This work was performed on behalf of the International Federation of Clinical Chemistry Working Group on CSF Proteins, which has the following composition: Kaj Blennow (Chair) and Henrik Zetterberg, Gothenburg University, Sweden; Les Shaw and Magdalena Korecka, University of Philadelphia, PA, USA; Ingrid Zegers, Institute for Reference Materials and Measurements, Geel, Belgium; Piotr Lewczuk, Universit&#228;tsklinikum Erlangen, Germany; and Rand Jenkins, PPD Bioanalytical Laboratory, Richmond, VA, USA; Randall Bateman, Washington University, MO, USA; and H Vanderstichele, Innogenetics NV, Ghent, Belgium. This work was also part of the Global Consortium for Biomarker Standardization CSF Reference Methods Working Group, which is led by Maria C. Carrillo, Ph.D., Senior Director, Medical &amp; Scientific Relations, Alzheimer's Association. The study was supported by The Swedish Research Council (grants #14002, #521-2011-4709, and #2013-2546); the Knut and Alice Wallenberg Foundation; Demensf&#246;rbundet; and Emil and Wera Cornell's, Aina Wallstr&#246;m and Mary-Ann Sj&#246;blom's, Gun and Bertil Stohne's, Magnus Bergvall's, and Gamla Tj&#228;narinnor's Foundations.

注：このプロトコルは、出発物質として、各Aβペプチドのために50μg/ mLの濃度で、少なくとも50μLのアリコートを必要とします。 Aβペプチドは、脱イオン水中の20％アセトニトリル（ACN）、4％濃アンモニア溶液（V / V）に溶解し、80℃で保存されるべきです。 注意：安全情報については、 表1を参照してください。 ソリューションの調製<ol><li>脱イオン水中でACN、濃縮アンモニアの4mLの（〜25％）の20 mLで希釈し、脱イオン水中の20％ACN、4％濃アンモニア溶液（v / v）を100mLのを準備します。脱イオン水で100mLへの最終的な音量を調整します。新鮮な毎日を行います。 </li><li> 50 mLの最終容量まで脱イオン水にグアニジン塩酸塩の26.08グラムを溶解することにより、5 Mグアニジン塩酸50mLのを準備します。 20℃で保存し、新鮮な毎月を作ります。 </li><li>脱イオン水中の4％リン酸（V / 200mLのを準備v）で脱イオン水中に濃リン酸（〜85％）の9.4 mLで希釈し。脱イオン水で200ミリリットルの最終容量を調整します。冷蔵庫に保管し、新鮮毎週行います。 </li><li>脱イオン水中でACN 37.5 mL及び濃アンモニア5mLの（〜25％）を希釈して、脱イオン水中で75％ACNおよび10％濃アンモニア溶液50ml（V / V）を調製します。脱イオン水で50 mLで最終容量を調整します。新鮮な毎日を行います。 </li><li>日常の臨床分析から匿名化された残りの試料から得られたキャリブレータのためのヒトCSFの少なくとも2.5mLのを、解凍します。 </li><li> 150mMのナトリウム、3.0 mMのK、1.4 mMのカルシウム、0.8 mMのマグネシウム、1.0 mMのP、および脱イオン水で155 mMののClを含む人工CSFを準備し、4ミリグラム/ mLの最終濃度にウシ血清アルブミンを追加します。唯一の1 mLを分析ごとに必要ですが、将来の使用のために大容量、一定分量を、およびストアを準備されています。 </li></ol>キャリブレーターの調製<ol><li> 4 / mlの1の0.5ミリリットルを準備20％のACNおよび0.5 mLのマイクロ遠心チューブ中の4％の濃アンモニアを50μg/ mLの15NAβ142 0.46ミリリットルの40μLを添加することにより5NAβ1-42ペプチド 。 1分間ボルテックスミキサーで混合します。 </li><li> 20％ACNおよび2 mLのマイクロ遠心チューブ中で4％の濃アンモニアの1.95 mLに50μLの4μg/ mLの15のNAβ142を追加することにより、100ng / mlの15NAβ1-42ペプチドの2ミリリットルを準備します。 1分間ボルテックスミキサーで混合します。 </li><li> 表2に示した各溶液の体積を混合することにより、6キャリブレーター溶液（AF）を準備します。 0.5、1.5、および2 mLのマイクロ遠心チューブを使用してください。 1分間ボルテックスミキサーで混合します。 </li><li> 1分間ボルテックスミキサーに、表3ミックスに応じて対応するキャリブレーションソリューションと人間のCSFを追加することにより、0.5 mLのマイクロ遠心チューブ中の最終キャリブレータ（二重に）を準備します。 </li></ol>内部標準の調製<ol><li> 20％ACNおよび2 mLのマイクロ遠心チューブ中の4％の濃アンモニアの1.968 mLに32μLを50μg/ mLの13のCAβ142を追加することにより、0.8μgの/ mLの13CAβ1-42ペプチドの2ミリリットルを準備します。 1分間ボルテックスミキサーで混合します。 </li><li> 5 mLのマイクロ遠心チューブ中の20％ACNおよび4％濃アンモニアの4.9 mLに0.8μgの/ mLの0.1mLのを追加することにより、16 ngの/ mLの13CAβ1-42ペプチドの5ミリリットルを準備します。 1分間ボルテックスミキサーで混合します。 </li></ol>レスポンスファクタのサンプルの4準備注：応答因子（RF）決意を較正するために使用標識ペプチドの濃度（15NAβ1-42）を決定するために実行されます。これは、天然Aβ1-42ペプチドの濃度は、アミノ酸分析（AAA）を用いて決定されていることを必要とします。このように、ボリュームおよびネイティブAβ1-42ペプチドのアリコートの濃度AAAの要件を満たす必要があります。 <ol><li> 20％のACNおよび0.5 mLのマイクロ遠心チューブ中で4％の濃アンモニアの0.46 mLに50μg/ mlのネイティブAβ1-42の40μLを追加することで、4μgの/ mLのネイティブ（非標識）Aβ1-42 0.5mLのを準備します。 1分間ボルテックスミキサーで混合します。 </li><li> 20％の1.96 mLに4μgの/ mLの15NAβ1-42の4 / mlのネイティブAβ1-42および20μLの20μLを追加することで、ネイティブと15NAβ1-42の2-mLの40 ngの/ mLのミックスを調製ACNおよび4％が2 mLのマイクロ遠心チューブ中のアンモニアを濃縮しました。 1分間ボルテックスミキサーで混合します。 </li><li> 0.5mLのマイクロチューブに人工CSFの0.38 mLに40 ngの/ mLの混合物の20μLを追加します。重複を準備し、1分間ボルテックスミキサーで混ぜます。 </li></ol> 5.サンプル調製注：ローラー上、室温で測定されるサンプルを解凍します。 <ol><li>（使用している場合、品質管理を含む[QC]サンプル） 図1（完全版が使用されていると仮定）によると、96ディープウェルプレート1 mLのタンパク質に、各キャリブレータの0.18 mLを加え、応答率、および未知のサンプル。でサンプルを追加してください、またはウェルの底に近いです。 </li><li>各ウェル（ すなわち、キャリブレータ、応答因子のQC、及び未知数）に内部標準の20μLを追加します。ピペットチップを浸漬することなく、試料の表面に十分に近い側のドロップを解放することが重要です。 </li><li>各ウェルに0.2 mLと5 Mグアニジン塩酸塩を追加します。 </li><li>マイクロプレートシェーカー上のサンプルプレートを配置し、1100 rpmで45分間サンプルを混ぜます。最適な周波数は、計装に応じて異なる場合があります。ソリューションが完全に混合され、内部標準またはCSFのない滴をウェルの側に混合されていない残っていないように、ミキサーの周波数と振幅を設定します。 </li><li> 4％リン酸0.2 mLを加え各ウェルに。渦を簡単に混ぜます。 </li></ol> 6.固相抽出注：すべての洗浄、ロード、および溶出工程では、溶液を添加した後、可能な限り低い真空を適用し、ロードまたは溶液を溶出するために、必要に応じて徐々に増加します。各ローディングおよび溶出工程の間に真空を無効にします。 <ol><li>混合モード下での廃棄物のための貯蔵トレーを入れ、抽出プレートマニホールド室における陽イオン交換、96ウェル固相抽出（SPE）プレート。 </li><li>各ウェルにメタノール0.2mLのを追加することにより、SPE吸着剤を調整します。 </li><li>各ウェルに、4％のリン酸を0.2mLを添加することにより吸着剤を平衡化します。 </li><li> SPEプレートにディープウェルプレートから全てのサンプル（各ウェル中に約0.62ミリリットル）を転送します。 SPEプレートにディープウェルプレートからサンプルを転送するときに8チャンネルピペットを使用します。サンプルはインターンが含まれているため、すべてのサンプルの全体または同等のボリュームを転送することが重要ではありません変動を補償しますアル標準。 </li><li>サンプルを各ウェルに4％リン酸を0.2mLを添加することによって通過した後の吸着剤を洗浄します。 </li><li>洗浄溶媒は、吸着剤から溶出した後、捕集板や管をリザーバートレイを交換してください。 </li><li>二回75％ACN / 10％濃アンモニアの50μLを添加することにより、吸着剤から試料を溶出し、そしてこの溶液が吸着剤を通過することは非常に低真空を必要とすることに注意してください。各添加の間に真空を無効にすることを忘れないでください。 <ol><li>オプション：コレクションプレートまたはチューブを密封し、-80℃でそれらを凍結。 6.8.2に進む前に、コレクションプレートまたはチューブからシールを取り外します。 </li><li> （熱を加えることなく）真空遠心分離を使用して溶出液を乾燥させます。これは、真空遠心分離に応じて、数時間に1から取ることができます。 </li><li>容器を密封し、-80℃でそれらを凍結。 </li></ol></li></ol> 7.液体クロマトグラフィー<ol><li> 、およびニードル洗浄（50％ACN（ACN [V / V]で4％の脱イオン水および0.1％濃アンモニア）、B（脱イオン水[V / V]中の5％ACNおよび0.3％濃アンモニア）移動相Aを準備4％の脱イオン水にアンモニアを濃縮[v / v]）。 <ol><li>移動相Aの500ミリリットルのために、脱イオン水にACNの25mLの濃アンモニアの1.5 mLを加え。脱イオン水で500 mLで最終容量を調整します。 </li><li>移動相Bの500ミリリットルのために、濃アンモニアの500μLとACNへの脱イオン水25 mLを加え。 ACNと500ミリリットルへの最終的な音量を調整します。 </li><li> ACN 120mLの脱イオン水に濃アンモニアの10ミリリットルを追加することにより、ニードル洗浄の250ミリリットルを準備します。脱イオン水で250 mLで最終容量を調整します。 </li><li> LCシステムで使用する前に20分間の超音波処理浴中にオープン移動相AとBとニードル洗浄瓶を入れて</li></ol></li><li> 20％のACNの25μLと、4％concentraで各サンプルを溶解テッドアンモニア溶液と20分間シェーカー上に置きます。遠心分離機のサンプルダウンして、オートサンプラーに配置します（7℃に保ちます）。 </li><li> 50℃に維持し、1×250ミリメートル2ポリスチレン-ジビニルベンゼン（逆相）モノリシックカラム上のサンプルの20μLを注入します。 <ol><li> 0.3 mL /分の流速を用いて、表4に示すLC勾配を使用します。質量分析計の汚染を低減するために迂回バルブを使用して（ポストカラム）を無駄に2最初と最後の5分をそらします。 </li></ol></li></ol> 8.質量分析注：これらのパラメータはaheatedエレクトロスプレーイオン化源を備えた四重極オービトラップハイブリッド質量分析計のために使用しました。 <ol><li> 表5に記載のイオン源のパラメータを設定します。 </li><li> （ネイティブAβ1-42の4+充電状態を分離するために1,129.48質量をMS機器を設定します2.5メートル/ zのの分離幅と四重極型質量分析計における電荷比[ のm / z]）、1-42 15NAβ（1,143.00 のm / z）、および13CAβ1-42（1,179.50 のm / z）。 </li><li> 17.0の正規化衝突エネルギー（NCE）との衝突セル内の単離されたペプチドを断片化します。 （このようなトリプル四重極MSなどの器具の他のタイプを、使用している場合は特に）であっても、同じタイプのこれは、各楽器に合わせて調整する必要がある場合があります。 </li><li> 2×10 5の電荷の自動利得制御対象と250ミリ秒の最大噴射時間で、17.500の分解能でフラグメントスペクトルを記録します。 </li></ol> 9.データ処理<ol><li>各ペプチドのためのクロマトグラフィーの領域を計算するために、 表6に（±250ミリ質量単位[MMU]の質量公差）プロダクトイオンの合計を使用してください。イオンの種類と数は、ネイティブAβのために示されていることに注意してください<suB&gt; 1-42プロダクトイオン、彼らは15NAβ1-42と13CAβ1-42の両方で同じであるため。 </li><li>ネイティブAβ1-42の曲線下面積を持つ15NAβ1-42の曲線（クロマトグラフピーク）の下の領域を分割して2つの応答因子サンプルの平均応答係数を決定します。 </li><li>ステップ9.2で算出された応答因子でそれを掛けることによって、キャリブレーションのために使用される15NAβ1-42の濃度を調整します。 </li><li>濃度に対するキャリブレータの2セットから13CAβ1-42に15NAβ1-42の面積比をプロットして検量線を作成します。 </li><li>線形回帰を用いて検量線の傾きと切片を計算します。 </li><li>アンのための内部標準（13CAβ1-42）にネイティブAβ1-42の面積比を計算します既知のサンプル。 </li><li>ステップ9.5で得られた傾きと切片を用いて、検量線から未知のサンプルの濃度を外挿する。 </li></ol>

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<li>Pannee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reference+measurement+procedure+for+CSF+amyloid+beta+%28A%CE%B2%291-42+and+the+CSF+A%CE%B21-42+%2FA%CE%B21-40+ratio+-+a+cross-validation+study+against+amyloid+PET%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Reference measurement procedure for CSF amyloid beta (Aβ)1-42 and the CSF Aβ1-42 /Aβ1-40 ratio - a cross-validation study against amyloid PET.</a> J Neurochem. 139 (4), 651-658 (2016).</li>
<li>Thienpont, L. M., Van Houcke, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traceability+to+a+common+standard+for+protein+measurements+by+immunoassay+for+in-vitro+diagnostic+purposes%5BTitle%5D)+AND+%22Clin+Chim+Acta%22%5BJournal%5D)">Traceability to a common standard for protein measurements by immunoassay for in-vitro diagnostic purposes.</a> Clin Chim Acta. 411 (23-24), 2058-2061 (2010).</li>
<li>Vesper, H. W., Thienpont, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traceability+in+laboratory+medicine%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Traceability in laboratory medicine.</a> Clin Chem. 55 (6), 1067-1075 (2009).</li>
</ol>

JP and EP reports no disclosures. HZ has served on the advisory boards of Roche Diagnostics, Eli Lilly, and Pharmasum Therapeutics. KB has served as a consultant or on the advisory boards for Fujirebio Europe, IBL International, and Roche Diagnostics. HZ and KB are co-founders of Brain Biomarker Solutions in Gothenburg AB, a GU Venture-based platform company at the University of Gothenburg.

記載された方法のために、代わりに代理母材を使用して、我々は、ヒトCSF中にキャリブレーションを可能にする、代用分析物アプローチ13、14、15、16を用います。サロゲートの検体のアプローチは、二つの異なる同位体標識された規格を必要とします。別の（13CAβ142）を内部標準?...

