Name: fast enzymatic processing of proteins for ms detection with a flow-through microreactor
Uploaded: 2016-04-06T13:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

This work was supported by NSF/DBI-1255991 grant to IML.

모세관 마이크로 반응기 1. 준비 <ol><li> 100 μm의 내경 (ID)이 360 ㎛의 외경 (OD) 7-8cm 길이로 모세관 유리 모세관 칼 3-5 센티미터 20 μm의 ID X 0.09의 OD 모세관 X 컷; 어떤 돌출 버없이, 모두 모세관 끝이 깨끗하고 스트레이트 컷을 현미경으로 확인합니다. </li><li> ~ 6mm의 길이, 100 μm의 ID X 360 μm의 외경 모세관의 한쪽 끝으로 20 μm의 ID X 90 μm의 외경 모세관을 삽입; 필요한 경우에, 현미경이 동작을 관찰한다. </li><li> 면봉의 끝이 90 μm의 OD / 100 μm의 아이디 모세 혈관의 교차점 주위에 접착제 E6000의 작은 방울을 적용하고 RT에서 접착제 경화 O / N을 할 수 있습니다. </li><li> ~ 10-15mm의 길이에 삽입 된 20 μm의 ID X 90 μm의 외경 모세관을 잘라; 이것은 전기 분무 이온화 방출 될 것입니다. </li><li> 미리 잘라 1/32 &quot;OD의 PEEK, 1/16&quot;OD의 PEEK 및 1/16 &quot;OD의 PTFE 욕조길이 4-5cm의 조각 보내고; 이러한 누출이없는 연결을 제공하기 위해 모세관 조합에 사용되는 소매 될 것입니다. </li><li> 까꿍 조합에 100 μm의 ID X 360 μm의 외경 모세관의 반대쪽 끝을 연결합니다; 누출이없는 연결을 제공하기위한 1/32 &quot;PEEK 슬리브를 사용합니다. </li><li> 삽입 / 노조의 반대쪽에 5cm 길이 1/16 &quot;PTFE 튜브 ~의 조각을 조입니다. </li><li> 사전 세척 / 2 ㎖의 유리 병을 건조에 C18 (5 μm의) 입자 ~ 4 mg의 무게; 0.5 ml의 이소프로판올을 추가 바이알을 닫고 초음파 목욕 슬러리를 분산. </li><li> , 250 μL 주사기 약 200 ㎕의 슬러리를 철수 PEEK 조합의 1/16 &quot;PTFE 튜브에서 주사기 바늘을 삽입하고 천천히 100 μm의 ID X 360 μm의 외경 모세관에 슬러리를 분배하며로서 현미경으로 관찰 모세 혈관이 달성된다 2-3mm 포장의 길이까지 입자로 채우고, 이것은 마이크로 반응 할 것이다 (그림 1) 20 μ.m의 ID 모세관은 키스톤 효과를 통해 마이크로 반응기에서의 입자를 유지한다. (15) </li><li> H 2 O / CH 3 CN 50:50 v / V를 50 μl의 솔루션 ~와 마이크로 반응기를 씻어하고 H 2 O / CH 3 CN (98)의 50 μl의 솔루션 ~와 : 2 v / V를. </li></ol> 샘플 솔루션의 2. 준비 주 : 유기 용매 및 산 및 용액 제제의 운반 작업 것을 포함 흄 후드에서 수행되어야한다. 고글, 장갑 및 보호 복을 착용 할 것. <ol><li> CH 3 OH 씻어 몇 유리 병 (4 ㎖) 및 폴리 프로필렌 튜브 (15 ml)에 건조. </li><li> H 2 O / CH 3 CN 용액 10 ㎖를 준비 (98 : 2 v / v)로 15 ml의 폴리 프로필렌 튜브에 200 μL의 CH 3 CN로 9.8 ml의에게 H 2 O를 혼합하여; 초음파로 섞는다. </li><li> 용액 10 ㎖를 준비 H 2 O / CH 3 CN (50:50 v / v)로 15 ml의 폴리 프로필렌 튜브에 5 ML의 H 2를 혼합하여 &lt;/ 서브&gt; O 5 ml의 CH 3 CN; 초음파로 섞는다. </li><li> H 2 O / CH 3 CN 4 ml의 4 μL TFA (10 %)를 추가하여 산성화 된 수용액 (TFA 0.01 %의 v / v)의 4 ml의 준비 : 4 ㎖의 유리 병에 (98 2 v / v)의; 초음파로 