저자는 에너지의 미국학과 (DEFG02 - 01ER15129)에 의해 부여 번호 0719232 아래에있는 미국 국립 과학 재단 (National Science Foundation)에 의해이 작품의 재정 지원에 감사하며, 프로 테아 Biosciences, 주식 회사 (모건, WV)에 의해. 저자는 또한 프로토콜 찍고하는 동안 그녀의 도움 제시카 A. Stolee 감사하고 싶습니다.

다음 프로토콜은 레이저 박리 전기 분무 이온화 (LAESI) 실험의 주요 단계를 설명하고 동물과 식물 조직 샘플에 대한 측면과 입체 (3D) 영상을위한 대표적인 예제를 제공합니다. 추가 실험 및 기술 정보는 다른 곳에서 얻을 수 있습니다. 1-6는 1. 조직 준비 및 마운트 <ol><li> sectioning이 필요한 경우, 조직은 -20 -10에서 10-100 μm의 두께로 자른 섹션에 cryomicrotome를 사용 ° C 달리 특정 조직 유형에 대한 권장하지 않는 한. </li><li> 직접 화학 수정자없이 평평한 표면 (예 : 화학적 사전 청소 유리 슬라이드)에 섹션을 탑재합니다. 분석하는 동안 항상 냉동 조직을 유지하기 위해 해동 장착시 sectioned 조직은 섹션을 해동 장착 즉시 펠티어 냉각 단계에 샘플 홀더를 확보. 이 단계는 섹션에서 분자 마이 그 레이션을 최소화 / 방지하기 위해 필요합니다. </li><li> 필요한 경우, 조직은 냉동 유지하는 펠티어 단계에서 열 제거를 용이하게하기 위해 저전력 팬이 장착된 히트 싱크를 사용합니다. </li><li> 시간 (1-2시간)의 연장 기간 동안 습기 환경에서, 물 또는 조직 표면에 얼음의 응축을위한 검사. 조직에 물이 응축은 detrimentally LAESI 실험에서 이미징 성능에 영향을 미치는 1. </li><li> 필요한 경우, 룸 dehumidifier을 사용하거나 결로를 방지하기 위해 불활성 가스 (예 ​​: 건조 질소 가스)로 가득 환경 챔버의 냉각 샘플 장소 1. </li></ol> 2. LAESI 이온 소스의 최적화 LAESI 이온 소스는 중간 적외선 레이저, 가벼운 스티어링을위한 광학 요소의 시리즈와 초점을뿐만 아니라 추가 샘플 홀더, 냉각 부품, 번역 단계 및 용매 전달 시스템으로 구성되어 있습니다. 그림 1은 질량 분석계의 대기 압력 이온 소스의 입구에 관련하여 이러한 요소의 전형적인 배치를 보여줍니다. <ol><li> 그림 1, 위치 질량 분석기 샘플링 콘 (D 또는 - FP)의 구멍 아래의 샘플 15-20mm에 그림과 같이. </li><li> 2.94 μm의 파장과 10 Hz에서 반복 속도 중반 IR 레이저를 운영하고 있습니다. ~ 100 μJ / 펄스 에너지의 레이저 출력을 감쇠. </li><li> 몇 가지 일반적인 입사에서 샘플에 레이저 펄스 에너지 (샘플 표면에 대하여 바로 입사 각도를 볼 수있는 레이저 파장 (예, 평철의 카페 2 ZnSe 렌즈)에서 투명 금색 거울과 초점 렌즈의 조합을 사용 그림 1). </li><li> 질량 분석계 샘플링 콘의 구멍 앞 중간 적외선 빔 축 5~8밀리미터를 놓습니다. </li><li> 초점 렌즈 위치와 초점 자리에 조직 제거를 달성하기 위해 레이저 빔을의 펄스 에너지를 조정합니다. ablated 볼륨의 크기는 이미징 응용 프로그램에 대한 픽셀 (또는 3 차원 영상의 voxel) 크기를 확인합니다. </li><li> 질량 분석계의 입구 축과 일치하며 ~ ​​10 mm의 구멍 - 투 - 에미터 팁 거리 (그림 1 참조)에서 nanospray의 방사체를 놓습니다. </li><li> 전기 분무의 경우, 각각 0.1 % 초산 또는 긍정적이거나 부정적인 이온 모드 0.1 % 암모늄 아세테이트 첨가제로 50 % 메탄올 솔루션을 준비합니다. 