R ・ メンツェルとドイツ研究振興協会 (RU RU780/11-1) によって L. ・ ルッスの財政支援は、感謝します。R. Nehring RU 780/10-1 によって資金を供給されました。最後に、原稿の校正のため博士ハシバミ Ruvimbo Maboreke に非常に感謝しています。

<ol>
	<li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+lipid+analysis+to+understand+trophic+interactions+in+soil.">Application of lipid analysis to understand trophic interactions in soil.</a> Ecology. 86 (8), 2075-2082 (2005).</li><li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+composition+of+Collembola+and+their+food+resources+in+deciduous+forest+stands+-+Implications+for+feeding+strategies.">Lipid composition of Collembola and their food resources in deciduous forest stands - Implications for feeding strategies.</a> Soil Biology and Biochemistry. 39 (8), 1990-2000 (1990).</li><li>Chamberlain, P. M., Bull, I. D., Black, H. I. J., Ineson, P., Evershed, R. 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Isotopologue のプロファイル
FAs 13C 分布の定量的側面の詳細な分析では、炭素の食物網でのパーティション分割を割り当てるには最先端の技術を必要があります。現在の仕事は isotopologue 13C を評価するためにプロファイリングを採用/熱帯の相互作用のための共通の FA バイオ マーカー12C 比。このメソッドでは、液体クロマトグラフィー (LC MS) によるアミノ酸分析の確立しているし、病原細菌17,23炭素代謝の調査に適用しました。ごく最近、isotopologue プロファイルさらに開発された小さな土壌無脊椎動物の脂質の食事ルーティングを研究するためのツールとしてメンツェルらによって24このメソッドは13C 信号の上「キャリー」によって妨げられていないので、GC C IRMS32と比較して特定の同位体を高濃縮有機組織評価ため有利がしたがって。
このアプローチでは分子イオンとその isotopologues 電荷質量 (m/z) の値は、GC-MS によって決まります EI と SIM モードを使用して。フル スキャンと比較してのガスクロマト グラフ定量範囲33をも上回っている要因 100 について、によって SIM の感度を強化できます。さらに、所定の時間ウィンドウでイオンの指定したグループの買収は、検体は測定確実に共溶出ピーク30の背景にもかかわらず。Thurnhofer ・ フェッター34証明 SIM は FA の識別と利用可能な脂質の低い量とも食品サンプルの定量に適しています。
13C isotopologue プロファイリングの SIM を使用する場合、少なくとも 2 つの方法論的制約は考慮されなければなりません。まず、分子イオンが比較的低い豊富そして、isotopologues はさらに少ない。汚いイオン源、これらはより豊かになるし、結果として SIM 法のみ半定量的33なります。第二に、FA の 1 つ以上の炭素の位置に13C の高められた発生は、関連付けられている炭素原子の13C 自然豊かな減少します。ラベルとこれらの変更は非線型、現象はこれらの固まりの20で濃縮の過小評価につながることが豊かな自然の「ゆがみ」と呼ばれます。このスキューの効果を名声標準 (フェルナンデスらを参照) に基づく補正マトリックスを使用して、またはラベルなし実験生物から FAsなど自然なコントロールなど考慮する必要があります。後者は、同じ FA ラベル (日 1 およびそれ以降) と非標識トビムシ (0 日) に比較すると現在の仕事で行われました。
食事のルーティングと13C フラックス
Isotopologue プロファイリング FAs の13C を決定するための方法論的利点が明白な中、生物の体内代謝は生物学的手段によって妨げられます。同位体および代謝の定常状態が困難なときなどを達成するために、食事療法の使用または消費者の生理学的状態は知られていない絶対炭素フラックスの取得を妨げます。ただし、国会の消費者 FAs の13C isotopologues プロファイルを比較すると、FA 同化および代謝経路の相対的な割合を取得できます。このアプローチにより、次の食餌療法のルーティングを追跡することで、 13C ラベルに基づいて FA マーカーの特定の運命です。MS SIM を示したマーカー FA だった両方のトビムシ分析種24の NFLA 画分に主に配線されています。また、FA のマーカーは、唯一のマイナーまたは変更なしで中性脂質に格納されました。食事1318:0 C16:0 およびモノエン同等に不飽和化の伸長だけが検出された (図 4を参照)。C16 ・ C18 1 日目サンプルの FAs でイオン M+16の存在は、これらの FAs が、補足の子孫パルミチン酸を完全にラベルを確認します。この発見は消費者の FA パターンに基づいており、国会のみ5,35以前の研究を支えるし、マーカー FAs が全体の分子として消費者組織に組み込まれていることを示唆している (レビュー ・ ルッスを参照してくださいとチェンバレン8)。
一方、PLFAs の isotopologue 13C パターンは、時間をかけて13C20:46 の観察された増加は完全標識前駆体 FAs の伸長/不飽和化と後者のデノボ合成のためも含むを明らかに13M18の存在によって示されるアセチル coa ラベルの付いた C イオンの跡 C20 とも C18 FAs (図 4を参照)。図 5は、補われた13PLFAs と観察対象のトビムシ種の NLFAs 内にある C16:0 の食事ルーティングをまとめたものです。興味深いことに、1 つだけに限定の伸長および 2 つの不飽和化の手順、NLFAs の代謝変換が残っているに対し、PLFAs の13C パルミチン酸だけは強い変化です。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57110/57110fig5.jpg"> 消費者の脂質の食事13C パルミチン酸の図 5: 運命。流れ図調査トビムシ種法人13C16:0 の提案の運命を示します。矢印は、厚さの異なる代謝回転の別のレベルを象徴しています。<a href="/files/ftp_upload/57110/57110fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
要約すると、isotopologue ・ MS SIM によってプロファイリング栄養マーカーは食物網の生態学の堅牢で信頼性の高いメソッドに明らかに FA を使用するを示します。実験的なデザインの FA 代謝経路で、 13C フラックス (例えばatom %13C の補われた FA として) 別の手順の正確な定量はできませんでした、isotopologue プロファイルは便利なツール栄養マーカーとして脂肪酸を使用するすべての生態学的研究。将来の目標は閉じた小宇宙システムを使用して量的消費者 FA 炭素予算を取得する、脂肪酸の標識前駆体の量を定義する消費者を供給します。