섞는다. </li><li> 4 ml의 H 2 O / CH 3 CN (50:50 v / v)로에서 4 mL 유리 바이알에 4 μl를 TFA (10 %)를 추가하여 산성화 된 유기 용액 (TFA 0.01 %의 v / v)의 4 ml의 준비; 초음파로 섞는다. </li><li> 16 mg의 NH 4 HCO 3 무게 4 mL 유리 바이알에서 분석 마이크로 밸런스와; (: 2 v / V를 98) 솔루션 및 초음파에 의해 분산 2 O / CH 3 CN에게 H의 4를 가하여 이 수성 버퍼 NH 4 HCO 3 (산도 ~ 7.8)의 50 mM의 솔루션을 초래한다. </li><li> 표준 단백질의 5 mg의 체중 (예를 들면, 소 혈청 알부민, 헤모글로빈 α / β, 시토크롬 C, 탄산 탈수 효소, α-2-HS 당 단백질 등) 4 mL 유리 바이알에서 분석 마이크로 밸런스와; 4 ML의 DI의 물 공급을 추가연구 및 초음파에 의해 분산; 이것은 일반적으로 높은 수준 μM 농도 용액을 초래한다. </li><li> NH 4 HCO 3 (50 mM)을 수성 완충액 적절한 부피 μM 높은 수준의 농도로 용액을 희석하여 1 mL의 단백질 용액 (1-2 μM)를 준비; 초음파로 섞는다. </li><li> (50 mM의 께) NH 4 HCO 3 170 μL 수성 완충액을 20 μg의 서열 분석 등급 트립신 용해 트립신 용액 (5 μM)를 준비; 초음파에 의해 분산. </li></ol> 3. 실험 설정 주 : LTQ-MS 시스템은 다양한 샘플 입력 방식으로 질량 분석 장치의 인터페이스 수있는 조립식 XYZ 스테이지를 포함하는 변성 ESI 공급원이 장착된다. <ol><li> 픽 조합에서 모세관 마이크로 반응기를 분리하고 PEEK 티에 연결합니다. </li><li> 픽 티 옆을 팔에 ~ 2cm 긴 백금 와이어를 삽입; 를 제공하는 1/32 &quot;PEEK 슬리브를 사용누수없는 연결과 백금 와이어를 절연합니다. </li><li> 픽 티의 반대쪽에 (~ 0.5 m 길이) 샘플 전송 모세관 50 μm의 ID X 360 μm의 외경을 연결합니다; 누출이없는 연결을 제공하기위한 1/32 &quot;PEEK 슬리브를 사용합니다. </li><li> XYZ 단계에 PEEK 티를 안전하고 멀리 질량 분석기 입구 모세관에서 ESI 터 ~ 2mm의 위치. </li><li> 질량 분광기의 ESI 전원부에 백금 와이어를 연결한다. </li><li> 스테인리스 연합 샘플 이송 모세관의 대향 단부에 연결; 누출이없는 연결을 제공하는 1/16 &quot;PEEK 슬리브를 사용한다. </li><li> 스테인레스 스틸 노동 조합의 반대 끝에 4-5cm 길이 1/16 &quot;PTFE 튜브를 연결하고 PTFE 튜브에 250 μL 주사기 바늘을 삽입, 예를 들어 (펌프를 켜고 원하는 값에 유량을 설정, 2 μL / 분). </li><li> 이동국 구을 제어하는​​ 소프트웨어 패키지에 입력 탠덤 MS 데이터 수집 매개분석을 수행하기위한 준비를 설치하는 방법 파일로 저장 거라고; LTQ-MS 시스템에 대해 다음 데이터 분석 의존 파라미터를 사용 제외 질량 폭 180 초 동안 활성화 된 동적 배제를 1.5 m / z ±; 하나의 MS는 한 줌 다음 스캔과 MS는 상위 5 가장 강렬한 봉우리 2 스캔, 5m / Z ± 스캔 폭을 확대; 충돌 유도 해리 분리 폭 3m / Z, 정규화 된 충돌 에너지 35 %, 활성화 Q 0.25 및 활성화 시간 30 밀리 초 설정 (CID) 매개 변수를 설정합니다. </li></ol> 4. 