기타 유기 용제 이러한 acetonitrile, 이소프로판올, 등, 샘플에 따라 분석 작업에 대한 적절한 농도에서 메탄올을 대체할 수 있습니다. 전기 분무의 안정성을 성공적으로 영상을 위해 매우 중요합니다. 용매 선택에 따라 유량과 분사 전압 안정 스프레이를 달성하기 위해 조정해야합니다. </li><li> 반응 LAESI 6 이미징 애플 리케이션에서, electrosprayed 솔루션 reactants를 포함할 수 있습니다. </li><li> ~ 300 NL / 분 유량에 전기 분무 방출을 통해 전기 분무 솔루션을 제공하기 위해 주사기 펌프를 사용합니다. </li><li> 질량 분석계의 구멍은 낮은 전압 (&lt;500 V는 지상으로부터 측정)에 보관하는 경우, 전기 분무 방출기 (예, 3000 V)로 직접 고압을 적용하거나 금속 노조를 통해 전기 분무를 생성합니다. 그렇지 않으면, 전기 분무를 구축하기 위해 직접 전기 분무 방출기 또는 금속 노조를 통해 지상. </li><li> LAESI에 의해 가장 효율적인 이온 생성을위한 모드를 분사 콘 제트의 전기 분무 소스를 운영하고 있습니다. 질량 스펙트럼에서 분사 모드 및 효과에 대한 운영 변수의 효과를 보려면 다른 토론을 참조하십시오. 5,7,8은 </li><li> 조심스럽게 같은 비행기에 레이저 빔을, 방출 및 오리피스 도끼를 유지하면서 LAESI 이온 수율을 최적화하기 위해 LAESI 설정의 상대적 거리를 조정합니다. 다른 자세한 지침을 찾아 주시기 바랍니다 5. </li><li> 광학 현미경으로, 시료의 절제 분화구의 측면 크기를 결정합니다. </li><li> 입체 LAESI 이미징 실험을 위해, 개별 펄스와 절제를 수행하고 예를 들어, 사용 voxel의 깊이를 결정 광학 현미경의 Z - 스택 모드 3. </li></ol> 3. 분자 이미징 및 데이터 분석 이미징 실험에서 조직 샘플보다 크거나 박리 영역의 크기와 동일한 단계 크기 X와 Y 방향으로 레이저의 초점 평면에서 이동합니다. 공간 해상도는 입사 레이저의 초점에 의해 제한됩니다. <ol><li> 샘플 표면에 대한 관심의 영역을 선택하고 (X, Y)가 해당 경계 좌표 구하십시오. </li><li> 몇 군데로 구역이 지남에 따라 각 픽셀에 시간을 머물러 선택과 샘플 표면을 래스터로있는 gridding 알고리즘 (예, 적응 그리드, 선택된 지역 이미징, 직사각형 그리드, 나선형 패턴, Z 스캔 등) 선택할 수 있습니다. </li><li> 3 축 번역 무대와 정해진 그리드에 따라 샘플을 rastering 수있는 소프트웨어를 사용합니다. </li><li> 이미징에 필요한 총 시간을 계산합니다. </li><li> 질량 분석계의 데이터 수집 시간 제한을 해제합니다. 이것이 불가 능할 경우, 계산 이미징 시간 값을 데이터 수집 시간 제한을 설정합니다. </li><li> LAESI 측면 이미징 실험을 수행하기 위해 각 픽셀에 머물러있는 시간 내에 질량 스펙트럼에 충분한 신호 대 잡음 비율을 생산하기 위해 적절한 반복 속도 중간 적외선 레이저 소스를 시작합니다. 3D 분자 이미징을 위해 성공적으로 단일 레이저 펄스 내에서 생성된 이온을 대량 분석 레이저 소스 반복 속도보다 높은 스펙트럼 수집 속도를 사용합니다. 절제 시퀀스를 시작하려면 시작 신호를 기다립니다. </li><li> 전기 분무 소스를 켭니다. 