Name: identification of fatty acids in bacillus cereus
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著者は原稿の改訂のための彼の技術サポートのために、とレイチェルKopecにトーマスMisonに感謝しています。

1.細菌培養<ol><li> LB寒天培地のプレートの表面上に、LB（ルリア-ベルターニ培地）中で30℃でインキュベート株の一晩培養物100μlを広げることによって菌（ バチルス・セレウス菌株 ATCC 14579）の芝生を準備します。 30℃で一晩プレートをインキュベートします。 </li></ol> 2. ECL：等価チェーンの長さ<ol><li>次のようにECLを計算します。 <img alt="方程式" src="/files/ftp_upload/54960/54960eq1.jpg" />と： 私、興味のある溶質; nは、溶質の前に溶出し、直鎖飽和脂肪酸メチルエステルの炭素数I。 N + 1、溶質の後に溶出する直鎖飽和脂肪酸メチルエステルの炭素数I。 RTI、RTN、RT（N + 1）上述のFAMEピークの保持時間。 注：（標準の混合物を注入することにより直鎖飽和脂肪酸メチルエステルの保持時間を得るBAME）。 </li></ol> 3. FAMEの調製および分析<ol><li>脂質の脂肪酸を得るために、寒天プレートからコロニーを掻き取りにより採取細菌細胞とスクリューキャップとPTFEシールを有する10mlのガラス管に細菌を40mg（10 9の生存細胞に相当する新鮮重量）移します。 </li><li>以下に詳述エステル結合方法14,15を経由してエステル交換反応を行います。 <ol><li>新鮮な細菌細胞にメタノール中0.2M KOHの5ミリリットルを加え、37℃で1時間インキュベートします。この反応は、脂質中のエステル結合を破壊し、脂肪酸メチルエステルを生成する、アルカリ性メタノリシスから成ります。 </li><li> pHを7.0に低下させるために1 M酢酸を1ml加えます。 pH試験片を用いてpHを確認してください。 </li><li> FAMEを抽出するためにヘキサンの3ミリリットルを追加します。 </li><li>約200μLを得るために、きれいなチューブに上清（有機相）を移し、窒素の連続気流下、室温で蒸発させることによって集中します抽出物。インサート付きGCバイアルにサンプルを転送します。 </li><li>ガスクロマトグラフィー - 質量分析（GC-MS）システムに抽出物を注入します。 </li></ol></li></ol> 4. GC / MS条件<ol><li>キャピラリカラムZB-WAX（;直径0.25mmで、膜厚0.25μmの長さ30メートル）を備えたGC-MS機器にFAMEサンプルを注入します。 </li><li> （スプリットレスモードでの）注入口250°Cまで温度を設定します。 37センチメートル/秒の線速度で、キャリアガスとしてヘリウムを使用してください。 20℃/分の速度で190℃まで上昇し、2℃/分の速度で230℃の最終温度までさらに増加、1分間50℃のオーブン温度を保持します。 </li><li> MSについては、70 eVでの電子イオン化（EI）の質量スペクトルを記録し、50〜400原子質量単位（AMU）（2スキャン/秒）の間の総イオン電流の取得を設定します。 </li><li>必要な場合には、同じ条件EXCEP下DMOXとピコリニル誘導体を注入次のように温度プログラムオーブンT： DMOX：50℃（1分）、20℃/分で210℃から2℃/分まで、240°C（5分）まで。 ピコリニル：6°C（1分）、220℃まで20℃/分で250℃（20分）まで2℃/分。 </li></ol> FAME 16から5ピコリニルエステル準備<ol><li>窒素の流れ（少なくとも10mgの乾燥物質）と第3のFAME抽出物を蒸発させ、乾燥ジクロロメタン1ml中に溶解します。 </li><li>テトラヒドロフラン中のカリウムターt-ブトキシドの1.0M溶液を準備します。 </li><li>ステップ5.2で作られた溶液0.1 mlにFAMEエキス、0.2ミリリットル3ピリジンメタノールを追加します。 </li><li>密閉バイアル中で30分間、40°Cで溶液を加熱します。 </li><li>室温に冷却した後、精製された脱イオン水（2mL、 材料表を参照）、ヘキサン（4 ml）を加えます。相を分離させ、ボルテックスで混合し、有機相を収集します。 </li><li>博士Yその有機相が完全に透明になるまで無水硫酸ナトリウムを添加することによって。きれいなチューブにそれを転送します。その後、200μlに蒸発させます。インサート付きGCバイアルにサンプルを転送します。 </li></ol> FAME 17から6 DMOX準備<ol><li>窒素（少なくとも10mgの乾燥材料）の流れと第3のFAME抽出物を蒸発させます。 </li><li> FAME乾燥抽出物を、2-アミノ-2-メチル-1-プロパノールの250ミリグラムを加えます。 、窒素で容器をフラッシュストッパーを追加し、190℃で一晩加熱ブロックに配置します。 </li><li>室温まで冷却した後、精製された脱イオン水を試験管に3ミリリットルのジクロロメタンを追加し、5ミリリットル（ 材料表を参照してください）。 </li><li>相分離のためにシェイクした後、水相を除去します。 </li><li> 5ミリリットルの水で有機相を洗浄します。相分離のためにシェイクした後、水相を除去します。 </li><li>有機相が完全に明確であるとまでは無水硫酸ナトリウムを添加することによって、乾燥きれいなチューブにそれを転送します。 200μlの容量に達するまで、窒素気流下で蒸発させます。インサート付きGCバイアルにサンプルを転送します。 </li></ol>