土壌など不可解な生息地で栄養関係解決困難な動物のサイズが小さいによってさらに制限されます。最後の十年は、フィールド条件1,2,3の下の土壌動物相の供給戦略を定義するためのバイオ マーカーとしての脂肪酸の使用で特に、生化学的な生態学の進歩を見ています。これは全体の分子として消費者組織のリソースから脂肪酸を組み込むことができるという事実に基づく、食事ルーティング4と呼ばれる、プロセス。脂肪酸の転送は、以上 3 つ栄養のレベル、すなわち、菌類から粘管目5線虫に報告されています。最近では、捕食動物は、6、7を考慮され、土壌食物網における栄養マーカーとして脂肪酸の最初のレビューが公開された8,9をされています。
栄養相互作用の詳細については、脂肪酸の安定同位体プロービング (FA SIP) によって達成されます。13C の定量/ダイエットと消費することができます脂肪酸12C 比バイナリ リンクのせいと関連付けられている炭素フローを見積もるし、地上波で採用されている、淡水、海洋の食物網10,11 ,12,13。基本的な前提は、ルーティングされた脂肪酸が酵素プロセスの対象とはしたがっての13C 信号、すなわち、 13C/12C 消費者の脂肪酸の比はダイエット1に類似しています。ただし、食物連鎖を13C 署名の段階的な減少は栄養研究14,,1516FA SIP の広範なアプリケーションをそれにより妨げる水生システムで報告されています。さらに、地上の食物網におけるほとんどの無脊椎動物の脂質代謝の知識はまだ限られています。
栄養マーカー脂肪酸食物網生態学における定量的炭素フローの決定のための手段としての使用のための消費者における脂質代謝経路の理解が欠かせません。念頭、 13C isotopologue のプロファイリングは、原理的にはすべての生物学的システムの17、炭素代謝の調査のため適用できる有望な方法であります。13C 標識炭素基板、 13C 代謝ネットワーク内の分布の導入は消費者を特定の isotopologue 分布の生成された代謝産物からトレースできます。これは定量核代謝共鳴分光法18,19または質量20,は21、その高いため低バイオマス後者の支持された生体試料によって評価されることができます。感度。
ただし、isotopologue プロファイルがされて正常にアミノ酸に適用し、細菌性病原体17,22,23, 脂肪酸の実装の体内の炭素代謝に洞察力を提供酸が遅れています。安定同位体標識前駆体脂肪酸、その食物ルーティングや劣化、土壌無脊椎動物の消費者に β 酸化を介しての運命の最初の詳細な分析、最近メンツェルらによって実行されました。24. 13C 標識脂肪酸 isotopologue による頻繁な土壌無脊椎動物、トビムシ、キーの子孫の後で導入実験の方法論的基礎を提供するここでは。これらの植栽は、土壌食物網とがよく、栄養マーカー脂肪酸8,25の調査の重要なコンポーネントは、フォームの良いモデル グループです。
栄養マーカー脂肪酸食物網生態学における定量的炭素フローの決定のための手段としての使用のための消費者における脂質代謝経路の理解が欠かせません。現在のプロトコルは、トビムシから実験、および抽出および支配的な脂質画分 (中性脂質、リン脂質) のメチル化のための生化学的なプロシージャを供給所のデザインと設定を与えます。質量分析法による脂肪酸の isotopologue 組成を分析する方法について説明し、関連の数式と計算について説明します。この手順の結果: (i) 1 つ以上によって (すなわち、脂肪酸分子イオン M+) 親イオンの質量を超える isotopologues の比質量単位 (M+1、+2M、M+3、等) および (ii) 全体1313C 標識前駆体由来の脂肪酸で C 濃縮。このアプローチは、これらは、成功したラベルの吸収量を確保するための制御された条件下で十分な量の培養とその後を前提に他の捕食者-被食者相互作用に適用できる一般にトビムシに使用、検証します。

