We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

注：この方法は、以前に12を説明した。 1。タンパク質の単離好ましくは、フレンチプレス、ビーズビーティング、または超音波のような物理的方法により、細胞を溶解することによって細胞タンパク質1mgを準備します。酵素は、質量分析の測定を混乱されますので、リゾチーム媒介細胞溶解を避けてください。 タンパク質の2。TCA沈殿タンパク質を沈殿さ10分間氷上で4倍量のタンパク質と寒さに1ボリュームトリクロロ酢酸（TCA）を追加します。 4℃で5分間、12,000×gで遠心分離し、上清を取り除く。 4℃で5分間、12,000×gで氷冷アセトン及び遠心分離機の1ml中にペレットを再懸濁上清を除去し、15分間、ペレットを乾燥させるためにベンチにチューブを反転させる。 -80℃での店舗タンパク質ペレット 3。変性タンパク質ジスルフィド結合を還元リタンパク質をサスペンド500μlの変性および還元緩衝液中で約2 mg / mlの（50mMのHEPES pH8.5で、5mMのDTT中4 M尿素または3％SDSのいずれか）である。任意選択で、緩衝液中のプロテアーゼ阻害剤が挙げられる。室温で10分間、続いて56℃で30分間、タンパク質をインキュベートする。 4。Aklylate無料スルフヒドリル基が不可逆的ジスルフィド結合の形成を破壊する水中での新鮮な0.3Mのヨードアセトアミドを準備します。注意！ヨードアセトアミドは、非常に毒性が強い。 500μlのタンパク質に25μlの0.3 Mヨードアセトアミド（15mMの最終濃度）を添加し、室温で暗所に20分間インキュベートする。 300のDTT（5 mMの最終DTT濃度）を10μlを添加することにより、ヨードアセト急冷。 -80℃でアルキル化されたタンパク質を保存する 5。タンパク質消化 TCAは、タンパク質を沈殿させる（工程2に記載のように）および50 mMのHEPES（pHは8.2）1mlを、1 M尿素中で再懸濁する。シルendoproteinの原液を準備し2μgの/μLの濃度のタンパク質溶液に5μLを加え、水中でのASE（のLys-C）。室温で16時間混合物をインキュベートする。最終の確保のLys-C濃度は10ngの/μlのタンパク質と対のLysC比である（w / w）の50分の1〜200分の1である。 、50 mMの酢酸を40μlの20μgのシーケンシンググレードのトリプシンを再懸濁のLys-C消化物に5μL（10μgのトリプシン）を追加し、37℃で6時間インキュベートトリプシンに使用したのLys-Cに同じプロテアーゼ濃度およびプロテアーゼとタンパク質との比を使用してください。 6。逆相ペプチド抽出<ol><li> 0.5％の最終濃度（pH試験≈2）にトリフルオロ酢酸（TFA）を加えることによりペプチドを酸性化する。抽出マニホールドにC18カラムを取り付けます。ペプチドのローディングおよび溶出を除くすべてのステップのための可能な限り最高の流量を使用してください。 </li><li> 6ミリリットルのアセトニトリル（ACN）で濡れ列。 6ミリリットル80パーセントのACN、0.1％TFAでカラムを洗浄し、6ミリリットルの0.1％TFAで平衡化させます。許可されていませんステップ間で枯渇する列。 </li><li>真空圧を停止し、約1ml /分の流速でカラムにペプチド500μgのを読み込む。ペプチドは、カラム、再始動真空に結合し、次いで（0.09 Mクエン酸、0.23 MのNa 2 HPO 4、pH5.5）中のクエン酸緩衝液3mlで6 mlの0.1％TFAで洗浄した後。 注：より高いペプチドの量は、標識することができるが、C18カラムの結合能力は、試料の損失を回避するために、ペプチドの量よりも少なくとも2倍高くなければならない。 </li></ol> 7。オンカラム還元ジメチル化によるペプチドの標識（REDIラベリング） シアン化水素は、ラベリング処理中に低濃度で放出された化学フードの下で、この手順を実行します。 <ol><li>ペプチド遊離アミンをメチル化するために「光」と「重い」REDIバッファの12ミリリットルを用意します。光のRedi緩衝液は0.8％ホルムアルデヒド及び番目に水素を運ぶ0.12 Mシアノ水素化ホウ素ナトリウムから成りクエン酸緩衝液中のEIR天然の同位体分布。重鎖のRedi緩衝液は0.8％重水素化ホルムアルデヒド、クエン酸緩衝液中の0.12 M重水素化シアノ水素化ホウ素ナトリウムからなる。 </li><li> 1ミリリットル/分の流速でペプチド含有列に10ミリリットル光や重いREDIバッファのいずれかを追加することにより、ペプチドを含む列をインキュベートし、完全なラベル付けを確実にするために繰り返します。ウォッシュ6ミリリットル0.1％TFAでカラム、次いで1ミリリットルの0.5％酢酸を含む。 </li><li>真空を停止し、1mlの80％ACN、約0.5ml /分の流速を用いて0.5％酢酸で、次いで1mlの40％ACN、0.5％酢酸を有する第1の標識されたペプチドを溶出させる。所望であれば、重鎖および軽鎖のサンプルを混合する前に、質量分析により個々の試料の標識効率を測定する（「代表的な結果」を参照）。質量分析によって定量化することが1:1重鎖及び軽標識化ペプチドのサンプルを混合する。 </li></ol>塩基性pH逆相（BPRP）クロマトグラフィー&lt;によって8。セパレートペプチド混合物/ pの&gt; 塩基性pHは、独立して、プロテオームカバー率を増加させるためにLC-MS/MSにより分析する複数の画分へのペプチド混合物を分離するために相（BPRP）クロマトグラフィーを逆転させた。 <ol><li> 10mMの重炭酸アンモニウム（pH8）に増加ACNの濃度勾配を適用することにより、C18-HPLCカラム上でペプチド混合物を分画する。 