이 작품은 에너지 Biosciences 연구소 부여 OO0G01에 의해 투자되었다.

OLIMP 방법은 glycosylhydrolase으로 endoglucanase를 사용하여 dicot 식물, xyloglucan의 세포 벽에있는 주요 hemicellulose에 exemplified입니다. 방법은 식물 조직 소스로 전체 Arabidopsis 모종로 증명됩니다. 효소와 세포외 매트릭스 물질은 동일한 절차를 사용하여 원하는 분석에 따라 대체하실 수 있습니다. 1. 세포 벽 절연 <ol><li> 하베스트 오 5 일 이전 전체 Arabidopsis 모종이나 원하는 자료의 상당 금액 및 1.5mL 반응 관에 그들을 전송합니다. 자료가 튜브의 아래쪽에 배치되었는지 확인합니다. </li><li> 액체 질소의 샘플 및 스냅인 동결 위에 반응 관에 두 3mm 금속 공을 추가합니다. </li><li> 볼 - 밀 (2시 반 분, 25Hz)를 사용하여 냉동 샘플을 분쇄. </li><li> 샘플에 1mL 70% 수성 에탄올을 추가합니다. 자석을 사용하여 금속 공을 제거합니다. 철저히 와동. </li><li> 펠렛에 10 분 세포 벽 재료가 들어있는 알코올 불용성 잔류물을 위해 14,000 RPM에서 샘플을 원심 분리기. </li><li> 조심스럽게 흡인하여 뜨는을 제거하십시오. 펠렛을 방해하지 않도록하십시오. </li><li> 클로로포름 / 메탄올 (1:1 V / V) 솔루션의 1mL를 추가합니다. 펠렛을 resuspend에 철저 와동. </li><li> 10 분 14,000 RPM에서 샘플을 원심 분리기 조심스럽게 흡인하여 뜨는을 제거하십시오. 펠렛을 방해하지 않도록하십시오. </li><li> 농축기를 사용하여 샘​​플을 건조시킵니다. </li></ol> 2. oligosaccharides의 Solubilization <ol><li> solubilization에 대한 oligosaccharides의 형태로 특정 다당류는 적절한 버퍼 25μl의 건조 펠렛을 resuspend. endoglucanase 50mM 질산 편대, pH4.5을 ​​위해 사용됩니다. endoglucanase의 0.2U를 추가합니다. 아래 소용돌이 서스펜션과 스핀 액체. </li><li> 37 16h에 대한 샘플을 품어 ° C를 인큐베이터에서 300rpm에서 떨고. </li><li> 농축기를 사용하여 소화 (물)의 용매를 제거합니다. </li></ol> 3. 출시 oligosaccharides의 든 - TOF 분석 <ol><li> BioRex MSZ 501의 양이온 교환 수지 비즈는 소화 샘플에서 소금과 효소를 제거하는 데 사용됩니다. 빈 열에 나누어지는을 배치하고 물을 풍부한 양의으로 구슬을 씻는하여 조건 비즈 있습니다. </li><li> 소화와 말린 예제 5-10 BioRex 비즈를 추가합니다. 그런 다음 물 재료 5μl 작은 양의 사용할 수 있습니다위한 물 10μl를 추가합니다. 간단히 튜브를 회전하여 솔루션 비즈 젖어. 상온에서 적어도 10 분에 대해 품어. </li><li> 든 대상 플레​​이트에 자리 2μl 매트릭스 (2,5 - dihydroxybenzoic 산성 (DHB), 물에 10mg/ml). 동질 매트릭스 화학 크리스탈로 이어지는 진공 하에서 용매 매트릭스를 증발. </li><li> 대상 접시 매트릭스 결정 위에 desalted 샘플 솔루션의 스팟 2μl. 이 샘플의 많은 지점하려는 경우에만 3min의 최대 야생 계속. 이 시간 프레임은 최초의 발견 샘플 아직 건조되지 않도록합니다. 마지막으로 발견 샘플에서 다시 용해 매트릭스와 샘플 올리 고당 분자의 철저한 혼합을 보장하기 위해 추가 2min 나올 때까지 기다리는 것이 좋습니다. 다음 진공에서 대상 번호판을 건조. 샘플 / 매트릭스 믹스는 동질적인 결정을 활성화 1min 이하에서 구체화한다. </li><li> 든 - TOF 질량 분석기로 대상 플레​​이트 플레이스. 기계 20000V의 가속 전압과 350 nm의의 지연 긍정 reflectron 모드로 설정되어야하며 선택된 질량 범위 (수가 예상 oligomers에 따라 변경) 500-3000 다해야합니다. 레이저를 쏘면서 시작하고 대표적인 평균 스펙트럼 (그림 2A)를 생성하는 컴파일 샘플 당 100-200 스펙트럼을 수집합니다. 특정 oligosaccharides를 나타내는 이온은 요금 (M / Z) 비율을 통해 그들의 질량에 의해 확인할 수 있습니다. 관심의 이온의 이온 농도 (최대 높이)는 샘플의 각 올리 고당의 상대적인 풍부 (그림 2B)로 인해 100 %까지 추가되어야합니다. </li></ol> 4. 원위치 OLIMP 분석 OLIMP는 모든 벽 준비 단계를 생략 조직에 직접 사용될 수 있습니다. 식물 조직 소스가 사용하는 같은 예제는 Arabidopsis의 hypocotyls을 etiolated으로. 다시 endoglucanase는 hemicelluloses의 xyloglucan의 구조를 결정하는 데 사용됩니다. 