この作品は自然科学基金、中国のによって部分的に支えられた (許可番号 31471703、A0201300537、J.V.、l. l.、31671854 許可番号 31470435 G.Y.)、および 100 外国の才能計画 (合弁会社許可番号 JSB2014012)。

1. サンプル コレクション
<ol><li>新鮮な大根 (ダイコン) の異なる品種を購入します。</li>
</ol>2. 大根から蛋白質の分離
<ol><li>10 分間キッチン ミキサーで新鮮な大根の約 100 g をホモジナイズしてください。</li>
	<li>50 mL の遠心管と不溶性物質を削除する 20 分のための 4 ° C で 14,000 × gで遠心スラリーに転送します。</li>
	<li>新しい 50 mL 遠心チューブに上清を慎重に転送し、2 M トリクロロ酢酸 (TCA) ソリューションの等しい量を追加します。 注: TCA を追加は、水溶性 (糖) タンパク質を沈殿させます。</li>
	<li>4 ° C、30 分で 14,000 × gで遠心分離機、ペレット (糖) 蛋白質から上澄みを取り外します。</li>
	<li>14,000 × g水溶性オリゴ糖や多糖類を削除する 5 分の 4 ° c で 20 ml の脱イオン水と遠心分離機のペレットを洗浄します。</li>
	<li>手順 2.5 を繰り返します。4 回。</li>
	<li>再 1 mL の脱イオン水と新鮮な 1.5 mL 遠心チューブに転送でペレットを中断します。</li>
</ol>3. N型糖鎖の調製
<ol><li>手順 2.7 からタンパク質溶液のミックス 50 μ L。(新鮮な大根の 5 g に等しい) 組換え PNGase h+ 10 mM 酢酸 0.2 mu。</li>
	<li>12 h の 37 ° C で反応混合物を孵化させなさい。、孵化後余分な蛋白質および酵素を削除する 5 分 14,000 × gで遠心分離機します。</li>
	<li>新鮮な 1.5 mL 遠心チューブに上清を転送します。</li>
</ol>4 N型糖鎖の精製
<ol><li>N型糖鎖を豊かにし、 N型糖鎖の蛍光 2 aminobenzamide (2 AB) ラベル エージェントの選択性を増やす反応混合物から塩および他の不純物を削除する固相抽出 (SPE) の列を準備します。誘導体化。<ol>
<li>3 mL の脱イオン水を洗い流すを追加します。</li>
		<li>アクティブにトリフルオロ酢酸 (TFA, 0.1 %v/v) を含む 80% アセトニ トリル溶液 3 mL SPE カラムを追加します。</li>
		<li>SPE コラムを平衡させ脱イオン水 3 mL を追加します。</li>
	</ol>
</li>
	<li>ステップ 3.3 列上からサンプルを転送し、流れを破棄します。</li>
	<li>1.5 mL の脱イオン水コラムを洗浄し、流れを破棄するを追加します。</li>
	<li>1.5 mL の 0.1% を含む 20% アセトニ トリル水溶液を使用して、列からリリースされたN型糖鎖を溶出 TFA (v/v) 2 mL の遠心管に。</li>
	<li>室温で遠心蒸発溶剤を削除します。このサンプルは、完全に乾燥するまでに蒸発させます。</li>
</ol>5 N型糖鎖の蛍光誘導体化
<ol><li>35 mM 2 AB と 0.1 M ナトリウム シアノ水素化ホウ素ジメチル ジメチルスルホキシド/酢酸酸溶液 (7:3 v/v) から成る、2 AB 溶液 1 mL を準備します。</li>
	<li>六つの異なる大根由来の乾燥サンプル (ステップ 4.5.) 2 AB ソリューションの 5 μ L を追加します。渦各サンプルまで完全に分解します。</li>
	<li>65 ° C で 2 h の混合物を孵化させなさい</li>
	<li>5 分間室温までサンプルの涼しい許可し、5 μ L の脱イオン水とアセトニ トリル 40 μ L を追加します。3 分間 14,000 x g でチューブを遠心します。</li>
	<li>300 μ L 高回復高速液体クロマトグラフィー バイアルに上澄みから 48 μ L を転送します。1 ヶ月の-20 ° C で derivatized のN型糖鎖のサンプルを格納します。</li>
</ol>6. 構築高精度のN型糖鎖のプロファイリング