미세 유체 설치 <ol><li> 유리 또는 폴리머 기판으로부터 마이크로 유체 장치 준비, 입구에 접속 16-18 장치의 레이아웃이도 2a에 제공되고, 마이크로 반응기로 이루어진 (1) (0.13 mm 폭 × 2 mm 길이 × 50 μm의 깊이) (2 ), 배출구 (3) 측의 포트 (4); 유체 조작에 대한 상호 연결 채널은 ~입니다110 μm의 폭 ~ 50 μm의 깊이; 다중 채널 구조는 (5)로 이루어진 ~ 1.5 ㎛의 깊이 × 10 ㎛ 폭 X 위치 100 μm의 길이 25 ㎛의 미세 간격, 마이크로 리액터에 입자를 유지하기위한 필터로서 작용한다. </li><li> 또는 유체 전달 (그림 2B)에 대한 피팅을 수용하는 고분자 스탠드를 고안하여 입구 포트에 특수 커넥터를 접착하여 칩에 유체 전달 연결을 사용하도록 설정합니다. (16) </li><li> 단계 1.8에 기재된 바와 같이 입자 C18 (5 μm의)의 슬러리를 조제; 칩 측 포트에 접속 1/16 &quot;PTFE 튜빙을 사용하여 주사기와 마이크로 반응기에 상기 슬러리를 제공하는 것은 (4) 90 ºC 오븐에서 에폭시 접착제와 1 시간 동안 경화와 함께 입자 로딩 후 포트를 밀봉 (16). </li><li> 경화 O / N을 E6000 접착제로 밀봉하고,하자, 출구에 모세관 (20 μm의 ID X 90 μm의 OD × 10 mm 길이)를 삽입; 이합니다 (ESI의 터가 될 것이다 <strong&gt; 6). </li><li> 50㎛의 ID X 360 μm의 지름을 연결 (~ 0.5 m 길이) 칩 유입구 샘플 이송 모세관 (2); 3.6-3.7에 기재된 바와 같이, 250 μL 주사기 및 주사기 펌프 모세관의 대향 단부를 연결한다. </li><li> 흡기구 직전 샘플 전송 라인상의 경량 PEEK 티 커넥터 배치 (2) 및 티 측 아암 편에 와이어를 삽입 3.2에 기재된 바와 같고; 이것은 ESI 전극 될 것입니다. </li><li> 질량 분석기 입구 모세관에서 ~ 2mm 거리에 위치하는 ESI 이미 터와 XYZ 스테이지에 미세 유체 소자를 고정; 3.8에 기재된 바와 같이 분석을 위해 MS를 준비한다. </li></ol> 5. 샘플로드, MS 분석을위한 단백질 분해 소화 및 용출 모든 용액 / 샘플 전송 단계를 주사기 펌프의 도움으로 수행되고, 데드 있습니다 volum 보상하기 위해, 완료하기 위해 몇 분 동안 추가로 허용한다 참고및 상기 마이크로 반응기에서 이송 라인과 관련된 ES; 필요한 시간이 포함 된 유량에 따라 달라집니다. <ol><li> 마이크로 반응을 씻어 2 O / CH 3 CN H의 수성 NH 4의 솔루션 HCO 3 (50 mm)를 주입하여 분석 준비를 5 분 동안 2 μL에서 (98 2 v / v)의 / 분; 250 μL 주사기를 사용합니다. </li><li> 2 μL의 시료 용액을 주입하여 미세 반응기에 단백질 샘플 (1 μM)을로드 / 분, 5 분; 250 μL 주사기를 사용합니다. </li><li> 1-3 분 / 2 μL에서 분 마이크로 리액터에 흡착 된 단백질을 통해 트립신 용액 (5 μM)을 주입. </li><li> TFA의 산성화 수용액으로 마이크로 반응기를 세척하여 단백질 분해 소화 과정을 끄다 (0.01 % v / v)의 H 2 O / CH 3 CN에서 (98 : 2 v / v)의 2 μL / 분 5 분; 250 μL 주사기를 사용합니다. </li><li> (단백질의 TFA 산성화 된 유기 용액을 주입하여 미세 반응기로부터 0.01 % 다이제스트 용출 시작v / v)로 H 2 O / CH 3 CN에서 (50:50 v / v)의 300 NL / 분. </li><li> 단말기 데이터 수집 과정과 ESI 전압 (~ 2000 V)를 켭니다; 단계 3.8에 제공된 데이터 수집 매개 변수를 사용하여 MS 데이터를 획득; 트립신 펩타이드의 용출을 관찰합니다. </li></ol> 6. 데이터 처리 <ol><li> 원시 MS 데이터 포맷과 호환되는 검색 엔진 탠덤 MS 데이터를 처리한다. <ol><li> 생체의 특정 최소 중복 단백질 데이타베이스를 사용하여 LTQ-MS 원시 파일을 처리; 검색 엔진의 원시 데이터를 업로드, 각각 2 및 1 다에 부모 및 조각 이온 질량 공차를 설정하고, 최대 두 개의 검색에 대한 분열을 놓친 만 완전히 트립신 단편을 사용한다; 번역 후 변형을 허용하지 않습니다 각각 1 %와 3 %로 높은 및 중간 신뢰 거짓 발견 비율 (FDR)를 설정하고, 번역 후 변형을 허용하지 않습니다. </li></ol></li><li> 만 높은 신뢰를 선택하기 위해 데이터를 필터링펩타이드는 데이터베이스의 단백질과 일치; 단백질 및 펩타이드 수준의 결과를 평가합니다. </li></ol>