<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&#214;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 &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>P717.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Hexan</td>
			<td>Carl Roth</td>
			<td>7339.1</td>
			<td>HPLC &#8805; 98 %</td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether (MTBE)</td>
			<td>Carl Roth</td>
			<td>T175.1</td>
			<td>HPLC &#8805; 99,5 %</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Carl Roth</td>
			<td>7328.2</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth</td>
			<td>6771.1</td>
			<td>p.a. &#8805;99 %, in pellets</td>
		</tr>
		<tr>
			<td>di-Natriumhydrogenphosphat</td>
			<td>Carl Roth</td>
			<td>P030.1</td>
			<td>p.a. &#8805;99 % , water free</td>
		</tr>
		<tr>
			<td>Na-dihydrogenphosphat Dihydrat</td>
			<td>Carl Roth</td>
			<td>T879.1</td>
			<td>p.a. &#8805;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&#228;paratengl&#228;ser Duran</td>
			<td>Glasger&#228;tebau Ochs</td>
			<td>135215</td>
			<td>&#216; 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&#160;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&#160;mg/mL in methyl caproate, analytical standard</td>
		</tr>
		<tr>
			<td>Methyl nonadecanoate</td>
			<td>Sigma-Aldrich</td>
			<td>74208</td>
			<td>analytical standard &#8805; 98.0 %</td>
		</tr>
		<tr>
			<td>Hexadecanoic acid-1-13C (Palmitic)</td>
			<td>Larodan Fine Chemicals</td>
			<td>78-1600</td>
			<td>GC &#8805; 98.0 % (13C: 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+</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

通常完全な分子として同化、マイナーまたはなしの変更は、異なる栄養段階の間食事ルーティングできるように消費者組織に転送されるため、脂肪酸 (FAs)、食物網の生態学的に有用なバイオ マーカーです。しかし、FA 栄養マーカー アプローチはまだ土壌動物相の脂質代謝の限られた知識によって妨げられます。この調査は、脂肪酸代謝経路の 2 つの広範な土壌トビムシ、 Protaphorura fimataとHeteromurus 光沢苔のトレーサーとして完全ラベル パルミチン酸 (13C16:0、99 の atom %) を使用しました。運命、この前駆体の新陳代謝の変更を調査するために isotopologue のプロファイリングの手法を提案する単一イオンのモニタリングを用いた質量分析法によって行います。さらに、上流の研究室実験を供給、抽出および支配的な脂質画分 (中性脂質、リン脂質) と関連数式および計算のメチル化して説明します。Isotopologue プロファイリング13C 標識前駆体由来の脂肪酸で全体の13C 濃縮収量のみではなく、また親イオン (すなわちFA の分子イオンの質量を超える isotopologues のパターンを生成M+) それぞれのラベルの 1 つまたは複数の質量単位(M+1、+2M、M+3等)で FA。この知識は、デノボ合成と比較して完全に消費された FA の栄養経路の比率に結論をことができます。Isotopologue のプロファイリングは、栄養相互作用を分離する土壌動物における脂肪酸代謝の評価のための便利なツールとして使用することをお勧めします。

