Name: identification of fatty acids in bacillus cereus
Uploaded: 2016-12-05T15:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

저자는 자신의 기술 지원 토마스 Mison에게 감사하고, 원고를 수정하기위한 레이첼 한전합니다.

1. 세균성 문화 <ol><li> LB 한천 배지의 플레이트의 표면에, LB (루리아 - 베르 타니 배지)에 30 ° C에서 배양 한 균주의 하룻밤 배양 100 μl를 분산시켜 박테리아 (바실러스 세레 우스 균주 ATCC 14579)의 잔디를 준비한다. 30 ℃에서 하룻밤 접시를 품어. </li></ol> 2. ECL : 등가 체인 길이 <ol><li> 다음과 같이 ECL을 계산합니다 : <img alt="방정식" src="/files/ftp_upload/54960/54960eq1.jpg" /> 와: 내가 관심있는 용질; N, 용질 전에 용출 직쇄 포화 지방산 메틸 에스테르의 탄소수 I; N + 1, 용질 후 용출 직쇄 포화 지방산 메틸 에스테르의 탄소수 I; RTI, RTN,있는 걸 (N + 1) 상기 FAME 피크의 유지 시간. 주 : (표준 혼합 주사 B가 직쇄 포화 지방산 메틸 에스테르의 체류 시간을 구하는AME). </li></ol> 3. FAME 준비 및 분석 <ol><li> 한천 플레이트에서 콜로니를 긁어 지질 지방산, 수확물 균체를 얻고 스크류 캡과 PTFE 씰 10 mL 유리 튜브에 박테리아 (109 생세포 등가 신선 중량) 40 mg을 전송하기 위해. </li><li> 아래에 설명 에스테르 링크 방법 14, 15을 통해 에스테르 교환 반응을 수행합니다. <ol><li> 신선한 박테리아 세포에 메탄올 0.2 M KOH의 5 ML을 추가하고 1 시간 동안 37 ° C에서 품어. 이 반응은 지질에 에스테르 연결을 끊고 지방산 메틸 에스테르를 생산하고, 알칼리 메탄 구성된다. </li><li> 7.0 pH를 낮추기 위해 1 M 아세트산 1 ㎖를 추가한다. 산도 테스트 스트립 pH를 확인합니다. </li><li> FAMES를 추출 헥산 3 ㎖를 추가합니다. </li><li> 약 200 μL를 얻는 질소의 연속 흐름하에 실온에서 증발시켜 깨끗한 튜브에 상청 (유기상)을 전송하고 집중추출물. 삽입과 GC 바이알에 샘플을 전송합니다. </li><li> 가스 크로마토 그래피 질량 분석 (GC-MS)로 추출 시스템을 주입한다. </li></ol></li></ol> 4. GC / MS 조건 <ol><li> 모세관 컬럼 ZB-WAX (직경 0.25 mm, 필름 두께 0.25 ㎛의 길이, 30m)를 구비 한 GC-MS 기기 FAME에 샘플을 주입. </li><li> (비분 모드) 주입구 250 ° C까지 온도를 설정합니다. 37cm / sec의 속도로 선형, 캐리어 가스로서 헬륨을 사용한다. 20 ° C / 분의 속도로 190 ℃로 증가시키고, 2 ℃ / 분의 속도로 230 ℃의 최종 온도로 점진적 증가, 1 분 동안 50 ° C의 오븐 온도를 잡아. </li><li> 상기 MS를 들어 70 eV로에서 전자 이온화 (EI)에 의하여 질량 스펙트럼을 기록하고, 50 내지 400 원자 질량 단위 (AMU) (2 스캔 / 초) 사이에 총 이온 전류의 인수를 설정한다. </li><li> 필요한 경우, 동일한 조건 excep에서 DMOX 및 picolinyl 파생 상품을 주입다음과 같이 온도 프로그램 오븐 T : DMOX : 50 ° C (1 분), 20 ° C 210 ° C까지 / 분, 2 ° C / 분 240 ° C (5 분)까지; Picolinyl : 6 ° C (1 분), 220 ° C까지 20 ° C / 분, 250 ° C (20 분)까지 2 ° C / 분. </li></ol> FAME (16) 5. Picolinyl 에스테르 준비 <ol><li> 질소의 흐름 (적어도 10 mg의 건조 물질)와 함께 제 3 항 중 FAME 추출액을 증발 건조 디클로로 메탄 1 ㎖에 용해. </li><li> 테트라 히드로 푸란 칼륨 터 t- 부톡 사이드의 1.0 M 용액을 준비합니다. </li><li> 명성 추출물 단계 5.2에서 만든 용액 0.2 ml의 3 피리딘 메탄올 0.1 ML을 추가합니다. </li><li> 밀폐 된 바이알에서 30 분 동안 40 ° C에서 상기 용액을 가열한다. </li><li> 실온으로 냉각시킨 후, (재료 표 참조, 2 ㎖)와 헥산 (4 mL) 중 정제 된 탈 이온수를 추가한다. 와류 혼합 상을 분리 할 수 ​​있도록하고, 유기상을 수집한다. </li><li> 박사유기상까지 무수 황산나트륨을 첨가하여 Y 완벽하게 알 수있다. 깨끗한 튜브로 전송합니다. 그런 다음 200 μl를 증발. 삽입과 GC 바이알에 샘플을 전송합니다. </li></ol> FAME (17) 6. DMOX 준비 <ol><li> 질소 (최소 10 mg의 건조 물질)의 흐름을 제 3의 FAME 추출물을 증발. </li><li> 명성 건조 추출물에 2- 아미노 -2- 메틸 -1- 프로판올 250 ㎎을 추가. 질소 용기를 세척하는 스토퍼를 추가하고, 190 ℃에서 하룻밤 가열 블록에 넣습니다. </li><li> 실온으로 냉각 한 후, 튜브를 3 ml의 디클로로 메탄을 추가하고, 5 ml의 탈 이온수 (재료 표 참조) 정제 하였다. </li><li> 상 분리 흔들어 후, 수성 상을 제거합니다. </li><li> 5 ㎖의 물, 유기 상을 세척한다. 상 분리 흔들어 후, 수성 상을 제거합니다. </li><li> 유기상까지 무수 황산나트륨을 첨가하여 건조 완벽하고 명확한깨끗한 튜브로 전송할 수 있습니다. 200 μL의 부피에 도달 할 때까지 질소 기류 하에서 증발시켰다. 삽입과 GC 바이알에 샘플을 전송합니다. </li></ol>