<ol>
	<li>Petersen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Bioreactors.">Microfluidic Bioreactors.</a> Encyclopedia of Microfluidics and Nanofluidics. , (2014).</li><li>Matosevic, S., Szita, N., Baganz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+and+applications+of+immobilized+microfluidic+enzymatic+reactors.">Fundamentals and applications of immobilized microfluidic enzymatic reactors.</a> J. Chem. Technol. Biotechnol. 86 (3), 325-334 (2011).</li><li>Liu, Y., Liu, B., Yang, P., Girault, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+enzymatic+reactors+for+proteome+research.">Microfluidic enzymatic reactors for proteome research.</a> Anal. Bioanal. Chem. 390 (1), 227-229 (2008).</li><li>Wu, H., Zhai, J., Tian, Y., Lu, H., Wang, X., Jia, W., Liu, B., Yang, P., Xu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+enzymatic-reactors+for+peptide+mapping:+strategy,+characterization,+and+performance.">Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.</a> LabChip. 4 (6), 588-597 (2004).</li><li>Jin, L. J., Ferrance, J., Sanders, J. C., Landers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchip-based+proteolytic+digestion+system+driven+by+electroosmotic+pumping.">A microchip-based proteolytic digestion system driven by electroosmotic pumping.</a> LabChip. 3 (1), 11-18 (2003).</li><li>Asanomi, Y., Yamaguchi, H., Miyazaki, M., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-immobilized+microfluidic+process+reactors.">Enzyme-immobilized microfluidic process reactors.</a> Molecules. 7 (16), 6041-6059 (2011).</li><li>Aravamudhan, S., Joseph, P. J., Kuklenyik, Z., Boyer, A. E., Barr, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microfluidic+enzyme+reactor+mass+spectrometry+platform+for+detection+of+anthrax+lethal+factor.">Integrated microfluidic enzyme reactor mass spectrometry platform for detection of anthrax lethal factor.</a> , 1071-1074 (2009).</li><li>Liu, X., Lo, R. C., Gomez, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+a+microfluidic+enzyme+reactor+utilizing+magnetic+beads.">Fabrication of a microfluidic enzyme reactor utilizing magnetic beads.</a> Electrophoresis. 30 (12), 2129-2133 (2009).</li><li>Liu, Y., Xue, Y., Ji, J., Chen, X., Kong, J., Yang, P., Girault, H. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticle+assembly+microfluidic+reactor+for+efficient+on-line+proteolysis.">Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis.</a> Mol. Cell. Proteomics. 6 (8), 1428-1436 (2007).</li><li>Wu, H., Tian, Y., Liu, B., Lu, H., Wang, X., Zhai, J., Jin, H., Yang, P., Xu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titania+and+alumina+sol-gel-derived+microfluidics+enzymatic-reactors+for+peptide+mapping:+design,+characterization,+and+performance.">Titania and alumina sol-gel-derived microfluidics enzymatic-reactors for peptide mapping: design, characterization, and performance.</a> J. Proteome Res. 3 (6), 1201-1209 (2004).</li><li>Ji, J., Zhang, Y., Zhou, X., Kong, J., Tang, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+protein+digestion+through+the+confinement+of+nanozeolite-assembled+microchip+reactors.">Enhanced protein digestion through the confinement of nanozeolite-assembled microchip reactors.</a> Anal. Chem. 80 (7), 2457-2463 (2008).</li><li>Hustoft, H. K., Brandtzaeg, O. K., Rogeberg, M., Misaghian, D., Torsetnes, S. B., Greibrokk, T., Reubsaet, L., Wilson, S. R., Lundanes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+enzyme+reactor+and+high+resolving+chromatography+in+&amp;#34;sub-chip&amp;#34;+dimensions+for+sensitive+protein+mass+spectrometry.">Integrated enzyme reactor and high resolving chromatography in &amp;#34;sub-chip&amp;#34; dimensions for sensitive protein mass spectrometry.