효소 및 조직 재료는 동일한 절차를 사용하여 원하는 분석에 따라 대체하실 수 있습니다. <ol><li> etiolated 묘목을 수확하고 비어 든 대상 플레​​이트에 그것을 장소와 묘목이 건조하게. </li><li> 원하는 위치에 말린 세들링에 0.5μl endoglucanase을 (50mM 질산 편대에 녹아있는 0.4U/μl, pH4.5)을 추가합니다. 효소 드롭 타겟 플레이트 부분적으로 접촉되었는지 확인합니다. </li><li> 피하기 위해 포화 습도와 닫힌 용기에 든 대상 플레​​이트를 삽입하는 효소 드롭 마른 아웃. 1 챔버에서 번호판을 품어37 6h가 ° C. </li><li> 식물 조직과 진공에서 효소 드롭으로 든 대상 플레​​이트를 건조시킵니다. </li><li> 각 건조 효소 자리 위에, 0.5μl 매트릭스 (10mg / L DHB) 추가합니다. 그것이 2min에 대한있게 해줘요. </li><li> 진공에서 든 대상 플레​​이트를 건조시킵니다. </li><li> 든 - TOF와 샘플을 분석할 수 있습니다. 든 - TOF 질량 분석기로 조직과 효소 / 매트릭스 지점 (S)와 타겟 플레이트 플레이스. 기계 20000V의 가속 전압과 350 nm의의 지연 긍정 reflectron 모드로 설정되어야하며 선택된 질량 범위 (수가 예상 oligomers에 따라 변경) 500-3000 다해야합니다. </li><li> 조직 옆에 매트릭스 / 샘플 결정에 레이저를 발사 시작합니다. 이 같은 분자의 비행 -의 시간이 끝날거야 이런 경우에, 조직 자체에 충돌하지 않도록합니다. 대표 평균 스펙트럼 (그림 3)을 생성하는 컴파일 장소, 당 20-50 주위 스펙트럼를 수집합니다. 특정 oligosaccharides를 나타내는 이온은 요금 (M / Z) 비율을 통해 그들의 질량에 의해 확인할 수 있습니다. 이온 농도 (이온 높이)를보고 수 있으며, 관심있는 모든 이온이 예제에서 각 oligosaccharides의 상대적인 풍부 결과 100 %까지 추가되어야합니다. </li></ol> 5. 대표 결과 Arabidopsis 모종에있는 hemicelluloses의 xyloglucan의 OLIMP 분석의 예제는 그림 2에 표시됩니다. 이온의 질량 차이 및 알려진 잘 특징 효소로 인해 (endoglucanase) 특이성은 이온은 특정 올리 고당 구조 (그림 2A)에 할당할 수 있습니다. 분명히, 구조 isomers는 구별되지 않습니다. 다양한 oligosaccharides의 상대적인 풍요의 결정에 대한 기본 가정은 (그림 2B)은 질량 분석기를 응답 계수 이러한 oligosaccharides 매우 비슷하다고합니다. 여기에 그림과 같이, OLIMP 부량는 매우 재현할 수 있습니다. 그러나, 방법의 견고는 각종 올리 고당 이온의 잡음 비율로 신호에 크게 의존된다는 점에 유의하시기 바랍니다. 예를 들면 소금이나 oligosaccharides의 낮은 금액과 오염은 비율을 줄일 수 있습니다. 조직 자체 (현장 분석)에 OLIMP 분석은 매우 작고 정의된 영역의 연구를 가능하게하고 아주 작은 샘플 준비 포함됩니다. 예제에 정성 (동일 이온)는 (그림 3) 여기에 제시 아니지만 양적 차이는 (이온 농도의 변화) Arabidopsis 묘목의 촬영 및 루트 - 조직 사이에 관찰되었다. OLIMP - 방법 Permutations 따라서 조직 &quot;이미지&quot;가 발생할 수 있습니다. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="그림 1" /> 그림 1. 플랜트 원본으로 전체 Arabidopsis의 모종을 사용하여 OLIMP 절차의 플로우 차트. 첫째, 조직이 분해되고 세포 벽 재료 (사진 후지노 외에서 수정되었습니다. 10) 준비가되어 있습니다. 그런 다음 특정 벽면 다당류의 oligosaccharides은 특정 하이드롤라제를 사용하여 발표하고 있습니다. 마지막으로, 가용성 oligosaccharides의 상대 abundances이 든 - TOF 질량 분광법을 사용하여 결정됩니다. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="그림 2" /> 그림 2. OLIMP에 의해 결정으로 Arabidopsis에서 etiolated 모종의 xyloglucan의 oligosaccharides의 상대 풍부. A) 대표 xyloglucan 올리 고당 질량 스펙트럼, 각 이온이 특정 올리 고당 구조를 나타내고, 구조 isomers는 구별되지 않습니다. 상대 올리 고당 풍요의 결정에 대해 B) 해당 막대 다이어그램. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="그림 3" /> 그림 3. 예로 Arabidopsis의 etiolated 모종을 사용하여 현장 OLIMP 분석. 각 효소 / 소화 소적 (유색 원)는 독립적으로 분석할 수 있으며 해당 질량 스펙트럼을 얻을하고 분석할 수 있습니다. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="그림 4" /> 그림 4. xylanase (Megazyme)와 Miscanthus 리프 자료를 소화하여 얻은 hemicellulose의 크실란의 OLIMP 스펙트럼.