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The authors declare that they have no competing financial interests.

표 1에 나타낸 FA를 크로마토 프로필 한천 플레이트 표면에 성장 B. 세레 우스 ATCC 14579에 대응한다. 세균을 동일 온도에서 8 통기 액체 배지에서 성장했을 때 유사한 프로파일을 얻었다. 액체 배지에서 성장 세균의 경우 세균 미생물이 성장 배지를 원심 분리에 의해 회수하고, 성장 조건에 따라 8,19 전술 한 프로토콜에 따라 세척 될 수있다. B. 세레 우스에 ?...

지방산 메틸 에스테르 (FAME) 기체 크로마토 그래피 (GC)의 지질 특성에 필수적인 메소드이다. 이는 신속 분리 짧은 추출 공정 후의 시료의 각종 지방산 (FA들)을 정량화한다. 메틸 에스테르의 유도체는 크로마토 그래피 컬럼을 향해 매우 휘발성 안정하고 불활성시켜 미행 피크를 회피. 크로마토 그래피 프로파일 중 하나를 게시 또는 기준에 비교하기 때문에 샘플이 잘 알려진 FA를 구성 할 때 그들의 인식은 오히려 간단합니다. 또한, 각종의 FA 정량 검정 표준의 반복 분사 불꽃 이온화 검출 (FID) 한 그들의 거의 일정한 응답 주어진 요구되지 않는다. FID에 더하여, 질량 분광 (MS) 검출 FAMES를 확인하는 내용의 상보 세트를 제공한다. FAMES가 전자 이온화 (EI)를 이용하여 충전하는 경우에는, 결과적인 스펙트럼은 항상 일을 허용하지FA 미세 구조의 전자 식별. 진단 이온 하나를 감지하기 어렵고 대상 이온 풍부 특성 변화가 질량 스펙트럼 라이브러리 (2)의 사용을 방지하는 시스템에 의존하기 때문에 예를 들어, 위치를 분기 (즉, 측쇄 메틸기)을 예측하는 것은 곤란하다. 또 다른 문제는 EI는 이중 결합의 이동을 야기하기 때문에 이중 결합의 위치를 ​​식별에있다. 따라서, 이중 결합의 위치를 ​​변화와 FA 이성질체들은 질량 스펙트럼에 의해 구별 될 수 없다. 다행히도 다른 도구 FA 식별을 위해 개발되었다. 예를 들어, 존재 및 준하여 이중 결합 또는 분지의 위치는 해당 체인 길이 (ECL) (3)을 계산함으로써 추측 할 수있다. 기타 유도체 화 방법은 이중 결합 또는 측쇄 메틸기의 위치에 따라 서로 다른 질량 스펙트럼 결과. 4,4- 디메틸 옥사 졸린 유도체 (DMOX) 4 EAS 허용불포화 지방산의 이중 결합의 위치의 Y 식별. 에스터 (picolinyl 에스테르) 메틸 유도체의 위치의 명확한 식별이 가능 -3- pyridylcarbinyl는 FA를 5 분지. 핵 자기 공명 (NMR) 스펙트럼 구조 특성 1의 uncontestable 방법에 필요한 정제의 복잡한 방법을 사용하지 않고도 크로마토 유지율 (ECL) 질량 스펙트럼 (DMOX 및 picolinyl) 정보는 대부분의 FA의 식별을 가능하게 결합 . 일부 인간과 동물의 병원체를 포함 바실러스의 박테리아는 매우 다양한 틈새 시장에 정착 할 수 있으므로 널리 환경 (6)에 배포됩니다. 바실러스 속 중에서, FA 조성물 (환경 변화의 넓은 범위에 적응 FA 패턴의 변조와 종의 생태적 틈새에 의해 영향을 받는다 예를 들어, 성장 배지, 온도,산도 등) 7-9. 이 때문에 표준 조건에서 성장 동안 바실러스 종의 FA에 걸쳐 패턴의 상대적 균질성에, FA 조성물의 결정은 바실러스 종을 정의하는 데 사용되는 중요한 조건 중 하나이다. 