Name: peptide and protein quantification using automated immuno-maldi (imaldi)
Uploaded: 2017-08-18T12:30:00.000+00:00
Duration: 8 min 57 s
Description: Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

ゲノム技術革新ネットワーク (ジン) をゲノム カナダ、操作 (204PRO) および技術開発 (214PRO) のゲノム ブリティッシュ コロンビア州からの資金援助に感謝いたします。撮影用飛行時間測定器の使用のマギル大学健康センター研究所薬剤発見プラットフォームをありがちましょう。H.L. 国立科学工学研究協議会のカナダ (レベル) からポスドク研究員プログラムからサポートに感謝です。C.H.B は最先端基金基金 (葉っぱ) からのサポートに感謝しています。えざるはマギル大学 (モントリオール, ケベック州, カナダ) 分子腫瘍学のシーガル マギル椅子からのサポートに感謝します。M.X.C. とえざるがユダヤ人の総合病院 (モントリオール, ケベック州, カナダ) にウォーレン Y. ソーパー慈善信託とアルヴィン シーガル家族財団から支援に感謝。

下記試薬の量は 20 の患者血漿検体の測定に基づいています。プロトコルはビクトリア大学のガイドライン以下提示 &#39; s 人間研究倫理委員会.
 1 アン世代ひと血漿中私 <ol><li>血漿検体を解凍 (&#8805; 200 μ l) の部屋で温度水は 5 分のお風呂と、完全に解凍するまで氷の上にサンプルを置く。</li>。
	<li>1.1 mL ディープウェル プレート (サンプル プレート) の井戸を分離し、遠心分離機の 2 ° C および 1278 x g で 10 分間遠心でプレートに手動で各血漿サンプルの転送 200 μ l</li>
	<li>リキッドハンド リング システムを使用して、連続希薄と鶏卵白アルブミン 6 校正ソリューションを準備する 500 fmol/μ L Ang I NAT の標準溶液 (0.1、0.2、0.6、1.9 5.7, 17.2 fmol/μ L).</li>
	サンプル プレートに <li>世代のも、125 μ L を各キャリブレータのピペット 200 μ L バッファーします 。
	注: CEWA のリン酸バッファー生理食塩水 (PBS) バッファーは実験の日に新たに作成される必要があります</li>。
	<li>リキッドハンド リング システムを使用して、新しいプレートのミックス プラズマの培養上清中の 125 μ L または 125 μ L の PBS の CEWA のアンの 25 μ L で私世代バッファー 1 M トリス、0.2 mM エチレンジアミン四酢酸 (EDTA)、1 mM を含む特異フッ化物 (PMSF). 
	注: 準備、Ang 私 1 M トリス/0.2 mM EDTA 水系バッファーをミキシングによって生成バッファー (pH 5.5 に調整酢酸を使用して) そして PMSF ソリューション (メタノール 100 mM)。両方のソリューションで、実験の日に 4 ° c. のミックス 2 つのソリューションで 1 ヶ月格納できます</li>。
	<li>は、96 ウェル プレート、1 ソリューションにつき 3 複製、34 μ L/ウェルにソリューションを自動的に転送します。3 h の 37 ° C でプレートを孵化させなさい</li>
</ol> 2。アンの免疫強化私を使用して抗体をコートしたビーズ <ol><li>蛋白質 G 磁気ビーズに抗体の共役 注: ビーズを抗体の共役が 3 h アンの中に手動で実行される私の発生期、実験の日。その他の検体の共役ビーズ プロパティおよび抗体の安定性に応じて 3 ヵ月以上、4 ° C で 0.015% チャップス (きて) を含む PBS バッファーに格納することができるかもしれない。
	<ol><li>(20 のサンプルを測定し、6 点の標準曲線を作る十分な) ビーズ スラリーの転送 110 μ L 1.5 mL チューブに。ビーズ 7 回 1 mL 25% アセトニ トリル/きてときて 1 mL で 3 回洗います。各洗浄工程間ビーズをペレットにマグネット スタンドを使用します。最後のワシントン州後に洗浄バッファーを削除 