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The authors declare that they have no competing financial interests.

表1に示したのFAクロマトグラムプロファイルは、寒天プレートの表面上に成長させたB.セレウス ATCC 14579に対応します。細菌が同じ温度8で通気し、液体培地中で増殖させた場合にも同様のプロファイルが得られました。液体培地中で増殖させた細菌の場合には、細菌バイオマスの増殖培地の遠心分離によって回収し、成長条件8,19によって以前?...

脂肪酸メチルエステル（FAME）、ガスクロマトグラフィー（GC）は、脂質の特性評価に不可欠な方法です。それは急速に分離し、短い抽出工程後の試料の様々な脂肪酸（FAS）を定量化します。メチルエステルの誘導体は、クロマトグラフィーカラムに向かって高度に揮発性の安定かつ不活性であり、それによってテーリングピークを回避します。クロマトグラフィーのプロファイルはいずれかの公開や標準に比較されるため、サンプルは、周知のFAで構成されている場合、それらの識別はかなり簡単です。加えて、様々なのFAの定量化のための較正標準の反復注射は、炎イオン化検出器（FID）1へのほぼ一定の応答を考えると、必要とされません。 FIDに加えて、質量分析（MS）検出のFAMEを確認するために情報の補完的なセットを提供します。 FAMEは、電子イオン化（EI）を使用して充電されたときしかし、得られるスペクトルは、常に目のために許可されていませんFA微細構造の電子識別。診断イオンが1を検出することが困難であり、ターゲットイオン存在量の特性変化は、質量スペクトルライブラリー2を使用することを防止する、機械に依存するので、例えば、位置分岐（ すなわち、分岐したメチル基）を予測することは困難です。もう一つの課題は、EIは、二重結合の移動が発生するため、二重結合の位置を特定することにあります。このように、二重結合の位置を変化させたFA異性体は、その質量スペクトルによって区別することはできません。幸いなことに、他のツールは、FAの同定のために開発されています。例えば、存在およびFASにおける二重結合のまたは分岐の位置は、等価な鎖長（ECL）3を計算することによって推測することができます。 他の誘導体化方法は、二重結合または分枝鎖のメチル基の位置に依存する異なる質量スペクトルをもたらします。 4,4-ジメチルオキサゾリン誘導体（DMOX）4 EASを可能に一価不飽和脂肪酸の二重結合の位置のyの識別。 3-pyridylcarbinylエステル（ピコリニルエステル）誘導体は、Fas 5分枝鎖メチルの場所の明確な同定を可能にします。核磁気共鳴（NMR）スペクトルは、構造的特徴1争う余地のない方法の必要に応じて、精製の複雑な方法を使用することなく、クロマトグラフィーの保持（ECL）および質量スペクトル（DMOXとピコリニル）情報は、ほとんどのFAの同定を可能にする合成。 いくつかのヒトおよび動物の病原体を含むバチルス属の細菌は、非常に多様なニッチにコロニーを形成することが可能であるため、広く環境6に分布しています。 バチルス属の中でも、FA組成物は、環境の変化（ 例えば 、増殖培地、温度の広い範囲に適応するためにFAパターンの変調を有する種の生態的地位に影響されますpHは、 等 ）7-9。