바실러스 속의 독특한 특성은 12-17 탄소 ISO 및 환경 조건에 대한 적응의 주요 결정 인 안테 이소 이성체의 비율로 10-12 분지 쇄의 FA 풍부하다. 바실러스 종은 불포화 지방산의 비율을 변경함으로써, 환경의 변동에 적응. 이러한 바실러스 세레 우스와 같은 일부 종에서는,이 지방산 desaturases 적응 9 다른 역할로 알킬 체인 (13)의 다른 위치에 이중 결합을 만들 수 있습니다. 바실러스 속의 예 정확하게 이중 결합의 위치를 식별하고 FA 분지의 중요성을 나타낸다. 수집ively, 바실러스 FA 패턴의 식별은 몇 가지 유용한 응용 프로그램이 있습니다. 여기서, 우리는 고전 GC-MS 분석의 고유 한 한계를 극복 바실러스 FA 패턴을 식별하기위한 신규 GC-MS 방법을 제안한다. 이 혁신적인 접근 방식은 원시 생물학적 물질에 직접 사용하고, 기존 기술의 조합으로 구성 할 수 있습니다 체류 시간 (ECL) 및 다른 FA들 유도체 (FAME, DMOX 및 picolinyl 에스테르)의 질량 스펙트럼에 대한 정보를 제공합니다. 우리는 다음과 FA 명칭을 사용합니다. a 및 n은 ISO를 나타낸다 난, 안테 이소 메틸 각각 분지 및 직쇄 지방산. 불포화의 FA는 C로 명명 하였다 : C는 탄소 지방산 원자 및 (D)의 수이고, D는 이중 결합의 수이다. Δ의 X는 이중 결합이 카르 복실 말단으로부터 계수, x 번째의 탄소 - 탄소 결합에있는 이중 결합의 위치를 나타낸다.

<table><tbody><tr><td>GC/MS</td><td>Shimadzu</td><td>QP2010</td><td></td></tr><tr><td>capillary column ZB WAX</td><td>Phenomenex</td><td>7HG-G007-11</td><td>30 m x 0.25 mm x 0.25&amp;nbsp;&amp;micro;m</td></tr><tr><td>Methanol Lichrosolv</td><td>VWR</td><td>1.06018.2500</td><td></td></tr><tr><td>potassium hydroxide</td><td>Aldrich</td><td>P1767</td><td></td></tr><tr><td>THF</td><td>Hipersolv Chromanorm</td><td>28559.320</td><td></td></tr><tr><td>Dichloromethane</td><td>Hipersolv Chromanorm</td><td>23373.320</td><td></td></tr><tr><td>Hexane</td><td>Hipersolv Chromanorm</td><td>24575.320</td><td></td></tr><tr><td>3-pyridinemethanol</td><td>Aldrich</td><td>P6-680-7</td><td></td></tr><tr><td>potassium tertiobutoxide</td><td>Aldrich</td><td>156671</td><td></td></tr><tr><td>2-amino-2-methyl-1-propanol</td><td></td><td>A-9879</td><td></td></tr><tr><td>MilliQ Academic</td><td>Millipore</td><td>ZMQS50001</td><td></td></tr><tr><td>Bacterial Acid Methyl Ester (BAME) Mix</td><td>Sigma-Aldrich</td><td>47080-U Supelco</td><td></td></tr></tbody></table>