<ol><li>それぞれ 330 nm/420 nm の励起/蛍光波長オンライン蛍光 detectorset に接続されている標準の高精度システムを使用してサンプルを分析します。</li>
	<li>疎水性相互作用クロマトグラフィー (構築) を使用して-分析のための 60 ° C のカラム温度の高精度糖鎖列。</li>
	<li>50 mL の原液 (1 M, ギ酸アンモニウム溶液、pH 4.5) の液体クロマトグラフィー質量分析計 (LCMS) 950 mL で希釈することによって溶剤 A を準備-水のグレードします。とおり、在庫ソリューションを準備します。<ol>
<li>LCMS グレード H2O を 700 mL にギ酸の 43 mL を追加します。</li>
		<li>(奥行 25% アンモニア水を滴下して 4.5 に pH を調整します。</li>
		<li>溶媒をメスシリンダーに転送し、3 ヵ月の LCMS グレード H2o. ストア 4-6 ° c と 1000 mL を入力します。</li>
	</ol>
</li>
	<li>溶剤 B として LCMS グレード アセトニ トリルを使用します。</li>
	<li>N型糖鎖を次のグラジエントで区切ります。<ol>
<li>まず 0 から 44.5 分溶媒 A. セット 0.5 mL/min の流量に溶剤 B の 95% を追加します。</li>
		<li>0 分から 6 分、線形のグラデーションの to78% の溶媒 B の割合を徐々 に減少します。</li>
		<li>6 分から 44.5 分、55.9% に線形グラデーションの溶媒 B の割合を徐々 に減少します。</li>
		<li>46.5 分 44.5 分からには、急速に 55.9% から 0% への線形グラデーションで溶剤 B の割合を減らすと 2 分 0% を保持し、列の洗濯を開始します。55 分 44.5 から 0.25 mL/分への流量を減らします。</li>
		<li>50.5 分 48.5 分から 95% に溶剤 B を増やすことによってさらに列を洗って、4.5 分の 95% を保持します。</li>
		<li>再度 0.5 mL/min で 95% 溶剤 B の開始条件にコラムを平衡させ、57 分 50.5 から。</li>
	</ol>
</li>
	<li>高精度システムに 45 μ L のサンプルを注入します。</li>
	<li>15 と 40 分間保持時間と、高精度での糖鎖を溶出します。</li>
	<li>2 mL 遠心管を使用して各観測の高精度ピーク分数を収集し、遠心蒸発によってサンプルを乾燥します。</li>
	<li>2 AB デキストラン標準 (2−20 グルコース単位) 工場 -N型糖鎖プロファイリング、調整し、標準化されたグルコース単位への溶出時間を割り当てるをラベルの約 1 pmol を注入します。</li>
</ol>7. MALDI-TOF 質量分析
<ol><li>LCMS のステップ 5 で 6.8 μ L のサンプルを溶解-水のグレードします。ミックス 1 μ L の試料溶液及び 1 μ L 6 字-2 thiothymine (ATT) ソリューション (0.3 %w/v 70 %v/v アセトニ トリル水溶液中で)、およびピペット MALDI-TOF サンプル キャリアの上に混合物の。</li>
	<li>[スタート] ボタンを押すことによって肯定的なイオン モードで MALDI-TOF 質量分析計の計測器を使用してサンプルを分析します。</li>
	<li>付属の MALDI TOF MS 解析ソフトウェアを使用して質量スペクトルを分析します。</li>
	<li>オープン ソース糖解釈ソフトウェア19を使用してスペクトルを解釈します。2 AB のラベル、[M+na]+の検索パラメーターを設定し、2.0 da 精度パラメーターを設定します。</li>
</ol>

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我々 はここで提示しているプロトコルは、さまざまな品種の大根のN型糖鎖のプロファイルの比較をできます。既存のプロトコルと比較してこの方法の重要な利点は、 N型糖鎖の酵素的リリースと 2 AB と誘導体化反応のバッファーの変更が必要ないことです。この手順の最も重要なステップは塩を削除する失敗として SPE カラムを用いたN型糖鎖の精製や反応混合物の他の不...