5分間、5％（v / v）のACNで開始し、60分で35％ACNへ増大し、1分間で、次いで90％ACN。 9分間の再平衡化の列に5％ACNを削減する前に4分間、90％ACNを保持します。 96ウェルプレート（H12にA1）に、等量の96画分を収集する。ペプチドは、（ここに記載された条件のために10〜70分）でカラムから溶出している間に220 nmでのUV検出器を用いて分画を監視します。 </li><li>井戸A2、C2、E2、およびG2（フラクションA2）から、それに応じてのために、ウェルB1、D1、F1、およびH1（フラクションB1）から、井戸A1、C1、E1、およびG1（フラクションA1）から分画を組み合わせ残りの画分。 solvenを削除真空遠心機を用いてtを画分からの再懸濁し、ペプチドが、A1、B2、A3、B4、A5、B6、A7、B8、A9、B10、A11、およびB12、1M urea/0.5％のTFAを130μL中、ステップ9で説明したようにStageTipsを使用して浄化する。ストア画分B1、A2、B3、A4、B5、A6、B7、A8、B9、A10、B11、および-20 A12ºCの</li></ol> 9。ストップアンドゴーの抽出によって精製し、ペプチド（StageTips） 1.07ミリメートルの内径（ID）が2 C18ディスクを備えた200μlのピペットチップを充填することにより、C18-StageTip 7マイクロカラムを準備します。エッペンドルフチュー​​ブにステージのヒントを置く。メタノール130μL、その後130 UL 80パーセントACN、0.5％の酢酸でのヒントを洗浄するためのマイクロを使用しています。 130μlの0.1％TFAでStageTipsを平衡化させます。 StageTipsにペプチド混合物を移し、40μlの0.5％酢酸、その後、40μlの0.1％TFA、その後、130μlの0.1％TFAで洗浄する。 20μlの40％ACN、0.5％酢酸、次いで、20μlの80％ACN、0.5％酢酸を有する第1のペプチドを溶出させる。溶出液A組み合わせるND真空濾過により乾燥させます。 10。マイクロキャピラリーLC-MS/MS <ol><li>約1μgの/μLの濃度に1-5μlの5％ギ酸、5％ACN中のペプチドを溶解させる。 0.125％ギ酸中の6から22パーセントACNの勾配で100ミクロン×20センチC18-逆相HPLCカラム上の解決〜1μgのペプチドは、〜300リットル/分の流量で75又は100分間にわたって適用した。 </li><li> LTQ Velos 12又はオービトラップ質量分析計は、高分解能、高質量精度を提供することで同様の液体クロマトグラフィー-質量分析プラットフォームを用いてペプチドを同定する。オービトラップアナライザで取得されたフルMSスキャン（60,000の分解能）とデータ依存モードでの質量分析装置を操作します。完全なMSスペクトルで検出された20の最も豊富なイオンのための線形イオントラップMS / MSスペクトルを生成する。 MS / MSのための完全なMSのための1×10 6と2000に自動利得制御（AGC）の目標を設定します。千ミリ秒、最大イオン蓄積時間を設定するMSおよびMS / MS用150ミリ用。 20〜60秒のためのMS / MSからのさらなる選択から断片化されたペプチド前駆体イオンを除外します。 </li></ol> 11。、MS / MSデータ収集これらのパラメータ（ 表1）を用いて、このようなSEQUEST 8などのアルゴリズムを用いて理論的なデータベースへのMS / MSスペクトルのRAWファイルを比較することにより、ペプチドを同定。 表1。ペプチドデータベース検索のパラメータを。 <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> 一般的なパラメータ </td><td>最大2と完全にトリプシン消化は、切断を逃した</td></tr><tr height="19"><td> 25 ppmの前駆イオントレランス</td></tr><tr height="19"><td> 1.0ダフラグメントイオンの許容範囲</td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> 静的な変更 </strong&gt; </td><td>システイン、カルボキシアミドメチル化に関する57.02146ダ</td></tr><tr height="19"><td>リジンおよびペプチドのN末端上28.03130ダ、光ジメチル化ラベル</td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> 動的変更 </td><td>メチオニン、酸化に対する15.99491ダ</td></tr><tr height="19"><td>リジンおよびペプチドのN末端、重いジメチル化ラベルに6.03766ダ</td></tr></tbody></table><ol><li>実際、逆向きでオープンリーディングフレームのデータベースを使用して、ターゲット·デコイ9戦略としてのメソッドを使用して1％の偽発見率にフィルタペプチド。 </li></ol> 12ペプチドの定量化計算MS1の重鎖および軽鎖のペアの領域は、イオンクロマトグラム（MS1ピーク面積）およびペプチドシグナル対ノイズ（S / N）比が10を抽出した。ペプチドを含むペアた場合にのみ、その平均信号NOIそれ比は5以上である。同じペプチド（MS1ピーク面積比）の重鎖および軽鎖のバージョンMS1ピーク面積の比が、2つの試料中のペプチドの相対存在量を定量する。タンパク質中の全てのペプチドの中央値MS1ピーク面積比と相対タンパク質量を計算します。