トビムシの新鮮な重量と脂質含量 記述されている実験の過程で NLFAs および PLFAs の内容変わりませんでした大幅時間をかけて大幅24が、供試体の重量がわずかに増加したに対し。両方のパラメーターは、トビムシ標本の体力の良いレベルを示します。脂肪酸組成と同位体分析のためのサンプリング日に対応する実験中のトビムシの新鮮な重量および脂質コンテンツを調査するため注意してください。重量の損失および/または実験期間中の脂質含量の減少を示す試験生物の減少フィットネス派生データは大幅に意義を失うことに注意してください。
Isotopologue 検出 プロジェクトのプロファイル isotopologue の特別な意味は個々 の検出と分子イオンと特定の脂肪酸ですべて isotopologues の定量化であります。たとえば、16:0 の場合イオン 286 (isotopologue M+16 - 重い13C で完全に置換炭素鎖) を 270、すなわち、分子イオン M+ (C スケルトン主体12C 原子) から及ぶ。図 2 aは、純粋な 16:0 パルミチン酸の SIM MS スペクトルを示します。この化合物の13C の豊かな自然は、M+1と分子イオン (M+) に加えて M+2 isotopologues の存在によって検出可能になります。比較では、図 2 bは、この研究で使用される完全ラベル パルミチン酸 (99 原子13C) のスペクトロ グラムを示しています。ここでは、イオン M+16の唯一の出現は、この総合的標識化合物の高純度を反映しています。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57110/57110fig2.jpg"> 図 2: 純粋なラベル (A) と (B) 完全ラベル パルミチン酸の代表 SIM スキャンします。完全標識脂肪酸 (99 原子 M+16 isotopologue のラベルのない c 16:0 (A) 排他的な存在が、M+1と分子イオン (M+) に加えて M+2 isotopologues の豊かな自然を注意してください。13C)。<a href="/files/ftp_upload/57110/57110fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
伸長/不飽和化対・ デ ・ ノボ合成 収穫試験生物, 脂質の抽出および誘導体化、生成された FAMEs GC と SIM MS で分析する準備が整います。Isotopologue データをプロファイリングの検討、によって今デノボ合成と不飽和化/伸びの全体のラベルの付いたマーカー脂肪酸のイベントを区別できます 1 つ。前者起こることができる、少なくとも部分的には13C アセチル β を介して標識前駆体の分解のプロダクトとしてに基づいて。図 3は、SIM スキャン ラベル後最初のサンプリング日にh. 光沢苔の PLFA の割合で 4 つの支配的な脂肪酸由来の代表的な例を示します。ラベル マーカー分子のパルミチン酸への同化は、まったく標識脂肪酸 (図 3 a - 16:0) を表すので、M+16 - isotopologue の豊かさに表示されます。さらに、16:0 (図 3 b - 16:1ω7) または伸長と不飽和化 (図 3 - 18:1ω9、図 3 の D - 20:4ω6) の不飽和化割り当てることができます。それによって、isotopologues M+16,+17 例えばM と M18よりも大きいの検出は13C2 フラグメントの使用によって標識前駆体 16:0 の鎖伸長を示しています。に基づいて13C アセチル coa 脂肪酸のde novoの生合成が示されます場合 isotopologues は分子イオンに近い、すなわち、 M+1 M+2で取得するよりも有意に多かったによって表されるコントロール0 日サンプリング。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57110/57110fig3.jpg"> 図 3: 代表 SIM (選択的イオン監視) スキャンHeteromurus 光沢苔(PLFA 分画、ラベリング後 1 日目) の脂肪酸のフラグメント イオンの。(A) パルミチン酸 (16:0) (B) のパルミトレイン酸 (16:1ω7) (C) (D) とオレイン酸 (18:1ω9) のアラキドン酸 (20:4ω6)。<a href="/files/ftp_upload/57110/57110fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
さらにこれを示す、図 4の比較 (PLFAs と NLFAs の両方分析) 5 つの最も重く標識脂肪酸の初期 isotopologue パターン0 日目と 1 日目にトビムシ種P. fimataからのデータを比較します。1 日目、C16 ・ C18 FAs に13C 信号はほぼ完全完全ラベル13C 16:0 食品に添加した結果、M+16分子イオンの発生に基づいていた。前述のように、18:0 と 18:1ω9 の M18の観察された痕跡は13アセチル coa C 18:0 16:0 から鎖伸長を介してのマイナー部分の導入をお勧めします。18:1ω9 のこれは PLFA 派でもっと発音されました。このような13C アセチル coa はラベル付けされた13C 16:0 分子の β 酸化から派生します。13C のアセチル coa も手順で発生しました、伸長鎖長 C18 から C20 に PLFA サンプルの 20:4ω6 によって割り当てられました。これにより、M+16と M18の13C 濃縮は、(図 4) を 14 日目までの時間が大幅増加しました。また、14 日目でこの PLFA 20:4ω6 の M+2増加 0 や 1 と比較されたとき。異なる isotopologues でこの13C 割り当てアセチル coa ラベル13C や豊富な自然13C での含めるとデノボ合成の両方 20:4ω6 の形成に基づいていることを示しますと、伸長/全体13C16:0 前駆体分子の不飽和化。対照的に、はっきりとラベル付けされた 20:4ω6 は、NLFA 画 (図 4) で表示されませんでした。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57110/57110fig4.jpg"> 図 4: Isotopologue パターン SIM (選択的イオン監視) で13C 定款。イオン M+1 M+3と M+16の相対部分は 5 つの最も頻繁にラベル付きの NLFAs と PLFAs の M+20まで、 Protaphorura fimataから提示 0 日目と 1 日目で推定されます。20:4ω6 のみのデータを 14 日間はさらに掲載されています。P< 0.05 (0 日目データ参照として使用される Dunnett のテスト)。この図は図 5メンツェルらで発行の復刻版です。24<a href="/files/ftp_upload/57110/57110fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