植物におけるN型糖鎖が注目増加近年、前の研究がアレルギー反応1,2 を引き起こすかもしれない免疫の内の潜在的なソースとしてN型糖鎖をハイライト表示します。.それが実証されて以前植物糖タンパク質糖鎖のNは、触媒活性3,4、耐熱性と折りたたみ式5,6または細胞レベル下のローカリゼーションに影響を与えることができ、分泌7。糖鎖構造をそれぞれの機能に関連付ける、するためにN型糖鎖最初から解放しなければならない糖タンパク質化学的にまたは酵素によって。N- とO型糖鎖を解放するための古典的な化学方法は β-除去するアルカリ サンプル治療と一緒に伴われる還元糖アルコール8を生成する水素化ホウ素。ただし、この手順は、蛍光でラベルを排除、糖鎖構造の還元末端から単糖類の単位の重要な崩壊を引き起こします。水酸化アンモニウム炭酸塩治療に基づいて化学糖鎖切り出しも一般的に使用される代替方法9です。これらの化学物質の放出方法のどちらも同じ質量範囲にラベルのない糖ペプチド フラグメントの干渉なしプールの質量分析が可能そのまま蛋白質が低下します。ただし、これらのメソッドの 1 つの欠点である α 1, 3 次N型糖鎖の増加の分解速度は共通の炭水化物構造植物10。また、ペプチドを用いた酵素リリース方法:N- 多糖分解酵素 (PNGases、EC 3.5.1.52) も広く適用されています。(病菌 meningosepticum) から組換え PNGase F は最も一般的な選択であり、コア α 1, 3 フコース11,12軸受構造を除いて、 N型糖鎖のすべてのタイプのリリースを許可します。したがって、PNGase A (アーモンドの種子から分離された) は通常植物13 N型糖鎖の解析に使用されます。しかし、この酵素 deglycosylates のみ生由来ペプチド、deglycosylate ネイティブ糖14ことができないと。したがって、詳細な分析、糖、低豊富15の特にそれらの広汎な損失を引き起こす前にマルチ ステップ サンプル精密検査が必要です。メソッドの全体的な目標は、 N型糖鎖のリリースとシンプルで堅牢な方法で蛍光のために最適化されたワークフローを提示することです。基になる根拠は PNGase H+ Terriglobus ニチニチソウの最近発見された recombinantly , 大腸菌で発現することができます、ことができる酸性のタンパク質足場から直接N型糖鎖を加水分解条件16。PNGase H+を使った代替方法の主な利点は、反応バッファー17,18を変更することがなく同じ反応管内の蛍光標識反応が可能です。簡単な調製条件と低豊富なオリゴ糖の高回収率このメソッドは、 N型糖鎖の解析で貴重なツールを作る。このプロトコルは、さまざまな植物の種からN型糖鎖の解析に適しています。