そのため標準化された条件で成長中のバチルス属の種間FAパターンの相対的な均質性、FA組成物の決意は、 バチルス種を定義するために使用される本質的な基準の一つです。 バチルス属のユニークな属性は、ISOおよび環境条件への適応の重要な決定要因であることアンテイソ異性体間の比で12-17個の炭素10-12を含む分岐鎖のFAの豊富さです。 バチルス種はまた、不飽和脂肪酸の比率を変えることにより、環境変動に適応します。このようなセレウス菌などのいくつかの種では、2脂肪酸デサチュラーゼは、適応9で異なる役割を持つアルキル鎖13の異なる位置に二重結合を作成します。 バチルス属の例は、正確に二重結合の位置を識別し、FAは、分岐の重要性を示しています。収集しますively、 バチルス FAパターンの同定は、いくつかの有用な用途を有します。ここで、我々は、古典的なGC-MS分析の固有の限界を克服するバチルス FAパターン識別のための新規なGC-MSの方法を提案します。 この革新的なアプローチは、生の生物学的物質に直接使用し、既存の技術の組み合わせで構成することができます。保持時間（ECL）の情報と異なるのFA誘導体（FAME、DMOXとピコリ - エステル）の質量スペクトル。 当社は、以下のFA命名法を使用しています。 I、およびnは、それぞれISO、アンテイソメチル分枝、直鎖脂肪酸を示します。不飽和のFAは、Cによって指定された：Cであり、Dは、脂肪酸およびD中の炭素原子の数は、二重結合の数です。 Δxは 、二重結合は、カルボン酸末端から数えて、x番目の炭素-炭素結合上に位置する二重結合の位置を示しています。

<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

バチルス種は、メチル分岐（イソまたはアンテイソ）の二重結合の多様な位置で分枝鎖不飽和脂肪酸（FAS）を含みます。 FA組成の変化は、環境への細菌の適応において重要な役割を果たしています。これらの変更は、イソ対アンテイソの比率の変化を伴う特定の位置に作成された二重結合を有する、FAの分枝、飽和のFAへの不飽和のFAの割合インチFAプロファイルの正確な同定は、 バチルス種の適応メカニズムを理解する必要があります。 バチルスからのFAの多くは市販されていません。脂肪酸メチルエステル（FAME）、4,4-ジメチルオキサゾリン：本明細書で提案された戦略は、FA誘導体の3種類の質量スペクトルと保持時間（等価な鎖長（ECL）の計算によって）情報を組み合わせてのFAを識別するデリバティブ（DMOX）、および3 pyridylcarbinylエステル（ピコリニル）。この方法は、未知のFAを精製する必要なしのFAを識別することができます。 標準の商業用混合物とセレウス菌から調製したFAMEのクロマトグラフィープロファイルを比較すると、他のFAのアイデンティティにするFA、ECLの計算、および仮説を飽和直鎖の同定を可能にします。分岐飽和のFA、イソまたはアンテイソのFAMEを、同じ数の炭素を有する直鎖飽和のFAに比べECLに一定の負のシフトを、表示します。不飽和のFAのFAMEをその分子イオンの質量によって検出され、対応する飽和のFAに比べECLにおける正のシフトをもたらすことができます。 FAの分岐位置及び不飽和のFAの二重結合の位置は、それぞれ、ピコリニルとDMOX誘導体の電子イオン化マススペクトルによって同定することができます。このアプローチは、すべての未知飽和の分岐を識別するEDのFA、不飽和直鎖状のFAおよびセレウス菌の抽出物から不飽和分枝状のFA。

