낸다. 통풍 운동 프로파일은 생성되고, 또한 하나의 지수 ?...

Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

구조적 유연성은 단백질 기능에 중요한 역할을한다. 여기서, 우리는 주문과 무질서 단백질의 기능을 구동 급속한 구조적 변화를 프로빙 수소 - 중수소 교환 결합 시분 전기 분무 이온화 질량 분석법의 사용을 설명합니다.

시간 해결 전기 분무 이온화 수소 - 중수소 교환 질량 분석 단백질 구조와 역학 연구를위한

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

time-resolved-electrospray-ionization-hydrogen-deuterium-exchange

Research

JoVE Journal

Biochemistry

9.8K 조회수.  York University.  구조적 유연성은 단백질 기능에 중요한 역할을한다. 여기서, 우리는 주문과 무질서 단백질의 기능을 구동 급속한 구조적 변화를 프로빙 수소 - 중수소 교환 결합 시분 전기 분무 이온화 질량 분석법의 사용을 설명합니다. 

비디오: Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

연구

화학

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

생화학

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

양적 질량 분광법 분석을위한 지질 물방울 절연

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

Bio3D-web을 이용한 단백질 염기 서열 구조 역학 관계 조사

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

질량 분석은 동결 건조 분말에서 단백질의 구조와 상호 작용을 연구하는 접근

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

펩타이드 기반 매트릭스 샘플을 고정 하는 포 르 말린의 최적의 준비 지원 레이저 탈 착/이온화 질량 분석 이미징 워크플로

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

펩타이드 생합성 효소를 조사하기 위한 수소-신투륨 교환 질량 분광법(HDX-MS) 플랫폼

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

단백질과 수화수의 피코초-나노초 역학을 연구하기 위한 고분해능 중성자 분광법

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

생리학적 조건하에서의 알파-시뉴클레인 구조역학 연구를 위한 밀리초 수소/중수소-교환 질량 분광법

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...