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	<tbody>
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			<td height="21" style="height:21px;width:347px;">Chemicals:</td>
			<td style="width:123px;"></td>
			<td style="width:207px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid&#160;</td>
			<td>SCR, Shanghai</td>
			<td>80132618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid glacial</td>
			<td>Huada, Guangzhou</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>General-reagent</td>
			<td>G80988C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Energy chemical</td>
			<td>W810031</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-aminobenzamide</td>
			<td>Heowns, Tianjin</td>
			<td>A41900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cyanoborohydride</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>314162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Huada, Guangzhou</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2AB-labeled dextran ladder, 200 pmol</td>
			<td>Agilent Technologies</td>
			<td>AT-5190-6998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Aza-2-thiothymine&#160;</td>
			<td>Sigma</td>
			<td>275514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kitchen blender</td>
			<td>Bear, Guangzhou</td>
			<td>LLJ-A10T1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Techcomp</td>
			<td>CT15RT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifugal Evaporator</td>
			<td>Hualida, Taicang</td>
			<td>LNG-T120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column</td>
			<td>Supelco</td>
			<td>Supelclean ENVI Carb SPE column</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>&#160;# 5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Column oven</td>
			<td>Hengxin</td>
			<td>CO-2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Waters</td>
			<td>Acquity UPLC BEH glycan column</td>
			<td>2.1 &#215; 150 mm, 1.7 &#956;m particle size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Water</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td># 100029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Aladdin</td>
			<td>F112034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia solution</td>
			<td>Aladdin</td>
			<td>A112080</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

近年、植物の糖鎖は、十字反応、アレルギー誘発の免疫応答の潜在的なソースは、かなりの注目を受けた。さらに、炭水化物の構造はまた植物の代謝に重要な役割を果たします。簡便・迅速な準備と、 N型糖鎖大根 (ダイコン) の品種から植物由来の炭水化物構造の解放のためのN- glycanase 固有を使用しての分析法を紹介します。これを達成するには、大根ホモジネートの原油のトリクロロ酸析出された PNGase H+で扱われ、蛍光タグとして 2 aminobenzamide を使用してラベル付け。ラベル付きのN型糖鎖のサンプルを超高性能液体クロマトグラフィー (高精度) 分離して飛行 (MALDI-TOF) 質量分析法による詳細な構造のマトリックス支援レーザー脱離イオン化時間行った後評価と大根由来のN型糖鎖構造の相対的な abundancies を定量化します。このプロトコルは、他の各種の植物の種からN型糖鎖の解析も使用でき、関数のさらなる調査と人間の健康に及ぼすN型糖鎖の役に立つかもしれません。

図 1は、(糖-) 大根、 N型糖鎖の調製、高精度分析とこれらのコンポーネントの MALDI TOF MS 分析からの蛋白質の分離を含む記述のプロトコルの概要を示しています。誘導体N型分析ダイコン品種の糖鎖の代表的な高精度のクロマト グラムを図 2に示します。図 3は、MALDI TOF 質量分析を用いた 2...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

ダイコン品種使用 PNGase H+からN型糖鎖の解析

簡便・迅速な準備と大根 (ダイコン) の品種間におけるN型糖鎖の解析法について述べる。

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K ビュー.  Nanjing Agricultural University. 簡便・迅速な準備と大根 (ダイコン) の品種間におけるN型糖鎖の解析法について述べる。

ビデオ: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

レーザーマイクロダイセクションRNA-のSeqのための実験デザイン：トウモロコシ葉の開発の分析からの教訓

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

生化学

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIREを用いた電子冷凍顕微鏡画像からの高分解能単一粒子解析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

複数のマウス組織からの中間体フィラメントタンパク質の単離による老化に関連する翻訳後修飾の研究

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

プロテオーム解析のための人間の網膜と網膜色素上皮・脈絡膜の解剖

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

動的プロテオミクスおよび miRNA 蛍光灌流後分離されたマウス心臓ポリソームの分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

遺伝子組み換え生物のコンテキストで次の人間ミルク三糖類の分析エンコード バイオ センサー

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

茶由来の新たに確立された細胞懸濁液培養における6種の全身殺虫剤の代謝に関する研究(カメリアシネンシスL.葉

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

潜在的エピジェネティックマーカーのトップダウン質量分析プロファイリングのためのソルガム葉組織からのヒストンの分離

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

支持浮遊ブロックを用いた透過電子顕微鏡におけるサンプル支持膜の作製

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...