</a> Scientific Reports. 3 (3511), 1-7 (2013).</li><li>Pramanik, B. N., Mirza, U. A., Ing, Y. H., Liu, Y. H., Bartner, P. L., Weber, P. C., Bose, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-enhanced+enzyme+reaction+for+protein+mapping+by+mass+spectrometry:+A+new+approach+to+protein+digestion+in+minutes.">Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes.</a> Protein Sci. 11 (11), 2676-2687 (2002).</li><li>Olszowy, P. P., Burns, A., Ciborowski, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-assisted+sample+preparation+for+proteomic+analysis.">Pressure-assisted sample preparation for proteomic analysis.</a> Anal. Biochem. 438 (1), 67-72 (2013).</li><li>Lord, G. A., Gordon, D. B., Myers, P., King, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tapers+and+restrictors+for+capillary+electrochromatography+and+capillary+electrochromatography-mass+spectrometry.">Tapers and restrictors for capillary electrochromatography and capillary electrochromatography-mass spectrometry.</a> J. Chromatogr. A. 768, 9-16 (1997).</li><li>Lazar, I. M., Kabulski, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+LC+Device+with+Orthogonal+Sample+Extraction+for+On-Chip+MALDI-MS+Detection.">Microfluidic LC Device with Orthogonal Sample Extraction for On-Chip MALDI-MS Detection.</a> Lab Chip. 13 (11), 2055-2065 (2013).</li><li>Harrison, D. J., Manz, A., Fan, Z. H., Ludi, H., Widmer, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+and+sample+injection+systems+on+a+planar+glass+chip.">Capillary electrophoresis and sample injection systems on a planar glass chip.</a> Anal. Chem. 64 (17), 1926-1932 (1992).</li><li>Jacobson, S. C., Hergenroder, R., Koutny, L. B., Warmack, R. J., Ramsey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Injection+Schemes+and+Column+Geometry+on+the+Performance+of+Microchip+Electrophoresis+Devices.">Effects of Injection Schemes and Column Geometry on the Performance of Microchip Electrophoresis Devices.</a> Anal. Chem. 66 (7), 1107-1113 (1994).</li><li>Armenta, J. M., Perez, M. J., Yang, X., Shapiro, D., Reed, D., Tuli, L., Finkielstein, C. V., Lazar, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Proteomic+Protocol+for+Biomarker+Fingerprinting+in+Cancerous+Cells.">Fast Proteomic Protocol for Biomarker Fingerprinting in Cancerous Cells.</a> J. Chromatogr. A. 1217, 2862-2870 (2010).</li><li>Doucette, A., Craft, D., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometric+Study+of+the+Effects+of+Hydrophobic+Surface+Chemistry+and+Morphology+on+the+Digestion+of+Surface-Bound+Proteins.">Mass Spectrometric Study of the Effects of Hydrophobic Surface Chemistry and Morphology on the Digestion of Surface-Bound Proteins.</a> J. Am. Soc. Mass Spectrom. 14, 203-214 (2003).</li><li>Lazar, I. M., Trisiripisal, P., Sarvaiya, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Liquid+Chromatography+System+for+Proteomic+Applications+and+Biomarker+Screening.">Microfluidic Liquid Chromatography System for Proteomic Applications and Biomarker Screening.</a> Anal. Chem. 78 (15), 5513-5524 (2006).</li><li>Lazar, I. M., Karger, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Open-Channel+Electroosmotic+Pumping+System+for+Microfluidic+Sample+Handling,&amp;#34.">Multiple Open-Channel Electroosmotic Pumping System for Microfluidic Sample Handling,&amp;#34.</a> Anal. Chem. 74 (24), 6259-6268 (2002).</li><li>Lazar, I. M., Rockwood, A. L., Lee, E. D., Sin, J. C. H., Lee, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+TOFMS+Detection+for+Capillary+Electrophoresis.">High-speed TOFMS Detection for Capillary Electrophoresis.</a> Anal. Chem. 71 (13), 2578-2581 (1999).</li></ol>