		注意: 洗浄 7 回 25% アセトニ トリル/きてはビーズ スラリー、Tween 20 MS 互換性のない添加物を除去するため重要です。選択したビーズではこのような添加剤は、この広範な洗浄工程必要がありますできません</li>。
		<li>きて、110 μ のビードを再停止しなさい、反中央の 110 μ L を追加私抗体 (最終的な抗体濃度: 100 μ g/mL)。ミックス ビーズとソリューションをピペッティングと、それらを 8 rpm で回転、1 時間室温でインキュベートします</li>。
		<li>3 回きて、1 mL でビーズを洗ってきて 1100 μ で再懸濁します 。
		注: 場合はビード ソリューションは、インキュベーション中にチューブのキャップに流れ込んできた、スピンダウン 2680 x g にベンチトップ遠心分離機を使用して、ソリューション</li>
		<li>は、96 ウェル プレート (ビーズ標準プレート) にビーズ ソリューションを手動で転送します。リキッドハンド リングの因数にシステム 120 μ L/ウェル同じ 96 ウェル プレートにビーズを使用して、</li></ol></li> <li>ビーズの免疫強化 <ol><li>後 3 h アン発生期培養プレートに。アン I. の生成を終了する 10 分の氷</li>
		<li>は自動的に SIS ペプチド原液 (10 pmol/μ L) を 100 倍希釈 PBS で、バッファーに格納し、自動的に更に因数 96 ウェル PCR プレートに安定した isoptope 標準的なペプチド希釈。培養プレートの各ウェルに 1.5 μ L (100 fmol 含む) 安定同位体標準 (SIS) ペプチド溶液を転送し、血漿検体または PBS バッファーで CEWA ミックスします</li>。
		<li>ビーズのソリューションは、ウェルあたり 10 mL に培養プレートの内容を転送、ミックスを自動的にします</li>。
		<li>1 h 4 ° C でプレートを 8 回転で回転させながらインキュベートします</li>。
		<li>ビーズにて 3 回洗浄自動的に 5 mM 重炭酸アンモニウム (アンビック) ソリューションでは、100 μ L/ウェル洗浄あたり。最後の洗浄後 5 mM 各ウェルに碧のソリューションの 7 μ L でビーズを再懸濁します。最後のワシントン州の後下部にビーズをプルする磁石を使用</li>
	</ol></li></ol> 3。MALDI ターゲット プレートと追加するマトリックス ソリューションにビーズの転送 <li>2,600 のスポット サイズと MALDI ターゲット プレートの上に自動的にビーズ スラリーの転送 7 μ <ol>μ m。ビーズが乾くみましょう 。
	注: 小型 USB 電源ファンはビーズ乾燥過程を加速するため使用できます</li>。
	<li>は自動的に α-シアノ-4-ヒドロキシケイ皮酸 (HCCA) の 2 μ L を追加-各サンプル ターゲット プレート上のスポットにはうまく行列から行列ソリューション (3 mg/mL HCCA、クエン酸アンモニウム 1.8 mg/mL、70% アセトニ トリル、0.1% トリフルオロ酢酸を含む).</li>
</ol> 4。MALDI TOF MS とデータ解析による買収を信号 正反射モードを使用して MALDI-TOF 楽器 <ol><li>分析サンプル スポット。内部校正、データの平滑化、およびベンダー固有のソフトウェアで自動的に基線の減算を実行します 。
	注: (正/負、線形/リフレクター) 選択したモード MALDI-TOF 測定対象のペプチッドまたは蛋白質によって異なります</li>。
	<li>相対応答比 (Nat/SIS 強度比) を計算して、アンの私を決定する標準的なカーブに各サンプルの濃度を比較すること。計算式 (1) を使用して、プラ Δt アン私世代 Ang i. の生成に使用される時刻を表す 
	プラ = [アン私]/Δt アン私世代 (1)</li></ol>