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+irregular+xylem9+mutant+is+deficient+in+xylan+xylosyltransferase+activity.">The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity.</a> Plant and Cell Physiology. 48 (11), 1624-1624 (2007).</li><li>Iglesias, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+glycosidases+active+against+xyloglucan+oligosaccharides+of+Arabidopsis+thaliana.">Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.</a> Plant and Cell Physiology. 47 (1), 55-55 (2006).</li><li>Fujino, T., Sone, Y., Mitsuishi, Y., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cross-links+between+cellulose+microfibrils,+and+their+occurrence+during+elongation+growth+in+pea+epicotyl.">Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.</a> Plant Cell Physiol. 41 (4), 486-486 (2000).</li><li>Vanzin, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mur2+mutant+of+Arabidopsis+thaliana+lacks+fucosylated+xyloglucan+because+of+a+lesion+in+fucosyltransferase+AtFUT1.">The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan because of a lesion in fucosyltransferase AtFUT1.</a> PNAS. 99 (5), 3340-3340 (2002).</li><li>Cavalier, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+two+Arabidopsis+thaliana+xylosyltransferase+genes+results+in+plants+deficient+in+xyloglucan,+a+major+primary+cell+wall+component.">Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.</a> Plant Cell. 20 (6), 1519-1519 (2008).</li><li>Hilz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+liquid+chromatography,+capillary+electrophoresis,+and+mass+spectrometry+methods+to+determine+xyloglucan+structures+in+black+currants.">A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.</a> Journal of Chromatography A. 1133 (1-2), 275-275 (2006).</li><li>Pauly, 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+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214 (1), 67-67 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 제시 OLIMP 메서드는 세포외 매트릭스에 존재 폴리머 매우 민감하고 신속한 분석이 가능합니다. OLIMP는 이후 든 - TOF 분석 oligomers의 효소 릴리스를 결합한 제품입니다. 든 - TOF 스펙트럼의 세대 미만 분 소요, 따라서 OLIMP는 돌연변이 스크린으로 높은 처리량 연구를 포함한 어플 리케이션의 광범위한 적합합니다. OLIMP는 식물의 다당류에 제한된 것은 아니지만 잠재적으로 특정 가수 분해 효소의 가용성에 의해 제한 고분자의 다양한 적용할 수 있습니다. 그러나, OLIMP의 제한은 폴리머의 절대 풍부를 얻을 수 없다는 것입니다. OLIMP가 종 다양성의 세포외 기질에 존재 다당류의 다양한 구조를 연구하는 데 사용할 수있는 이전처럼 언급했다. 예를 들어, 그림 4는 잔디 종의 주요 hemicellulose, 크실란의 OLIMP 스펙트럼을 나타냅니다. 여기, 온대 잔디 Miscanthus에서 파생된 세포 벽 재료는 xylanase를 사용하여 소화했다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oligo mass profiling (olimp) of extracellular polysaccharides

