Name: fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers
Uploaded: 2018-04-17T13:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

R. Menzel과 도이치 가운데 (RU RU780/11-1)에 의해 L. Ruess의 재정 지원은 기꺼이 인정 했다. R. Nehring RU 780/10-1에 의해 투자 되었다. 마지막으로, 우리는 매우 감사 박사 헤이젤 Ruvimbo Maboreke에 우리의 원고 교정을 위해입니다.

<ol>
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Isotopologue 프로 파일링
FAs에 13C 유통에서 양적 측면의 상세한 분석 탄소 음식 거미줄에 분할 할당을 최첨단 기술이 필요로 한다. 현재 작업 고용 13C를 평가 하기 위해 프로 파일링 isotopologue / 트로픽 상호 작용에 대 한 일반적인 FA biomarkers에12C 비율. 이 방법은 액체 크로마토그래피 (LC-MS)에 의해 아미노산 분석을 위해 잘 설립 이며, 병원 성 ...

토양 등 암호 서식 지, 영양 관계 주소로 어려운 고 추가 동물군의 작은 크기에 의해 제한 됩니다. 지난 10 년간 필드 조건1,2,3토양 동물의 먹이 전략을 정의 하기 위한 생체로 지방산의 사용에 특히 생화학 생태학, 발전을 보이고 있다. 이것은 리소스에서 지방산은 전체 분자 소비자 조직에 통합 될 수 있다는 사실에 기반, 프로세스 식이 라우팅4되 나. 지방산의 양도 3 영양 단계, 즉, 곰 팡이에서 Collembola5선 충을 통해 보고 되었습니다. 최근, 육 식 동물6,7 여겨졌다 고 토양 음식 웹에서 영양 마커로 지방산에 대 한 첫 리뷰 게시8,9되었습니다.
영양 상호 작용에 대 한 더 자세한 정보는 지방산 안정 동위 원소 (FA-SIP) 조사에 의해 달성 했다. 13C의 결정 /12C 비율 지방산 다이어트와 소비자에에서 돌리다 이진 링크를 관련된 탄소 흐름을 예측 하 고 지상파에 고용 되어 신선한 물, 그리고 해양 식품 거미줄10,11 ,,1213. 기본 가정은 식이 라우트된 지방산 효소 프로세스; 적용 되지 않습니다. 따라서, 그들의 13C 신호, 즉, 13C /12C 소비자에서 지방산의 비율은 다이어트1에 비슷합니다. 그러나, 먹이 사슬의 13C 서명의 점진적인 고갈 영양 연구14,,1516에 FA SIP의 광범위 한 응용 프로그램을 방해 함으로써 수 중 시스템에서 보고 되었습니다. 또한, 지상파 식품 웹에서 대부분 무척 추 동물의 지질 대사에 대 한 지식을 여전히 제한 됩니다.
소비자에서 지질 물질 대사 경로의 이해 영양 마커 지방산 식품 웹 생태에 양적 탄소 흐름의 결정에 대 한 의미의 사용을 위해 필수적 이다. 이 고려해, 13C-isotopologue 프로 파일링, 유망한 방법은 어떤 생물 학적 시스템17, 탄소 물질 대사의 조사에 대 한 어떤에 원리를 적용할 수 있습니다. 13탄소 C 표시 기판, 13C에서 대사 네트워크의 배포의 도입에 따라 하는 것은 소비자 쇼 특정 isotopologue 배포에서에서 생성 된 대사 제품부터 추적. 이 양적 핵 대사 공명 분광학18,19 또는 질량 분석20,21그것의 더 높은 때문에 낮은 바이오 매스와 후자에 선호 생물 학적 샘플에 의해 평가 될 수 있다 감도입니다.
Isotopologue 프로 파일링 성공적으로 아미노산에 적용 되었으며 vivo에서 탄소 물질 대사 세균성 병원 체17,,2223, 지방산에서 구현에 대 한 통찰력을 제공 하지만 산 뒤에 느껴 지 있다. 안정 동위 원소 표시 전조 지방산, 식이 라우팅 또는 β-산화, 토양 무척 추 동물 소비자에 통해 저하의 운명에 첫번째 상세한 분석 최근 수행한 Menzel 외. 24. 여기, 13C 지방산 이어서 자주 토양 무척 추 동물, Collembola, 키 하위의 isotopologue 분석 분류 관 실험 방법론 기본 제공 됩니다. 이 microarthropods는 좋은 모델 그들은 중요 한 구성 요소는 잘 그들의 영양 마커 지방산8,25에 대 한 조사를 하 고 토양 먹이 그물의 형성.
소비자에서 지질 물질 대사 경로의 이해 영양 마커 지방산 식품 웹 생태에 양적 탄소 흐름의 결정에 대 한 의미의 사용을 위해 필수적 이다. 현재 프로토콜 먹이 실험, 그리고 추출 및 지배적인 지질 분수 (중립 지질, 인지질)의 메 틸 화에 대 한 생 화 확 적인 절차 Collembola에서 실험실을 위한 설계 및 설정 제공 합니다. 그것은 isotopologue 구성 지방산의 질량 분석에 의해 분석 되는 방법을 보여 줍니다 고 관련된 수식 및 계산에 설명 합니다. 이 절차에서 결과: isotopologues 초과 하나 이상 (즉, 지방산 분자 이온 M+) 부모 이온의 질량의 비율 (i) 질량 단위 (M+ 1, M2, M+ 3, 등) 및 (ii) 전체 13 C 농축 지방산에서 13C 이라는 전조에서 파생. 이 방법은 일반적으로 이들은 성공적인 레이블 통풍 관을 위해 제어 된 조건 하에서 충분 한 수량에 culturable 및 후속 전제에 다른 어떤 약탈자 먹이 상호 작용에 적용할 수 있습니다 Collembola 사용, 비록 확인입니다.