細菌細胞からFA特定の戦略は、図1に示されています。各ステップは、補完的なスペクトル情報またはクロマトグラフィー保持に関する情報を提供します。ステップ1は、標準液を用いた予備FA識別で構成されています。ステップ2は、暫定的に製品を識別するために、FAME EIスペクトルとそのECLの解釈を可能にします。ステップ3は、分岐鎖-のFAで正確な分岐?...

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).

脂肪酸の同定で<em&gt;セレウス菌</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

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

9.5K ビュー.  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). 

ビデオ: 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.

微細藻類中の脂肪酸含量および組成の分析

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

環境学

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.

脂肪酸13C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

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

生化学

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.

細菌病原体脂肪酸を膜リン脂質に取り込む研究のための鶏卵黄からのリポタンパク質粒子の単離

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.

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

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.

クリックケミストリーを用いた脂肪酸アシル化タンパク質の検出のためのアルキニル脂肪酸類似体の最適化された組み込み

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.

代謝標識と薄層クロマトグラフィーによる サッカロミセス・セレビシエ における中性脂質合成の解析

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とトップダウンプロテオミクス解析を用いた 、大腸菌 における抗菌性免疫タンパク質の同定

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

化学

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.

腸管病原体の胆汁酸塩によるバイオ フィルム形成: 同定及び定量化の手法

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

免疫・感染学

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.

薄層クロマトグラフィーによるマイコバクテリアの脂質組成の解析

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.

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

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.

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

脂肪酸の同定で

要約

さらに動画を探す

この動画の章

微細藻類中の脂肪酸含量および組成の分析

脂肪酸¹³C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

細菌病原体脂肪酸を膜リン脂質に取り込む研究のための鶏卵黄からのリポタンパク質粒子の単離

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

クリックケミストリーを用いた脂肪酸アシル化タンパク質の検出のためのアルキニル脂肪酸類似体の最適化された組み込み

代謝標識と薄層クロマトグラフィーによるサッカロミセス・セレビシエにおける中性脂質合成の解析

MALDI-TOF-TOF-MS/MSとトップダウンプロテオミクス解析を用いた、大腸菌における抗菌性免疫タンパク質の同定

腸管病原体の胆汁酸塩によるバイオフィルム形成: 同定及び定量化の手法

薄層クロマトグラフィーによるマイコバクテリアの脂質組成の解析

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

脂肪酸の同定で

要約

さらに動画を探す

この動画の章

微細藻類中の脂肪酸含量および組成の分析

脂肪酸13C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

細菌病原体脂肪酸を膜リン脂質に取り込む研究のための鶏卵黄からのリポタンパク質粒子の単離

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

クリックケミストリーを用いた脂肪酸アシル化タンパク質の検出のためのアルキニル脂肪酸類似体の最適化された組み込み

代謝標識と薄層クロマトグラフィーによる サッカロミセス・セレビシエ における中性脂質合成の解析

MALDI-TOF-TOF-MS/MSとトップダウンプロテオミクス解析を用いた 、大腸菌 における抗菌性免疫タンパク質の同定

腸管病原体の胆汁酸塩によるバイオ フィルム形成: 同定及び定量化の手法

薄層クロマトグラフィーによるマイコバクテリアの脂質組成の解析

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

脂肪酸¹³C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

代謝標識と薄層クロマトグラフィーによるサッカロミセス・セレビシエにおける中性脂質合成の解析

MALDI-TOF-TOF-MS/MSとトップダウンプロテオミクス解析を用いた、大腸菌における抗菌性免疫タンパク質の同定

腸管病原体の胆汁酸塩によるバイオフィルム形成: 同定及び定量化の手法