<ol>
	<li>Aebersold, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics.">Mass spectrometry-based proteomics.</a> Nature. 422 (6928), 198-207 (2003).</li><li>Nicol, G. R., 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=Use+of+an+Immunoaffinity-Mass+Spectrometry-based+Approach+for+the+Quantification+of+Protein+Biomarkers+from+Serum+Samples+of+Lung+Cancer+Patients.">Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients.</a> Mol. &amp; Cell. Proteomics. 7 (10), 1974-1982 (2008).</li><li>Webster, J., Oxley, D., Zanders, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Genomics and Proteomics: Reviews and Protocols. , 227-240 (2012).</li><li>Yergey, A. 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=De+novo+sequencing+of+peptides+using+MALDI/TOF-TOF.">De novo sequencing of peptides using MALDI/TOF-TOF.</a> J. Am. Soc. Mass Spectrom. 13 (7), 784-791 (2002).</li><li>Anderson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+decades+searching+for+meaning+in+the+proteome.">Six decades searching for meaning in the proteome.</a> J. Proteomics. 107, 24-30 (2014).</li><li>Landegren, U., 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=Opportunities+for+Sensitive+Plasma+Proteome+Analysis.">Opportunities for Sensitive Plasma Proteome Analysis.</a> Anal. Chem. 84 (4), 1824-1830 (2012).</li><li>Guo, A., 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=Immunoaffinity+Enrichment+and+Mass+Spectrometry+Analysis+of+Protein+Methylation.">Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation.</a> Mol. &amp; Cell. Proteomics. 13 (1), 372-387 (2014).</li><li>Florentinus-Mefailoski, A., Soosaipillai, A., Dufresne, J., Diamandis, E. P., Marshall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-linked+immuno-mass+spectrometric+assay+with+the+substrate+adenosine+monophosphate.">An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate.</a> Anal. Bioanal. Chem. 407 (4), 1119-1130 (2015).</li><li>Fredolini, C., 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=Immunocapture+strategies+in+translational+proteomics.">Immunocapture strategies in translational proteomics.</a> Expert Rev Proteomics. 13 (1), 83-98 (2016).</li><li>Tran, J. C., 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=Automated+Affinity+Capture+and+On-Tip+Digestion+to+Accurately+Quantitate+in+Vivo+Deamidation+of+Therapeutic+Antibodies.">Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies.</a> Anal. Chem. , (2016).</li><li>Razavi, M., Leigh Anderson, N., Pope, M. E., Yip, R., Pearson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+precision+quantification+of+human+plasma+proteins+using+the+automated+SISCAPA+Immuno-MS+workflow.">High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.</a> N. Biotechnol. 33 (5 Part A), 494-502 (2016).</li><li>Razavi, M., 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=High-Throughput+SISCAPA+Quantitation+of+Peptides+from+Human+Plasma+Digests+by+Ultrafast,+Liquid+Chromatography-Free+Mass+Spectrometry.">High-Throughput SISCAPA Quantitation of Peptides from Human Plasma Digests by Ultrafast, Liquid Chromatography-Free Mass Spectrometry.</a> J. Proteome Res. 11 (12), 5642-5649 (2012).</li><li>Anderson, N. 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=SISCAPA+Peptide+Enrichment+on+Magnetic+Beads+Using+an+In-line+Bead+Trap+Device.">SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device.</a> Mol. &amp; Cell. Proteomics. 8 (5), 995-1005 (2009).</li><li>Parker, C. E., Pearson, T. W., Anderson, N. L., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometry-based+clinical+proteomics+-+a+review+and+prospective.">Mass-spectrometry-based clinical proteomics - a review and prospective.</a> The Analyst. 135 (8), 1830-1838 (2010).</li><li>Reid, J. D., Holmes, D. T., Mason, D. R., Shah, B., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+Development+of+an+Immuno+MALDI+(iMALDI)+Mass+Spectrometry+Assay+for+the+Diagnosis+of+Hypertension.">Towards the Development of an Immuno MALDI (iMALDI) Mass Spectrometry Assay for the Diagnosis of Hypertension.</a> J. Am. Soc. Mass Spectrom. 21 (10), 1680-1686 (2010).</li><li>Mason, D. R., Reid, J. D., Camenzind, A. G., Holmes, D. T., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexed+iMALDI+for+the+detection+of+angiotensin+I+and+angiotensin+II.">Duplexed iMALDI for the detection of angiotensin I and angiotensin II.</a> Methods. 56 (2), 213-222 (2012).</li><li>Camenzind, A. G., vander Gugten, J. G., Popp, R., Holmes, D. T., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+immuno-MALDI+(iMALDI)+assay+for+angiotensin+I+and+the+diagnosis+of+secondary+hypertension.">Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension.</a> Clin. Proteomics. 10 (1), 20 (2013).</li><li>Jiang, J., 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=Development+of+an+immuno+tandem+mass+spectrometry+(iMALDI)+assay+for+EGFR+diagnosis.">Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosis.</a> Proteomics Clin Appl. 1, (2007).</li><li>Jiang, J., Parker, C. E., Fuller, J. R., Kawula, T. H., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Immunoaffinity+Tandem+Mass+Spectrometry+(iMALDI)+assay+for+Detection+of+Francisella+tularensis.">An Immunoaffinity Tandem Mass Spectrometry (iMALDI) assay for Detection of Francisella tularensis.</a> Analytica chimica acta. 605 (1), 70-79 (2007).</li><li>Popp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+assay+for+the+clinical+measurement+of+plasma+renin+activity+by+immuno-MALDI+(iMALDI).">An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI).</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1854 (6), 547-558 (2015).</li><li>Schoenherr, R. M., 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=Automated+screening+of+monoclonal+antibodies+for+SISCAPA+assays+using+a+magnetic+bead+processor+and+liquid+chromatography-selected+reaction+monitoring-mass+spectrometry.">Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry.</a> J Immunol Methods. 353 (1-2), 49-61 (2010).</li><li>Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+quantitation+and+identification+of+peptides+and+proteins.">Methods of quantitation and identification of peptides and proteins.</a> US patent. , (2010).</li><li>Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+quantitation+and+identification+of+peptides+and+proteins.">Methods of quantitation and identification of peptides and proteins.</a> Canadian patent CA. , (2011).</li><li>Wei&#223;, F., 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=Catch+and+measure-mass+spectrometry-based+immunoassays+in+biomarker+research.">Catch and measure-mass spectrometry-based immunoassays in biomarker research.</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1844 (5), 927-932 (2014).</li><li>Nelson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+fragments:+Hope+and+hype.">Antibody fragments: Hope and hype.</a> mAbs. 2 (1), 77-83 (2010).</li><li>Gupta, V., 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=An+evaluation+of+an+aptamer+for+use+as+an+affinity+reagent+with+MS:+PCSK9+as+an+example+protein.">An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.</a> Bioanalysis. 8 (15), 1557-1564 (2016).</li></ol>