The authors declare no competing financial interests.

이 작품에서 설명하는 마이크로 반응기가 제공하는 간편한 구현 미만 30 분에서 MS 분석 및 식별을 가능하게하는 단백질의 효소 소화를 수행하기위한 실험 장치를. 기존의 방법에 비해이 시스템의 뚜렷한 장점은 단순성, 속도, 낮은 시약 소모와 낮은 비용을 포함한다. 특히, 고가의 트립신 고정화 비드 카트리지가 필요 없다. 모세관 마이크로 리액터의 제조는 간단 (도 1a), 그리고 통상...

정제 된 단백질의 동정 및 특성화 자주 MS 기술을 사용함으로써 달성된다. 단백질은 효소로 분해되고 그 펩티드는 더 간단한 주입 실험 장치를 사용하여 MS로 분석된다. 단백질 분해 소화 가장 MS 분석기의 유효 질량 범위에 속하는 작은 펩티드 단편을 생성하기 위해 필요하고, 그 용이 아미노산 서열 정보를 생성하기 위해 저 에너지 충돌 유도 된 분해를 통해 단편화 될 수있다. 절연 단백질 또는 단순 단백질 혼합물, MS 검출하기 전에 펩티드의 크로마토 그래피 분리에 대한 추가적인 필요가 없다. 25-50 펩티드의 혼합물을 용이 MS 이온 소스에 직접 주사기 펌프 시료를 주입하여 분석 할 수있다. 질량 분석 장치는 분석을 수행하고, 짧은 시간 프레임 내에서 단백질의 서열을 확인할 수있다. 현대 데이터 수집 방법과,이 프로세스는 승 달성 될 수있다몇 분 또는 초 ithin. 단시간 스케일에 대한 전체 프로세스를 완료하는 제한 요인이 단백질 분해성 분해 공정이다. 일반적으로, 이것은 수 시간에 걸쳐 수행된다 (또는 O / N) 용액에 37 ºC에서 사용한 기판 (50-100) 효소의 비를 1. 설명 된 미세 반응기 또는 시판되는 카트리지의 형태로, 분, 초, 고정화 효소의 마이크로 리액터의 효소 소화 시간을 감소시킨다. 1-6 일반적으로 효소를 공유 결합, 비 - 공유 / 물리 흡착 복합체가 고정화 형성 또는 캡슐화, 효소 처리의 -3,6- 향상된 효율으로 사용되는 큰 표면 대 부피 및 효소에 대한 기판 비. 고정화 반응기의 추가 이점은 MS 분석에서자가 분해 효소와의 간섭을 감소 효소 안정성 및 재사용 성을 향상시킬 것을 포함한다. 접근법을 이용하여 유리 또는 중합 미세 다양한 장치가 설명되었지만항체 - 항원 상호 작용에 의해 자성 비드에 고정화 효소를 이용하여, 7,8- 9는 티타니아, 알루미나 졸 - 겔 10 nanozeolites 11에 캡슐화 또는 니켈 NTA 또는 그의 태그 복합체 형성을 통해 캡처 된 금 나노 입자의 네트워크에 포획. 6 대안 고정화 효소 개방 관형 모세관도 개발되어있다. (12) 또한, 향상된 단백질 분해 절단은 30-120에 대한 반응 시간을 감소시키기위한 제어 마이크로파 조사 (13) 또는 압력 보조 또는 압력 사이클링 기술 (PCT)을 사용하여 입증되었다 분. (14) 고정화 효소 반응기의 여러 장점에도 불구하고, 상업적 카트리지의 비용이 높고, 일상적인 사용을위한 미세 유체 장치의 유용성은 제한되어, 추가 계측을 필요로하는 전자 또는 PCT 기술 결과의 용도. 이 연구의 목적은 circumve 방법을 개발했다이러한 단점을 NTS, 그것은 쉽게 분 이내 MS 분석에 대비 단백질의 효소 적 분해를 수행하기위한 간단하고 효과적인 방법으로 연구자 강화할 각 실험실에서 구현 될 수있다. 접근법은 모세관 또는 미세 유동 장치에 사전로드 된 소수성 C18 입자의 사용에 의존하며 통해 효소의 주입 중에 효소 분해 한 다음 이들 입자에 관심 단백질 (들)의 흡착 충전 층 및 캡처 단백질 (들). 이 방법에서, 기판은 비 - 공유 상호 작용을 통해 고정되고, 상기 효소가 고정화 된 단백질을 통해 주입된다. 단백질 분해 소화 효율을 효소 처리, 환원 및 입자의 표면으로부터의 거리와, 확산 시간은 단백질을 노출 큰 입자 표면적이 증가 질량 전사 효소의 활성에 영향을 미칠 수없는 공유 결합, 기능 향상 빠르게 evaluat다른 효소 처리 성 및 다중화 E의 조합이 과정은 미세 형식으로 실행되는 경우. 이 방법은 표준 단백질 및 ESI-MS 검출 이전에 단백질 분해 소화 트립신 가장 일반적으로 사용되는 효소의 혼합물을 이용하여 설명된다. 본 연구에 사용 된 검출 질량 분석기는 선형 트랩 중극 (LTQ)기구이다.