환경에 세포의 직접 접촉은 세포외 기질로 많은 생물의 중재입니다. 세포외 매트릭스 중 하나는 일반적인 측면들은 복잡한 설탕 glycoproteins 형태의 moieties, proteoglycans, 및 / 또는 다당류를 포함한다는 것입니다. 예 원섬유의 단백질과 proteoglycans 또는 식물과 곰팡이의 다당류 기반의 세포 벽 주로 구성된 인간과 동물 세포의 세포외 기질과 proteoglycan / 박테리아의 glycolipid 기반의 세포 벽을 포함합니다. 이러한 모든 glycostructures는 휴대 전화 및 휴대 환경 통신 및 신호에서 중요한 역할을 재생합니다. 세포외 기질의 특별한 복잡한 예제는 높은 식물 세포의 벽에 속해 있습니다. 그들의 벽이 최대 75% 건조 중량, 당분의 거의 전적으로 만들어진,이 지구상에서 존재하는 가장 풍부한 biopolymers 구성되어 있습니다. 따라서, 연구는 화석 연료에서 파생 석유를 대체할 수있는 탄소 중립 재생 자원으로 최고 이러한 자료를 활용하는 방법을 실시합니다. 연료 전환의 주요 과제는이 독특한 biocomposite에있는 독특한 glycostructures에 의한 효소 또는 화학적 열화에 벽의 말을 안들음 남아 있습니다. 여기, 우리는 식물 세포 벽의 glycostructures의 신속하고 민감한 분석을위한 방법을 제시한다. 이 방법은 OLIgo 질량 프로파일 (OLIMP)은 (MALDI-TOF/MS 매트릭스를 이용한 레이저 탈착 / 이온화 시간의 기내 질량 분석법을 사용하여 특정 glycosylhydrolases 및 solubilized 올리 고당 혼합물의 후속 분석을 촉진 벽 자료에서 oligosaccharides의 효소 릴리스를 기반으로합니다 1) (그림 1). OLIMP은 전체 분석에 대해서만 5,000 세포의 벽이, 조직 자체 2 수행할 수 있어야하고, 높은 처리량 분석 3 의무가 있습니다. solubilized oligosaccharides의 절대 금액이 OLIMP에 의해 결정되지 않을 수 있지만 각종 올리 고당 이온의 상대적인 풍부가 벽에 현재 기본 다당류의 대체 패턴에 대한 통찰력을주는 질량 스펙트럼에서 delineated 수 있습니다. OLIMP은 특정 효소 4의 가용성에 의해 제한 벽 폴리머의 다양한 분석하는 데 사용할 수 있습니다. 예를 들어, 식물 세포 벽의 효소에있는 폴리머의 분석을 위해 엔도 - β - (1-4) - xylanase 6,7을 사용하여 xyloglucanase 5, 11, 12, 13, 크실란를 사용하여 hemicelluloses의 xyloglucan을 분석하실 수 있습니다 또는 polygalacturonase와 methylesterase 8 조합을 사용하여 pectic 다당류하십시오. 또한, OLIMP glycosylhydrolase 심지어 glycosyltransferase 활동의 동일한 원리를 사용하는 것은 감시하고 9 결정하실 수 있습니다.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

빠른 방법은 세포외 기질에서 다당류의 구조에 대한 통찰력을 얻을 수 설명되어 있습니다. 방법은 glycosylhydrolases의 특이성과 물질의 미세한을 분석 수 있도록 질량 분석계의 감도 활용합니다. 이 기술은 조직 자체에서 직접 사용할 수 적용할 수 있습니다.

세포외 다당류의 OLIgo 질량 프로파일 (OLIMP)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K 조회수.  University of California, Berkeley. 빠른 방법은 세포외 기질에서 다당류의 구조에 대한 통찰력을 얻을 수 설명되어 있습니다. 방법은 glycosylhydrolases의 특이성과 물질의 미세한을 분석 수 있도록 질량 분석계의 감도 활용합니다. 이 기술은 조직 자체에서 직접 사용할 수 적용할 수 있습니다.

비디오: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

루시페라제 Immunoprecipitation 시스템 (입술)에 의해 항체 프로파일

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

성인 마우스 Cardiomyocytes의 단세포 Transcriptional 프로파일

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polysom​​e 프로파일 링 포유 동물 세포에 선택적 mRNA의 번역 평가

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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 ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

고감도 현미경을 이용한 세포 산화 환원 프로파일 링

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 셀 형식 관련 Epigenome 프로 파일링

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...