C.H.B は、iMALDI 技術の特許を保持しています。

従来の MS を用いたタンパク質定量と比較して、iMALDI は、分析検体を豊かにし、したがって、それはタンパク質またはペプチド低濃度を定量化することが可能、複雑なサンプルからそれらを浄化する抗体を使用します。IMALDI プロトコルの重要なステップは、ターゲット ペプチドの免疫強化です。このため、高い特異性と親和性の抗体を選択してください。SISCAPA、内 10-9 M 以上で抗体?...

質量分析法による定量的プロテオミクスにおける必要不可欠なツールとなっています。質量分析法は、ターゲット蛋白質またはペプチッドの固まりを判断できます、したがって得られた試料信号することができます非常に具体的イムノアッセイと比較しています。エレクトロ スプレーと MALDI は、2 つのイオン化方法は、タンパク質・ ペプチド1,2,3、4を検出するためによく使用されます。MS を用いたタンパク質定量における大きな課題は、高豊富な蛋白質の存在下で ng/mL または pg/mL の濃度で複雑な試料中の低豊富なタンパク質の検出、ひと血漿中発見多く候補蛋白質バイオ マーカーこれの範囲の5を実行します。この問題は、本質的に広いダイナミック レンジおよび人間の proteome6の複雑さが主原因です。
ターゲット蛋白質または固体表面上にサンプル ソリューションからペプチドを豊かにする方法が免疫 MS 検出課題を克服するために開発されて、検体と MS 測定7,8の溶出順,9,10します。 複雑なサンプルから濃縮、免疫を介して、検体を精製し、他の分子からイオン抑制効果を最小化するため。固体の様々 なサポートの中では、扱いやすさと高い抗体結合能力の利点を有するので磁気ビーズは最も広く使用現在。別の functionalizations とサイズの磁気ビーズを開発し、免疫沈降実験のため商品化します。日には、ビーズに免疫強化をエレクトロ スプレー イオン化 (ESI) と MALDI MS タンパク質およびペプチドの測定のためインターフェースされてが。安定同位体標準および抗ペプチド抗体 (SISCAPA) 技術による捕獲は、免疫強化のための抗体をコートしたビーズと孵化によって続いて、試料中のタンパク質は消化されます。「古典的な」SISCAPA でキャプチャ proteotypic ペプチドが、ビーズから溶出し、測定による液体クロマトグラフィー-MS (LC-MS)、または直接注入 ESI 複数反応モニタリング MS (ESI MRM MS)11,12。免疫強化は、ng/mL の低範囲13に達する 3-4 桁 MRM アッセイ感度を向上します。
エレクトロ スプレー MS と比較して、MALDI MS の高速、かつを含まない洗浄と LC カラムの再平衡持ち越しと汚染の問題がないのでより高スループット研究14に適しています。ペプチッドおよび蛋白質 (proteotypic ペプチドの定量に基づく) の特異的かつ高感度定量 MALDI MS と免疫強化を結合する当研究室で開発した免疫 MALDI 技術15,16 ,17。免疫濃縮後 MALDI ターゲット プレートにビーズを堆積、マトリックス ソリューション ビーズに追加されます、乾燥後プレートは MALDI TOF MS による分析の準備ができて。ビーズからペプチドの溶出は別のステップとして実行されませんが、アフィニティ バインド analytes は MALDI のマトリックス ソリューションによって溶離されるビード スポット、サンプル準備を簡素化し、サンプルの損失を最小限に抑えることに追加するとき。IMALDI 技術適用されているさまざまなアプリケーション18,19で、最近自動化されアンジオテンシンを測定するために使用私 (Ang 私) 血漿レニン活性 (PRA)20を決定します。このプロトコルは、自動 iMALDI 分析を実行するための手順を説明します。例については、10% 未満の間日 CVs としてプラ アッセイを取って日20あたり 744 のサンプルを測定する機能を備えた、自動化によって達成されています。
本稿で示した iMALDI プラ アッセイはターゲット分子としてのタンパク質消化を必要としない (アン私) 1295.7 Da の分子量のペプチドです。C とそれを簡単に区別できるように 800 Da 以上の質量を持つ、ターゲット蛋白質に固有にする必要があります iMALDI の選択したペプチドその他試金蛋白質消化が必要あり、ペプチドはそのまま蛋白質の代理として使用される、MALDI のマトリックス ソリューションから 2・3 のノイズ。抗ペプチド抗体、ペプチドの免疫強化に必要です。PRA を測定 iMALDI 試金のためのプロトコルは 4 つのステップで構成されています: 1) アンの世代人間プラズマで2) 免疫-豊かなアンの私は抗体をコートしたビーズ; を使用して3) ビーズの MALDI ターゲット プレートと追加するマトリックス ソリューションへの転送・ 4) 信号集録 MALDI TOF MS とデータ解析20。