<table><tbody><tr><td>Ion trap ESI-MS</td><td>Thermo Electron</td><td>LTQ</td><td>The LTQ mass spectrometer is used for acquiring tandem MS data</td></tr><tr><td>XYZ stage</td><td>Newport</td><td>Multiple parts</td><td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td></tr><tr><td>Stereo microscope</td><td>Edmund optics</td><td>G81-278</td><td>The microscope is used to observe the microreactor packing process</td></tr><tr><td>Analytical balance/Metler</td><td>VWR</td><td>46600-204</td><td>The balance is used to weigh the protein samples</td></tr><tr><td>Ultrasonic bath/Branson</td><td>VWR</td><td>33995-540</td><td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td></tr><tr><td>Syringe pump 22</td><td>Harvard Apparatus</td><td>552222</td><td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td></tr><tr><td>Milli-Q ultrapure water system</td><td>EMD Millipore</td><td>ZD5311595&amp;nbsp;</td><td>The MilliQ water system is used to prepare purified DI water</td></tr><tr><td>Pipettor/Eppendorf (1,000&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-410</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (100&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-406</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (10&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-402</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Fused silica capillary (100 &amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP100375</td><td>This capillary is used for the fabrication of the microreactor</td></tr><tr><td>Fused silica capillary (20&amp;nbsp;&amp;micro;m ID x 100 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP020090</td><td>This capillary is used for the fabrication of the ESI emitter</td></tr><tr><td>Fused silica capillary (50&amp;nbsp;&amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP050375</td><td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td></tr><tr><td>Glass capillary cleaver</td><td>Supelco</td><td>23740-U</td><td>This is a tool for cutting fused silica capillaries at the desired length</td></tr><tr><td>Glue</td><td>Eclectic Products</td><td>E6000 Craft</td><td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td></tr><tr><td>Epoxy glue</td><td>Epo-Tek</td><td>353NDT</td><td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td></tr><tr><td>Reversed phase C18 particles (5&amp;nbsp;&amp;micro;m)</td><td>Agilent Technologies</td><td>Zorbax 300SB-C18</td><td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4&amp;nbsp;mm x 20 cm C18 LC column from Agilent</td></tr><tr><td>Syringe/glass (250 &amp;micro;l)</td><td>Hamilton</td><td>81130-1725RN</td><td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td></tr><tr><td>Internal reducing PEEK Union (1/16&amp;rdquo; to 1/32&amp;rdquo;)</td><td>Valco</td><td>ZRU1.5FPK</td><td>This union is used to connect the 250 &amp;micro;l syringe to the microreactor for loading the 5 &amp;micro;m particle slurry</td></tr><tr><td>Stainless steel union (1/16&amp;rdquo;)</td><td>Valco</td><td>ZU1XC</td><td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td></tr><tr><td>Microvolume PEEK Tee connector (1/32&amp;rdquo;)</td><td>Valco</td><td>MT.5XCPK</td><td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td></tr><tr><td>Tee connector (light weight)</td><td>Valco</td><td>C-NTXFPK</td><td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td></tr><tr><td>Pt wire (0.404 mm)</td><td>VWR</td><td>66260-126</td><td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td></tr><tr><td>PTFE tubing (1/16&amp;rdquo; OD)</td><td>Valco</td><td>TTF115-10FT</td><td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&amp;nbsp;</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/16&amp;rdquo; OD)</td><td>Upchurch Scientific</td><td>1565</td><td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50&amp;nbsp;&amp;micro;m ID transfer capillary</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/32&amp;rdquo; OD)</td><td>Valco</td><td>TPK.515-25</td><td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td></tr><tr><td>Clean-cut polymer tubing cutter</td><td>Valco</td><td>JR-797</td><td>This cutter is used to pre-cut the 1/16&amp;rdquo; and 1/32&amp;rsquo; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td></tr><tr><td>Amber vial (2 ml)</td><td>Agilent&amp;nbsp;</td><td>HP-5183-2069</td><td>The vials are used to prepare sample solutions and the C18 particle slurry&amp;nbsp;</td></tr><tr><td>Amber vial (4 ml)</td><td>VWR</td><td>66011-948</td><td>The vials are used to prepare sample solutions</td></tr><tr><td>Polypropylene tube (15&amp;nbsp;ml)</td><td>Fisher</td><td>12-565-286D</td><td>The vials are used to prepare buffer solutions</td></tr><tr><td>Cylinder (100 ml)</td><td>VWR</td><td>24710-463</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Cylinder (10 ml)</td><td>VWR</td><td>24710-441</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Pipette tips (1,000 &amp;micro;l)</td><td>VWR</td><td>83007-386</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (100 &amp;micro;l)</td><td>VWR</td><td>53503-781</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (10 &amp;micro;l)</td><td>VWR</td><td>53511-681</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Glass substrates</td><td>Nanofilm</td><td>B270 white crown, 3&amp;rdquo; x 3&amp;rdquo;</td><td>These are glass substrates for microchip fabrication</td></tr><tr><td>Male nut fitting (1/16&amp;rdquo;)</td><td>Upchurch</td><td>P203X</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Nanoport assembly</td><td>Upchurch</td><td>N-122H</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Protein standards</td><td>Sigma</td><td>Multiple #</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher</td><td>A955</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher</td><td>A452</td><td></td></tr><tr><td>Isopropanol, HPLC grade</td><td>Sigma</td><td>650447</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Aldrich</td><td>A6141</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Promega</td><td>V5111</td><td></td></tr></tbody></table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