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安価な標識試薬（試薬はサンプルあたり1ドル未満のコスト）、速い反応速度（〜10分）、副生成物が存在しない場合、高：いくつかのポイントが還元ジメチル化を使用して、ペプチドの安定同位体標識（REDIラベリング）定量的プロテオミクスのための魅力的な方法を作る再現性（ 図3、図4）、安定した反応生成物、任意のプロテアーゼを使用する能力、および標識されたペプチ?...

複雑なサンプル間の多くのタンパク質の濃度差を測定するプロテオミクスにおける中心的な課題である。ますます、このことは、異なる同位体タグを各試料中のタンパク質を標識するサンプルを結合し、濃度差を定量化するために質量分析法を用いて行われている。いくつかの方法がタンパク質およびペプチドの安定同位体標識のために存在しています。15 N標識の1やSILAC 2 ICAT 3、のiTRAQ 4、還元ジメチル化5は、タンパク質の抽出および消化後に安定同位体タグを追加する一方で、 生体内で代謝的に同位体標識を導入。これらの方法の中でも、還元ジメチル化（ラベリングのRedi）は、サンプルのほぼ任意のタイプのタンパク質濃度の違いを定量化するための安価な、再現性のある方法として人気を集めている。 REDI標識は、その後減少するシッフ塩基を形成し、ホルムアルデヒドとペプチドを反応させることを含むシアノ水素化による。この反応は、N-末端およびリジン側鎖およびmonomethylates N-末端のプロリンに遊離アミノ基をdimethylates。ここで説明するプロトコルは、重水素化ホルムアルデヒドとシアノ水素化ホウ素（ 図1）を使用して、「重い」のラベルとの天然の同位体分布とサンプル2の水素原子を有する試薬を使用した「光」ラベルでサンプル1中のペプチドをメチル化。光、質量分析計を用いて二つの形態間を区別するために使用される重質形態との間の6.0377 Daの質量差ペプチドの結果についての各ジメチル化アミノ基である。具体的には、ペプチドの相対存在量は、光のMS1抽出イオンクロマトグラムの面積の比（MS1ピーク面積比）と各ペプチドイオン対についての重鎖バージョンとして定量化される。タンパク質の相対存在量は、タンパク質中の全てのペプチドのうち、中央値MS1のピーク面積比として計算される。本稿では、行動のための堅牢なプロトコルを記述逆相ペプチド固相抽出、オンカラムのRedi標識、ペプチド分画塩基性pH位相（BPRP）クロマトグラフィーを反転し、StageTipsを用いてペプチド混合物を精製し（ 図2）を含むLC-MS/MSによってのRedi標識実験をING 。我々は定量的プロテオミクスのためREDIラベルを使用することの利点と制限について説明します。