<table><tbody><tr><td>Healthy control human plasma</td><td>Bioreclamation</td><td>HMPLEDTA2</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma Aldrich</td><td>09830</td><td></td></tr><tr><td>Ammonium citrate dibasic</td><td>Sigma Aldrich</td><td>09833</td><td></td></tr><tr><td>CHAPS (&gt;=98%)</td><td>Sigma Aldrich</td><td>C9426</td><td></td></tr><tr><td>Albumin from chicken egg white (&gt;98%)</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma Aldrich</td><td>EDS</td><td></td></tr><tr><td>Alpha-cyano-4-hydroxycinnamic acid</td><td>Sigma Aldrich</td><td>70990</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma Aldrich</td><td>78830</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Thermo Fisher Scientific</td><td>85172</td><td>LC-MS grade</td></tr><tr><td>acetonitrile</td><td>Fluka</td><td>34967</td><td>LC-MS grade</td></tr><tr><td>water</td><td>Fluka</td><td>39253</td><td>LC-MS grade</td></tr><tr><td>acetic acid</td><td>Fluka</td><td>320099</td><td>LC-MS grade</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Roche Diagnostics</td><td>3118169001</td><td></td></tr><tr><td>Dynabeads Protein G magnetic beads</td><td>Thermo Fisher Scientific</td><td>10003D</td><td>2.8 &lt;span class=&quot;font5&quot;&gt;&amp;mu;m, 30 mg/mL&lt;/span&gt;</td></tr><tr><td>anti-Ang I goat polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-7419</td><td></td></tr><tr><td>Nat and SIS Ang I</td><td>synthesized at the University of Victoria-Genome BC Proteomics Centre</td><td></td><td></td></tr><tr><td>Automated liquid handling system</td><td>Agilent</td><td>16050-102</td><td>Agilent Bravo robotic workstation</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td>Invitrogen DynaMag-2 magnet</td></tr><tr><td>Tube rotator</td><td>Theromo Scientific</td><td>400110Q</td><td>Labquake Tube Rotator</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td>DynaMag-96 side skirted magnet</td></tr><tr><td>Magnet</td><td>VP Scientific</td><td>771RM-1</td><td>used to pull the beads to the bottom of the well</td></tr><tr><td>MALDI-TOF</td><td>Bruker</td><td></td><td>Bruker Microflex LRF instrument</td></tr></tbody></table>