이미징에 필요한 전체 시간에 대한 충분한 해결책이 있는지 확인합니다. </li><li> 동시에 대량 스펙트럼의 인수 중반 IR 레이저 박리 및 표면 검사를 시작합니다. </li><li> 이미징 실행이 완료되면, 표면 검사, 중반 IR lasing 및 데이터 수집를 중지합니다. </li><li> 레이저 소스를 해제합니다. </li><li> 높은 전압의 전원을 끕니다. </li><li> 주사기 펌프를 전환합니다. </li><li> 대기 모드로 질량 분석기를 설정합니다. </li><li> 펠티어 냉각 전자를 끄십시오. </li><li> 사용하면 불활성 가스 흐름을 닫습니다. </li><li> 해당 검사 결과와 3 차원 분석 측면 영상이나 voxels에있는 픽셀의 절대 좌표를 연관시키는 소프트웨어를 사용합니다. </li><li> 수평 및 3D 분자 이미지를 얻기 위해 분석의 절대 좌표를 상대로 선택한 M / Z 값에 대한 이온 강도 신호를 플롯. </li></ol> 4. 대표 결과 그림 2는 몇 가지 주요 조직 유형 및 이미징 modalities에 대한 대표적인 결과를 제공합니다. 패널 탈수를 방지하기 위해 실험 기간 동안 냉동되어 동물의 조직 섹션에 대한 사건을 묘사. 또한 1, 샘플이 샘플 표면에 응축에서 주변 물 증기를 피하기 위해 건조 질소 가스 환경에 위치했다. 쥐 두뇌의 100 μm의 두께 코로나 섹션 (Rattus norvegicus)가 옆으로 LAESI와 몇 군데했다. 두뇌의 해부 지역 (패널에 광학 이미지를 볼 A) 분자 plasmalogens PC 용 얻은 이미지 (O - 33 : 3)와 좋은 상관 관계 표시 및 / 또는 PE (O - 36 : 3) M / Z 728.559과 함께합니다. 패널 B는 제브라 공장 (Aphelandra squarrosa) 잎 조직의 3D LAESI의 이미지를 보여줍니다. 단풍은 탈수에 대한 자연 방어 메커니즘을 가지고 있기 때문에 샘플은 주위 환경에서 심문 수 있습니다. 3 얻은 3D 분자 이미지 기본 및 보조 metabolites에 대한 유통 패턴의 다양한 밝혔다. 다른 사람들과 M / Z 285.076과 acacetin는 다른 균일한 분포로 위로부터 두 번째 및 세 번째 레이어의 노란 분야에서 높은 이온 카운트에서 검색되었습니다. 이 배포판은 광학 이미지에서 본 잡색의 패턴으로 동의했다. <img src="/files/ftp_upload/2097/2097fig1.jpg" alt="그림 1" /> 그림 1. LAESI 시스템의 개략도 (ES, 전기 분무의 에미터 팁, 또는, 질량 분석계 샘플링 콘의 구멍, FL, 렌즈를 집중, FP, 초점, P, 펠티어 냉각 단계, HS, 히트 싱크). 분자 및 샘플 이온 (녹색 점들)와 씨앗 충전 방울을 낼 수있는 전기 분무와 중반 IR 절제 (빨간색 점) coalesces 동안 퇴학 미립자 문제의 부분. 이러한 방울에서 발표 이온 분석 및 질량 분석기에 의해 기록됩니다. <img src="/files/ftp_upload/2097/2097fig2.jpg" alt="그림 2" /> 그림 2. 수평 및 3D imagi에 대한 대표적인 결과를. 또는 PE (O - 36 / : LAESI 질량 분석계와 함께 NG (A) 상단 패널은 쥐의 두뇌 (Rattus norvegicus) 코로나 섹션 plasmalogens PC 용 얻은 분자 이미지의 광학 이미지 (3 O - 33) 모습 : 3) M / Z 728.559. 흰색 규모 막대가 1mm에 해당합니다. 의 허가 (참고 1) 적응. 저작권 2010 미국 화학 학회. (B) 하단 패널은 잡색 제브라 공장 (Aphelandra squarrosa)에서 나뭇잎의 3D 이미지를 보여줍니다. M / Z 285.076과 Acacetin은 다른 사람의 균일한 분포로 위로부터 두 번째 및 세 번째 레이어의 노란 분야에서 높은 이온 카운트에서 검색되었습니다. 의 허가 (참고 3) Reprinted. 저작권 2009 미국 화학 학회.