질량 분석 (MS)의 대부분은 단백질 분석 방법은 일반적으로 트립신, 검출 효소 분해 단계 전에 수반 기반. 이 단계는 질량 분석 기기의 유효 주사 범위 내에 MW &lt;3,000-4,000 다 함께 일반적으로 분자량이 작은 펩타이드의 생성을 위해 필요하다. 종래의 프로토콜은 37 ºC에서 O / N 효소 소화를 포함한다. 최근 발전은 전형적으로 예를 들면, 전자 렌지 나 고 고정화 효소 또는 몇 분에 단백질 가수 분해에 필요한 시간을 단축 상보 물리적 처리 (범위의 마이크로 리액터의 이용을 포함하는 다양한 전략의 개발을 주도 압력). 본 연구에서는 단백질을 빠르게 분해 효소를 달성하기 위해 임의의 실험실에서 구현 될 수있는 간단하고 비용 효율적인 방법을 설명한다. 단백질 (또는 단백질의 혼합물)은 결합 된 C18 역상 높은 퍼포에 흡착모세관 컬럼에서 사전로드 된 액체 크로마토 그래피 (HPLC) 실리카 입자를 필요치 않는, 수성 완충액 트립신은 짧은 시간 동안 입자를 통해 주입된다. 온라인 MS 검출을 사용하려면 트립신 펩티드는 MS 이온 소스에 직접 증가 유기물 함량과 용매 시스템과 용출된다. 이 방법은 고가의 고정화 효소 입자의 사용을 회피하고, 처리를 완료하는 처치를 필요로하지 않는다. 단백질 소화 완전한 샘플 분석은 각각 ~ 30 분 이하 ~ 3 분 이상으로 달성 될 수있다.

단백질 분해 소화 공정의 대표적인 결과가 상기 마이크로 반응기 (도 1, 2) 표 1에 제공하여, 단백질의 혼합물을 동시에 수행 하였다.이 표는 특정 단백질을 식별하는 고유의 펩티드 서열을 포함하는 십자가 상관도 스코어 (Xcorr) (즉, 대응하는 탠덤 질량 스펙트럼 실험 투 이론적 매치의 품질 특징 스코어) 놓친 분열 횟수, 펩티드 충전 상태 (z...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

플로우를 통해 마이크로 반응기와 MS 검출을위한 단백질의 빠른 효소 처리

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K 조회수.  Virginia Tech. A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

비디오: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

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

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

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. ...

연구

생화학

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

연속 흐름 처리를 사용하여 수행 반응의 실시간 모니터링 : 예를 들어 3 Acetylcoumarin의 제조

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

화학

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

단일 시료의 대사 산물, 지질 및 단백질의 종합 분석을위한 간단한 분취 추출 방법

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

단백질 접기 유학을위한 Microfluidic 믹서

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

생체공학

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

자동화된 워크스테이션을 사용한 질량 분석법을 위한 플라즈마 시료 준비

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).

이동식 XY 단계가 있는 플랫폼 인큐베이터: 단백질의 세포 내 빠른 광화학 산화 를 구현하기위한 새로운 플랫폼

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

자동 Proteomic 처리를위한 디지털 Microfluidics

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

생물학

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

발달 중인 (개구리) 배아의 세포 계통 유도 질량 분석법 단백질체학

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...