Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
Uploaded: 2016-04-04T15:00:00
Description: Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

우리는 NIH 보조금 P30ES013508 및 T32ES019851의 지원을 인정합니다.

윤리 정책 : 인간의 샘플의 처리에 관한 모든 프로토콜은 대학의 펜실베니아의 인간 연구 윤리위원회의 지침을 따르십시오. 버퍼의 1. 준비 및 100 배 스톡 솔루션 <ol><li> 기본 타이로드의 버퍼 1 L를 준비합니다. 2 ∙ 2 H 2 O, 0.224 g의 KCl, 및 0.095 g의의 MgCl 2. DDH 2 O.과 1 L에 총 볼륨을 조정 8.123 g의 NaCl, 1.428 g의 수 NaHCO3, 0.466 g의 염화칼슘?...

<ol>
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여기에서 우리는 교란 미토콘드리아 대사 연구를위한 플랫폼으로 고립 된 혈소판의 유틸리티를 보여 주었다. 특히, 우리는 로테으로 복잡한 I 억제에 대한 응답으로 신진 대사 적응을 특징으로하고있다. 본 연구는 인간의 혈소판에 세포 라인에서 로테 논에 의해 복잡한 I 억제의 역할 이전에보고 된 연구 결과를 확장했다. 중요한 것은,이 또한 억제 로테 혈소판 석시 닐 -CoA?...

역기능 미토콘드리아 대사는 신경 변성, 암, 심혈관 질환 (30)을 포함하여 다양한 질환에 연루되어왔다. 따라서, 많은 노력이 질병의 발병에 기여하는 대사 결함을 특성화에 배치되었습니다. 액체 크로마토 그래피 - 탠덤 질량 분석 (LC-MS / MS)는 복잡한 생물학적 매트릭스에서 분석의 정량화를위한 ​​황금 표준으로 간주되며 종종 대사 연구 8 사용된다. 종종 인간의 질병과 관련이 접근하고 잘 정의 된 모델을 달성 생물 의학 연구의 경우입니다 그러나, 도전이다. 많은 연구는 세포 대사 7,9에 생체 이물질 또는 유전 적 이상의 영향을 프로빙을위한 세포 모델을 변형 사용한다. 혼란을 도입 할 수 암 세포에서 발생하는 대사 재 프로그래밍 (21) 요인 때문에 적합하지 않습니다. 이러한 문제는 circumve 될 수 있습니다대사 분석에 충분한 미생물을 얻는 것은 어려울 수 있지만, 일차 전지 모델 nted. 또한, 문화에 사용되는 항생제의 많은 양의 영향은 잠재적으로 혼란 미토콘드리아 연구 (16)과 같이 강조하고있다. 인간 혈소판 대사 연구 5,22,27,32 충분한 미토콘드리아 콘텐츠 일차 전지 모델을 이용할 수있는 기회를 제공. 혈액 공여자 개별 또는 혈액 은행에서 대량 흡입되므로 외부 요인 용이하게 제어 할 수있는 모델을 제공 내지 제 혈소판 쉽게 얻을 수있다. 둘째, 그들의 작은 크기 혈소판 용이에도 최소한 갖춘 실험실 5 최소 준비 작업과 다른 혈액 성분으로부터 분리 될 수있다. 참고로, 혈소판 핵을 포함하지 않는 때문에, 독립적으로 전사 조절 대사 변화를 연구하는 데 이용 될 수있다. 여기에 우리가 보여아실 - 조효소 A (COA) 티오 상대 정량 이외에 혈소판 격리 시스템은 탄​​소 대사를 검토 할 수있다. 특히, 우리는 중요한 대사 물질 아세틸으로 [(13) C] -label의 설립을 조사하기 위해 안정 동위 원소 (비 방사성) 표지 [13 C 6] 글루코스와 [13 C 16] -palmitate과 신진 대사 라벨의 사용을보고 CoA를 해당 작용 또는 지방산 산화를 통한. 이는 생화학 적 경로 13,24에서 아실 CoA를 종의 광범위한 참여와 같은 로테 3,33 복잡한 I의 억제와 같은 다른 변수를 테스트에이 시스템의 취급 용이성에 강력한 일반화하고, 다양한 플랫폼을 제공합니다. 아래의 프로토콜에서 제공하는 정보에 더하여, 동위 원소 표지하고 상기 LC-MS 기반 분석에 사용되는 방법의 광범위한 설명 바수 블레어 4에서 찾아 볼 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

우리가 노출 로테 논에 결과 이​​전에 설명한 보상 대사 적응의 일반화를 재현 한이 방법의 유용성을 입증합니다. 이 결과는 이전에 세포 배양 모델에서 발견되었으며,이 연구는이 대사 변화도 anuclear과 세포 배양과 같은 실험적 인공물 경향이없는 혈소판 발생하면 테스트하고자 하였다. 그들의 신진 대사 활동을 유지하지만,이 작품은, 인간의 주입을 위해 너무 오래된...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

여기에서 우리는 고립 된 인간의 혈소판 내가 로테를 억제제 복잡한에 대한 응답으로 신진 대사 적응을 연구하는 생체 접근으로 모델을 사용할 수 있습니다 보여줍니다. 이 방법은 액체 크로마토 그래피 질량 분석에 의해 추적 동위 상대적 정량을 채용하고 연구 다양한 디자인을 적용 할 수있다.

미토콘드리아 대사 유학을위한 플랫폼으로 인간 혈소판의 LC-MS 분석

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

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SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

나무 조직 내에서 절지 동물과 미생물 군집 유학을위한 기술

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

연구

환경과학

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

깁슨 조립 및 유전자 총을 사용하여 담배 일시적인 유전자 발현

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Chironomidae (파리목)의 사용 표면 부동 물 기관에 대한 신속한 생물학적 평가 프로토콜로 번데기 Exuviae을

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

수정 및 절지 동물의 필드 샘플링을위한 잎 송풍기 - 진공 청소기의 적용

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

공부 하 고 전도성 물리의 위상 동작을 제어 하는 일반 방법 젤로 Conductometry (TSC)를 검색 하는 열

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

공장 수용 성 단백질과 소화 탄수화물 콘텐츠를 사용 하 여 옥수수 (지 시 메이 스)를 표본으로 측정

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

제브라피시 유충에 대한 환경 오염물질의 신경행동 효과 연구

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

목재-폴리머 복합 특성에 대한 건설 및 철거 폐기물 플라스틱 분획의 효과

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

필로스피어와 채소 발효에서 미생물군유전체 조립을 연구하기 위한 노토바이오틱 시스템

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

15개의질소 분석을 위한 마이크로플롯 설계 및 식물 및 토양 샘플 준비

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...