<ol>
	<li>Nemes, P., Woods, A. S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Imaging+of+Small+Metabolites+and+Lipids+in+Rat+Brain+Tissues+at+Atmospheric-Pressure+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric-Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Anal Chem. 82, 982-988 (2010).</li><li>Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure,+in+vivo,+and+imaging+mass+spectrometry.">Laser ablation electrospray ionization for atmospheric-pressure, in vivo, and imaging mass spectrometry.</a> Anal Chem. 79, 8098-8106 (2007).</li><li>Nemes, P., Barton, A. A., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+metabolites+in+tissues+under+ambient+conditions+by+laser+ablation+electrospray+ionization+mass+spectrometry.">Three-dimensional imaging of metabolites in tissues under ambient conditions by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 6668-6675 (2009).</li><li>Nemes, P., Barton, A. A., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+molecular+imaging+and+depth+profiling+of+live+tissue+by+infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 80, 4575-4582 (2008).</li><li>Nemes, P., Vertes, A., Nemes, S. S., Rubakhin, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure+molecular+imaging+mass+spectrometry.+Mass+Spectrometry+Imaging.">Laser ablation electrospray ionization for atmospheric-pressure molecular imaging mass spectrometry. Mass Spectrometry Imaging.</a> Methods in Molecular Biology. , (2010).</li><li>Shrestha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+lipids+and+small+metabolites+in+mouse+brain+tissue+by+AP+IR-MALDI+and+reactive+LAESI+mass+spectrometry.">Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.</a> Analyst. 135, 751-758 (2010).</li><li>Nemes, P., Marginean, I., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spraying+mode+effect+on+droplet+formation+and+ion+chemistry+in+electrosprays.">Spraying mode effect on droplet formation and ion chemistry in electrosprays.</a> Anal Chem. 79, 3105-3116 (2007).</li><li>Nemes, P., Goyal, S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+and+noncovalent+complexation+changes+in+proteins+during+electrospray+ionization.">Conformational and noncovalent complexation changes in proteins during electrospray ionization.</a> Anal Chem. 80, 387-395 (2008).</li><li>Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+by+Mid-IR+laser+ablation+mass+spectrometry.">Molecular imaging by Mid-IR laser ablation mass spectrometry.</a> Appl Phys A-Mater Sci Process. 93, 885-891 (2008).</li><li>Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+metabolic+profiling+of+single+cells+by+laser+ablation+electrospray+ionization+mass+spectrometry.">In situ metabolic profiling of single cells by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 8265-8271 (2009).</li><li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, 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=Ambient+mass+spectrometry.">Ambient mass spectrometry.</a> Science. 311, 1566-1570 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