peptide and protein quantification using automated immuno-maldi (imaldi)

質量分析 (MS) は、各ターゲット分子の質量電荷比の直接検出の結果として優秀なアッセイの特異性と複雑な試料中のタンパク質を定量化するための最も一般的に使用される技術の一つです。しかし、他のほとんどの分析技術のような MS ベースのプロテオミクスは高豊富な検体を測定への偏り、検出限界は低い ng/mL のまたは pg/mL で複雑なサンプル、およびこれは多くの濃度範囲を達成するために困難ですのでひと血漿など生体の疾患関連蛋白質。低豊富な検体の検出を支援するには、免疫強化を集中し、MS の測定、アッセイの感度が大幅に向上する前に検体を浄化するアッセイに統合されています。この作品で、免疫-マトリックス レーザー脱離イオン化 (iMALDI) 技術がタンパク質及びペプチドの生体の定量化のために提示はビーズに免疫強化に基づく、事前の溶出なし MALDI MS 測定が続きます。抗ペプチド抗体は磁気ビーズの官能基化、サンプルと孵化させます。洗浄後、ビーズは MALDI ターゲット プレート上に直接転送し、マトリックス ソリューションがビーズに適用された後、飛行 (MALDI-TOF) 計器の MALDI 時間は信号を測定します。その他免疫 MS の試金に比較サンプルの準備の手順を簡略化し、MALDI の測定は高速。改善された分析の再現性と高スループット処理システム、液体全体サンプル準備が自動化されます。この記事で iMALDI 試金はペプチド アンジオテンシンを決定するため使用される私 (Ang 私) 原発性アルドステロン症 (PA) のスクリーニングのため血漿レニン活性の読み出しとして臨床的に使用されている血漿中濃度。

アンを測定するための自動 iMALDI 手順は図 1にされます。ターゲット ペプチド (内因性ペプチドまたは消化タンパク質由来のペプチド)、抗ペプチド磁気ビーズの濃縮し、MALDI 測定用ターゲット プレートにビーズを転送するし。追加ペプチド溶出のステップを必要とする他の免疫 MS 技術と比較して、全体の手順を簡略化します。IMALDI アッセイの?...

Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

自動免疫 MALDI (iMALDI) 技術を使用して複雑な体液でプロテインの定量のためのプロトコルが表示されます。

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 ...

peptide-and-protein-quantification-using-automated-immuno-maldi-imaldi

Research

JoVE Journal

Chemistry

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

7.9K ビュー.  University of Victoria-Genome BC Proteomics Centre. 自動免疫 MALDI (iMALDI) 技術を使用して複雑な体液でプロテインの定量のためのプロトコルが表示されます。

ビデオ: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

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 ...

化学

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

バイオマーカー探索のためのメソポーラスシリカ薄膜の低分子量蛋白質の濃縮

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工学

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

パーキンソン病における神経ペプチドのMALDIイメージングマススペクトロメトリー

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

医学

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

ペプチド濃縮と質量分析によるタンパク質ユビキチン化部位の検出

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

生化学

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

機能的モチーフとその結合パートナーのペプチドベース識別

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

免疫・感染学

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 ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

免疫ペプチドミクス:マウスとヒトMHCクラスI-およびII関連ペプチドの分離による質量分析

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

生物学

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...