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

還元ジメチル化（ラベリングのRedi）によるペプチドの安定同位体標識を正確に質量分析を使用して試料間のタンパク質発現の差異を定量化する方法である。 REDI標識は、各フリーアミンに2個のメチル基を追加するために、ホルムアルデヒドおよびシアノ水素化ホウ素ナトリウムを定期的に（光）または重水素化（重）の形式のいずれかを使用して実行されます。ここでは、REDI標識および複雑なタンパク質混合物の定量的な比較のための堅牢なプロトコルを示しています。比較のためのタンパク質試料を光や重いメチルタグ、混合し、LC-MS/MSによって共同で分析のいずれかを実行するために標識されたペプチドの中に消化される。タンパク質の相対存在量は、全MSスペクトルから抽出された成分ペプチドの重鎖及び軽標識バージョンのイオンクロマトグラムのピーク面積を比較することにより定​​量される。ここで説明する方法は、塩基性のpH REVによる逆相固相抽出によるサンプル調製、ペプチドのオンカラムREDIラベル、ペプチド分画を含んでいるersed相（BPRP）クロマトグラフィー、およびStageTipペプチド精製。当社は、安定同位体の組み込みのための他の方法に関してREDIラベルの利点と制限について説明します。我々は、サンプルのほぼすべてのタイプの中のタンパク質の存在量を比較するために、高速で安価な、そして正確な方法としてREDIのラベリングを用いた新規のアプリケーションを強調表示します。

私たちは、Saccharomyces cerevisiaeを使用して正確さ、精度、およびREDIラベルの再現性を評価し、 クロストリジウムは、全細胞溶解物をphytofermentans。まず、CのミックスのRediの標識効率を定量化セルロース（重ラベル、H）とグルコース（光ラベル、L）の培養物からタンパク質溶解物をphytofermentans。 1％ペプチド偽発見率に濾過する場合、このサンプルは、98％のRed...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

還元ジメチル化（REDIラベリング）によるペプチドの安定同位体標識は、正確な質量分析に基づく定量的プロテオミクスのための迅速、安価な戦略である。ここでは、ほぼすべてのサンプルタイプに適用することができるのRediアプローチを使用してタンパク質混合物の調製および分析のためのロバストな方法を示す。

安定同位体標識するための還元ジメチル化を用いた定量的プロテオミクス

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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Chemistry

16.0K ビュー.  Genoscope. 還元ジメチル化（REDIラベリング）によるペプチドの安定同位体標識は、正確な質量分析に基づく定量的プロテオミクスのための迅速、安価な戦略である。ここでは、ほぼすべてのサンプルタイプに適用することができるのRediアプローチを使用してタンパク質混合物の調製および分析のためのロバストな方法を示す。 

ビデオ: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

超高速液体クロマトグラフィー - 質量分析（UPLC-MS）による血清中の大規模非標的メタボロームプロファイリング

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

化学

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

質量分析法による総合的なO-結合およびスルホグライコミクスのための改良されたゲル内還元β脱離

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

コロイドプローブナノスを使用して、粒子間相互作用の定量的および定性的調査

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量的グライコミクスおよびプロテオミクス複合精製戦略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

サイズ排除ICP-MSを用いた定量Metalloproteomic分析のための基準

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

ペプチドおよびタンパク質定量を使用して自動免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

キャピラリー ゾーン電気泳動タンデム質量分析法による大規模なトップダウン プロテオミクス

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

組み合わせ前駆体等位同位体ラベリングとアイソバリックタギング(cPILOT)を使用した自動サンプル多重化

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

チューニング可能なナノスケールの次元と組成を有するマンガンフェライトクラスターの安定した水性懸濁液

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

13C6-グルコース標識を用いた薬用植物における一次臓器の同定

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

著者スポットライト: 13C6-グルコース標識と LC-MS を使用した植物一次臓器プロファイリング(英語)

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13名LC-MSに関連するC6-グルコース標識:二次代謝物合成における植物一次器官の同定

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...