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

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。

すなわち、分子全体消費者組織への伝達と脂肪酸の同化脂肪酸の栄養マーカー アプローチ変更が小さい土壌無脊椎動物の脂肪酸代謝の知識のギャップによって妨げられて無またはマイナー。Isotopologue ・ プロファイリングは、栄養相互作用を分離するための貴重なツールとして提供されます。

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

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

7.9K ビュー.  Humboldt-Universität zu Berlin. すなわち、分子全体消費者組織への伝達と脂肪酸の同化脂肪酸の栄養マーカー アプローチ変更が小さい土壌無脊椎動物の脂肪酸代謝の知識のギャップによって妨げられて無またはマイナー。Isotopologue ・ プロファイリングは、栄養相互作用を分離するための貴重なツールとして提供されます。

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

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

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 ...

生化学

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

洗剤無料補完充電プロテオリポソーム融合ペプチドを用いた脂質膜に膜タンパク質の超高速再構成。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

リーシュ マニア、ひと細胞およびマウス精巣におけるプロファイリングをポリソーム

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

安定炭素・酸素同位体分析の炭酸塩のサンプリングと歯のエナメル質の前処理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

アンジオテンシンIIによるニトロペプチドプロファイリングと同定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

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 ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

脂質混合アッセイ用リポソームへの組換えショウジョウバエアラストシンの洗剤支援再構成

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

ユビキチンおよびユビキチン様依存性翻訳後修飾のプロファイリングと有意な変化の同定

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

タンパク質-脂質相互作用を測定するためのマイクロスケール熱泳動の使用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

脂質結合タンパク質およびアレルゲンからの内因性リガンドの除去と置換

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...