다른 조직 유형이 효과를 완화하기 위해 차례로, 샘플의 박리 특성에 영향을 미칠 수, 물, 컨텐츠 및 인장 강도. 9 다양한 전시, 그것은 사이에 변화하는 때 레이저 fluence, 샘플 처리 및 분석 프로토콜 개정 않는다 주요 조직 유형. 단일 셀 또는 고해상도 수사, 중간 적외선 대신 초점 렌즈의 둘레에 광섬유로 결합 수 있습니다. 조직에서 선택한 세포의 근접 이내에 섬유 팁을 위치하여 10, LAESI 분석을 수행할 수 단일 세포 수준. 주위 이온화 질량 분광법에 대한 레이블이없는 이온 소스로, 11 LAESI는 조직의 생화 학적 과정의 조사에 대한 잠재력을 보이지 않고있다. 직접 분석, 수평 및 3D 이미징의 추가 혜택과 함께, LAESI는 프로파일뿐만 아니라 이미징 어플 리케이션을위한 새로운 bioanalytical 도구입니다.

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		<th>Material Name</th>
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		<th>Comment</th>
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<td>Mass spectrometer</td>
<td>&nbsp;</td>
<td>Waters Co. (Milford, MA)</td>
<td>Q-TOF Premier</td>
<td>&nbsp;</td>
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<td>Mid-IR laser</td>
<td>&nbsp;</td>
<td>Opotek Inc. (Carlsbad, CA)</td>
<td>Vibrant IR</td>
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atmospheric-pressure molecular imaging of biological tissues and biofilms by laesi mass spectrometry

질량 분광법에서 대기 이온화 방법은 분석 조사가 모국어와 같은 실험 조건에서 조직이나 biofilm에서 직접 수행할 수있게합니다. 레이저 박리 전기 분무 이온화 (LAESI)는 하나의 개발이며, 특히 물이 포함된 표본 조사에 적합합니다. LAESI은 샘플의 물 분자를 자극 중간 적외선 레이저 빔 (2.94 μm의 파장)를 사용합니다. 절제 fluence 임계값이 초과되면, 샘플 자료 미립자 물질의 형태로 퇴학이 projectiles은 샘플 표면 위에서 mm 수만 여행입니다. LAESI,이 절제 플륨는 배출 샘플 자료의 일부를 캡처 및 가스 상 이온에의 화학적 성분을 변환하는 높은 비용이 청구 방울에 의해 포착됩니다. 대기 압력 이온 소스 인터페이스를 갖춘 질량 분석기는 시료의 탐지 영역 (픽셀)에서 발생하는 발표 이온의 구성을 분석하고 기록하는 고용됩니다. 픽셀의 배열을 통해 체계적인 심문은 microprobe 분석 모드에서 분자 이미징을위한 방법을 엽니다. LAESI 질량 spectrometric 이미징의 독특한 측면은 측면 이미지와 함께, 3 차원 (3D) 분자 이미징을 가능하게, 깊이 프로 파일링입니다. ~ 100 μm의 각각 ~ 40 μm의, 현재 수평 깊이 해상도로 LAESI 질량 spectrometric 영상은 생물 학적 조직의 분자 구조를 탐구하는 데 도움이됩니다. 여기에, 우리는 LAESI 시스템의 주요 요소를 검토하고 성공적인 영상 실험에 대한 지침을 제공합니다.

Watch this Scientific Journal Video about Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry at JoVE.com

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

레이저 박리 전기 분무 이온화 (LAESI)는 질량 분광법에 대한 대기 압력 이온 소스입니다. 이미징 모드에서 중간 적외선 레이저 프로브 조직 섹션 또는 biofilm에 걸쳐 분자의 배포판. 이 기술은 원래 실험 조건 하에서 수행 다양한 bioanalytical 연구에 대한 새로운 접근법을 제공합니다.

LAESI 질량 분석법에 의해 생물 학적 조직 및 Biofilms의 대기 압력 분자 이미징

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

atmospheric-pressure-molecular-imaging-biological-tissues-biofilms

Research

JoVE Journal

Biology

14.1K 조회수.  George Washington University. 레이저 박리 전기 분무 이온화 (LAESI)는 질량 분광법에 대한 대기 압력 이온 소스입니다. 이미징 모드에서 중간 적외선 레이저 프로브 조직 섹션 또는 biofilm에 걸쳐 분자의 배포판. 이 기술은 원래 실험 조건 하에서 수행 다양한 bioanalytical 연구에 대한 새로운 접근법을 제공합니다.

비디오: Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T - 파 이온 모빌리티 분석법 - 질량 : 단백질 복잡한 분석을위한 기본 실험 절차

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

대기압에서 질량 분석법에 의해 단일 ​​세포의 직접 분석

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L</sub캡처 컴파 운드 질량 분광법 (CCMS)에서> - 호모 시스테인 결합 단백질

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

동위 원소 태그 질량 분광법을 사용하여 Thiol 산화 환원 프로테옴 프로 파일링

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

단일 프로브의 응용 프로그램 : 주변 조건에서 질량 분석 이미징 및 단일 세포 분석

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

이미징 센터의 형광 질량으로 Golgi 단백질의 양적 지역화

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...