<table><tbody><tr><td>neoLab-Round jars</td><td>neoLab</td><td>2-1506</td><td>69 x 40 mm, 10 pacs/pack</td></tr><tr><td>Charcoal activated</td><td>Carl Roth</td><td>X865.1</td><td>p.a., powder, CAS No. 7440-44-0</td></tr><tr><td>Alabaster Dental</td><td>R&amp;Ouml;HRICH-GIPSE</td><td>---</td><td>http://www.roehrich-gipse.de/dentalgipse.php</td></tr><tr><td>Chloroform</td><td>Carl Roth</td><td>7331.1</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>P717.1</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>Hexan</td><td>Carl Roth</td><td>7339.1</td><td>HPLC &amp;ge; 98 %</td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butyl methyl ether (MTBE)</td><td>Carl Roth</td><td>T175.1</td><td>HPLC &amp;ge; 99,5 %</td></tr><tr><td>Aceton</td><td>Carl Roth</td><td>7328.2</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>NaOH</td><td>Carl Roth</td><td>6771.1</td><td>p.a. &amp;ge;99 %, in pellets</td></tr><tr><td>di-Natriumhydrogenphosphat</td><td>Carl Roth</td><td>P030.1</td><td>p.a. &amp;ge;99 % , water free</td></tr><tr><td>Na-dihydrogenphosphat Dihydrat</td><td>Carl Roth</td><td>T879.1</td><td>p.a. &amp;ge;99 %</td></tr><tr><td>Hypochloric acid (6 N)</td><td>VWR International</td><td>26,115,000</td><td>AVS TITRINORM vol. solution</td></tr><tr><td>Bond Elut (Columns)</td><td>Agilent Tech.</td><td>14102037</td><td>HF Bond Elut-SI, 500 mg, 3 mL, 50/PK</td></tr><tr><td>Pr&amp;auml;paratengl&amp;auml;ser Duran</td><td>Glasger&amp;auml;tebau Ochs</td><td>135215</td><td>&amp;Oslash; 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket</td></tr><tr><td>Supelco 37 Component FAME Mix</td><td>Sigma-Aldrich</td><td>47885-U Supelco</td><td>10&amp;nbsp;mg/mL in methylene chloride, analytical standard</td></tr><tr><td>FlowMesh</td><td>Carl Roth</td><td>2796.1</td><td>Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing</td></tr><tr><td>Bacterial Acid Methyl Ester (BAME) Mix</td><td>Sigma-Aldrich</td><td>47080-U Supelco</td><td>10&amp;nbsp;mg/mL in methyl caproate, analytical standard</td></tr><tr><td>Methyl nonadecanoate</td><td>Sigma-Aldrich</td><td>74208</td><td>analytical standard &amp;ge; 98.0 %</td></tr><tr><td>Hexadecanoic acid-1-13C (Palmitic)</td><td>Larodan Fine Chemicals</td><td>78-1600</td><td>GC &amp;ge; 98.0 % (&lt;sup&gt;13&lt;/sup&gt;C: 99.0 %)</td></tr><tr><td>RVC 2-25 CDplus</td><td>Martin Christ Gefrier-trocknungsanlagen</td><td></td><td>Compact benchtop midi concentrator</td></tr><tr><td>Alpha 2-4 LDplus</td><td>Martin Christ Gefrier-trocknungsanlagen</td><td></td><td>Drying manifold</td></tr><tr><td>MZ 2C NT</td><td>Vacuubrand GMBH</td><td></td><td>Vacuum pump</td></tr><tr><td>Roto-Shake Genie</td><td>Scientific Industries</td><td></td><td>Combined rocking and rotating device</td></tr><tr><td>XP64 Micro Comparator</td><td>Mettler Toledo</td><td></td><td>Super high precision balance</td></tr><tr><td>GC-System 7890A</td><td>Agilent Tech.</td><td></td><td>Gas chromatograph</td></tr><tr><td>7000 GC/MS Triple Quad</td><td>Agilent Tech.</td><td></td><td>Triple Quad mass spectrometer</td></tr><tr><td>7683B Series Injector</td><td>Agilent Tech.</td><td></td><td>Sample injector</td></tr><tr><td>Heraeus Multifuge 3SR&lt;sup&gt;+&lt;/sup&gt;</td><td>Thermo Scientific</td><td></td><td>Centrifuge with 10 ml tube rotor</td></tr></tbody></table>

fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

지방산 (FAs) 식품 웹 생태에 유용한 biomarkers는 그들은 일반적으로 완전 한 분자로 동화 하 고 미성년자 또는 다른 영양 수준 사이 식이 라우팅 수 있도록 수정, 소비자 조직으로 전송 때문에. 그러나, FA 영양 마커 접근 토양 동물의 지질 대사에 한정 된 지식에 의해 아직도 방해 된다. 이 연구는 지방산 물질 대사 경로 두 개의 광범위 한 토양 Collembola, Protaphorura fimata , Heteromurus nitidus에 추적 프로그램으로 완전히 이라는 팔 미트 산 (13C16:0, 99 원자 %)를 사용합니다. 운명과이 전조의 대사 수정, 조사 하기 위하여 isotopologue 프로 파일링 하는 방법 제시, 단일 이온 모니터링을 사용 하는 질량 분석에 의해 수행. 또한, 업스트림 실험실 실험 먹이 추출 및 지배적인 지질 분수 (중립 지질, 인지질) 및 관련된 수식 계산의 메 틸 화 설명 되어 있습니다. Isotopologue 프로 파일링 하지만 수익률 13C 전조를 분류에서 파생 된 지방산에서 전체 13C 농축 않지만 isotopologues 부모 이온 (즉, FA 분자 이온의 질량 초과의 패턴 생성 M+) 각각의 표시 된 하나 이상의 질량 단위 (M+ 1, M2, M+ 3, 등)에 의해 FA. 이 지식을 de novo 생 합성에 비해 완전히 소비 FA의 식이 라우팅의 비율에 결론을 수 있습니다. Isotopologue 프로 파일링은 풀 영양 상호 작용을 토양 동물에서 지방산 대사의 평가 위해 유용한 도구로 서 좋습니다.

Collembola의 신선한 무게와 지질 콘텐츠 설명된 실험 과정에서 NLFAs 및 PLFAs의 내용을 변화 하지 않았다 크게 시간이 지남에, 표본의 신선한 무게 하지 크게24하지만 약간 증가 하는 반면. 두 매개 변수 Collembola 표본의 체력의 좋은 수준을 나타냅니다. 지방산 및 동위 원소 분석을 위한 샘플링 일에 해당 하는 실험을 통해 Collembola의 신선한 무게...

Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

영양 탄소 전송 및 무척 추 동물 소비자의 지질 대사에 대 한 통찰력을 제공 합니다 지방산 13C Isotopologue 프로 파일링

지방산 영양 마커 접근, 즉, 전체 분자와 소비자 조직으로 전송으로 지방산의 동화 없이 또는 사소한 수정, 작은 토양 무척 추 동물의 지방 산 성 대사에 지식 격차에 의해 방해 된다. Isotopologue 프로 파일링 풀 영양 상호 작용 하는 귀중 한 도구로 제공 됩니다.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

fatty-acid-13c-isotopologue-profiling-provides-insight-into-trophic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

7.9K 조회수.  Humboldt-Universität zu Berlin. 지방산 영양 마커 접근, 즉, 전체 분자와 소비자 조직으로 전송으로 지방산의 동화 없이 또는 사소한 수정, 작은 토양 무척 추 동물의 지방 산 성 대사에 지식 격차에 의해 방해 된다. Isotopologue 프로 파일링 풀 영양 상호 작용 하는 귀중 한 도구로 제공 됩니다.

비디오: Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

제조 박층 크로마토 그래피 및 MALDI-TOF 질량 분석에 의해 박쥐 외피 지질의 Triacylglyceride 내용을 프로파일 링

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

연구

화학

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

추출 및 토양의 미생물 인지질 지방산의 분석

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

환경과학

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

생선 기름 보충제의 지질 프로필의 신속한 평가를위한 강력한 도구로 NMR 분광학

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Determination of Total Lipid and Lipid Classes in Marine Samples

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined &#39;dissolved&#39; and &#39;particulate&#39; factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

해양 샘플의 총 지질 및 지질 수업 결정

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

개발하고 성인 단계에서 중개 신진 대사 플럭스 안정 원소 프로파일 Caenorhabditis 엘레간스</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

생물학

안정 동위원소 라벨링을 사용한 지질 구조 스크리닝

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

저자 스포트라이트: Quantification of Complex Lipidomic Samples Using Stable Isotope Labeling (안정 동위원소 라벨링을 사용한 복잡한 지질도미 시료의 정량화)

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

을 통해 대사 경로 확인 및 검색 13</supProteinogenic 아미노산> C - 라벨링

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

영양 탄소 전송 및 무척 추 동물 소비자의 지질 대사에 대 한 통찰력을 제공 합니다 지방산 ¹³C Isotopologue 프로 파일링

요약

더 많은 비디오 탐색

이 비디오의 챕터

제조 박층 크로마토 그래피 및 MALDI-TOF 질량 분석에 의해 박쥐 외피 지질의 Triacylglyceride 내용을 프로파일 링

추출 및 토양의 미생물 인지질 지방산의 분석

생선 기름 보충제의 지질 프로필의 신속한 평가를위한 강력한 도구로 NMR 분광학

해양 샘플의 총 지질 및 지질 수업 결정

개발하고 성인 단계에서 중개 신진 대사 플럭스 안정 원소 프로파일 Caenorhabditis 엘레간스

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

을 통해 대사 경로 확인 및 검색^{13 C - 라벨링}

에서 지방 질량의 생화학 및 높은 처리량 미세 평가

영양 탄소 전송 및 무척 추 동물 소비자의 지질 대사에 대 한 통찰력을 제공 합니다 지방산 13C Isotopologue 프로 파일링

요약

더 많은 비디오 탐색

이 비디오의 챕터

제조 박층 크로마토 그래피 및 MALDI-TOF 질량 분석에 의해 박쥐 외피 지질의 Triacylglyceride 내용을 프로파일 링

추출 및 토양의 미생물 인지질 지방산의 분석

생선 기름 보충제의 지질 프로필의 신속한 평가를위한 강력한 도구로 NMR 분광학

해양 샘플의 총 지질 및 지질 수업 결정

개발하고 성인 단계에서 중개 신진 대사 플럭스 안정 원소 프로파일 Caenorhabditis 엘레간스

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

안정 동위원소 라벨링 LC-TIMS-TOF MS/MS를 사용한 새로운 지질 추적

을 통해 대사 경로 확인 및 검색 13 C - 라벨링

에서 지방 질량의 생화학 및 높은 처리량 미세 평가

영양 탄소 전송 및 무척 추 동물 소비자의 지질 대사에 대 한 통찰력을 제공 합니다 지방산 ¹³C Isotopologue 프로 파일링

을 통해 대사 경로 확인 및 검색^{13 C - 라벨링}