identification of fatty acids in bacillus cereus

바실러스 종 메틸 분기의 다양한 위치 체인 및 불포화 지방산 (FA들)을 포함 분지 (ISO 또는 안테 이소) 및 이중 결합. FA 조성물의 변화는 환경에 박테리아의 적응에 중요한 역할을한다. 이러한 수정은 안테 이소 FA들에 비해 분지 ISO의 비율의 변화를 수반하고, 특정 위치에 생성 된 이중 결합을 갖는 불포화 포화 FA를 FA에 대하여, 비율이다. 각 FA 프로파일의 정확한 식별은 바실러스 종의 적응 메카니즘을 이해하는 것이 필요하다. 바실러스에서의 FA의 대부분은 상업적으로 사용할 수 없습니다. 지방산 메틸 에스테르 (FAMES), 4,4- 디메틸 옥사 졸린 : 본 명세서에서 제안 된 전략은 세 FA 유도체의 종류의 질량 스펙트럼과 (등가 체인 길이 (ECL)의 연산에 의해) 보유 시간에 대한 정보를 조합하여 FA를 식별 파생 상품 (DMOX), 및3 pyridylcarbinyl 에스터 (picolinyl). 이 방법은 미지의 FA를 정제 할 필요없이 FA를 식별 할 수있다. 표준 상용 혼합물 바실러스 세레 우스로부터 제조 FAME의 크로마토 그래피 프로파일을 비교하는 다른 FA들 중 신원의 FA의 ECL의 계산 및 가설 포화 직쇄의 식별을 허용한다. FAMES의 탄소수가 동일한 수의 선형 포화 FA들에 비해 FA들, 또는 ISO 안테 이소에서, ECL에 일정한 마이너스 시프트를 표시 포화 분지. 불포화의 FA FAMES는 분자 이온의 질량을 감지하고 대응하는 포화 FA들에 비해 ECL의 양의 변화가 발생할 수있다. FA들의 분기 위치의 FA 불포화 이중 결합의 위치는 각각 picolinyl 및 DMOX 유도체의 전자 이온화 질량 스펙트럼에 의해 확인 될 수있다. 이 접근법은 모든 미지의 포화 지점을 식별ED의 FA 불포화 직쇄 FA들 및 B. 세레 우스 추출물의 불포화 측쇄의 FA.

박테리아 세포에서 FA 식별의 전략은 그림 1에 제시되어있다. 각 단계는 상호 보완적인 스펙트럼 정보 또는 크로마토 그래피 보존에 대한 정보를 제공합니다. 1 단계는 표준 용액을 사용하여 예비 FA 식별 구성된다. 단계 2 임시 제품을 식별하기 위해, FAME EI 스펙트럼 및 ECL의 해석을 허용한다. 3 단계는 분지 쇄-의 FA의 정확한 분기 위치를 식별합니다. 마지막으?...

Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

지방산의 확인 시험<em&gt; 바실러스 세레 우스</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

identification-of-fatty-acids-in-bacillus-cereus

Universidad de Cartagena UDEC

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JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

9.5K 조회수.  INRA, Université d'Avignon. We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl). 

비디오: Identification of Fatty Acids in Bacillus cereus

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

미세 조류의 지방산 콘텐츠 및 조성 분석

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

연구

환경과학

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

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

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

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

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

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

생화학

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

세균성 병원체 지방산 을 막 인지질로 통합하는 연구를 위해 닭 계란 노른자에서 지단백질 입자의 분리

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

클릭 화학을 이용한 지방 아실화 단백질의 검출을 위한 알키닐 지방산 유사체의 최적화된 혼입

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

대사 라벨링 및 얇은 층 크로마토그래피에 의한 사카로미세스 세레시아의 중립 지질 합성 분석

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

MALDI-TOF-TOF-MS/MS 및 하향식 프로테오믹 분석을 사용하여 대장균에 있는 항균 면역 단백질의 식별

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

화학

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

담 즙 소금 유도 Biofilm 형성 장 병원 체에서: 식별 및 정량화 기술

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

면역학 및 감염병학

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

얇은 층 크로마토그래피에 의한 진균의 지질 조성 분석

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

<meta charset="utf8"></head><body>Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Baces of Healthy Pigs(건강한 돼지의 신선한 배설물에서 바시트라신 생산 Bacillus lic

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Baces of Healthy Pigs(건강한 돼지의 신선한 배설물에서 바시트라신 생산 Bacillus licheniformis 의 분리, 정제 및 식별)

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

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