アルツハイマー病（AD）は、認知症の最も一般的な形態であり、全世界で約35万人1に影響を与えます 。広くADの病因の中心に位置するように考えられて疾患の神経病理学的特徴は、過リン酸化タウ蛋白質2の細胞内神経原線維変化および凝集したアミロイドβ（Aβ）ペプチド3からなる細胞外斑です。これに伴い、バイオマーカーによって、in vivoでのプラーク病変の評価は最近、AD 4のための研究診断基準に含まれています。 Aβ1-42のCSF測定のために、いくつかのイムノアッセイが利用可能であり、多くの臨床検査室5で使用されています。 CSF中のAβ1-42の濃度は、BRでのプラーク中のペプチドの沈着を反映し、認知正常な高齢者に比べてAD患者では約50％低くなっていますAIN 6、7。 これらのバイオマーカーは、主に免疫アッセイ（ すなわち、抗体ベースの技術）を用いて分析されているが、これらのアッセイは、マトリックス効果8によって影響され得ます。異なるテクノロジプラットフォームイムノアッセイおよびアッセイの標準化9の欠如の使用は、10は 11、12困難なグローバルなカットオフ濃度の導入を行います。分析的に検証さRMPは、理想的には、分析プラットフォーム間でより良い比較では、全体的な測定のばらつきの要因のより良好な制御が得られ、異なるアッセイプラットフォームの均一なキャリブレーションを可能にするであろう。 開発されたLC-MS / MS法を用いてAβ1-42の絶対的な定量化は、抗体ベースのTECHNに関連する問題の多くを克服しますiques。検査医学（JCTLMデータベース識別番号C11RMP9）におけるトレーサビリティのための合同委員会がRMPとしてリストされている方法は、CSFAβ1を調和させる認証標準物質（CRM）におけるAβ1-42の絶対濃度を決定するために使用されます技術および分析プラットフォーム間-42測定。説明ワークフローは、医学の他の領域内のペプチドおよびタンパク質のための候補参照方法の開発のための関連性のものでなければなりません。

<table><tbody><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 2 mL</td><td>Eppendorf</td><td>022431102</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 5 mL</td><td>Eppendorf</td><td>0030 108.310</td><td></td></tr><tr><td>Eppendorf Protein LoBind Deepwell Plates 96</td><td>Eppendorf</td><td>951032905</td><td></td></tr><tr><td>Micronic 0.75 mL polypropylene tubes, V-bottom</td><td>Micronic</td><td>MPW32071BC3</td><td>Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates.</td></tr><tr><td>Micronic Split TPE Capcluster for 96 ind. tubes</td><td>Micronic</td><td>MP53026</td><td>Caps for Micronic tubes.</td></tr><tr><td>Micronic Loborack-96 white w high cover bar coded</td><td>Micronic</td><td>MPW51015BC3</td><td>Holder for Micronic tubes.</td></tr><tr><td>Biohit Optifit tips 1.2 mL</td><td>Sartorius</td><td>791210</td><td>Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well.</td></tr><tr><td>Waters Oasis MCX 96-well &amp;micro;Elution Plate</td><td>Waters</td><td>186001830BA&amp;nbsp;</td><td></td></tr><tr><td>Waters Plate manifold reservoir tray</td><td>Waters</td><td>WAT058942</td><td></td></tr><tr><td>Waters Extraction Plate Manifold for Oasis 96-Well Plates</td><td>Waters</td><td>186001831</td><td></td></tr><tr><td>Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis</td><td>Sigma-Aldrich</td><td>30501-1L-D</td><td></td></tr><tr><td>Acetonitrile, Far UV HPLC Gradient grade, 2.5 L</td><td>Fisher Scientific</td><td>A/0627/17X</td><td></td></tr><tr><td>ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur</td><td>Merck Millipore</td><td>1005731000</td><td></td></tr><tr><td>Guanidine Hydrochloride, 500 g</td><td>Thermo Scientific</td><td>24110</td><td></td></tr><tr><td>Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer</td><td>Thermo Scientific</td><td>IQLAAEGAAPFALGMAZR</td><td>With bundled Dionex Ultimate 3000 LC &amp;amp; autosampler.</td></tr><tr><td>Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm)</td><td>Dionex</td><td>066640</td><td></td></tr><tr><td>Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated</td><td>Sigma-Aldrich</td><td>B6917</td><td></td></tr><tr><td>Beta-Amyloid (1-42), Ultra Pure, TFA</td><td>rPeptide</td><td>A-1002-2</td><td></td></tr><tr><td>15N Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1102-2</td><td></td></tr><tr><td>13C Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1106-2</td><td></td></tr><tr><td>Microplate shakers, TiMix 2</td><td>Edmund B&amp;uuml;hler</td><td>6110 000</td><td></td></tr></tbody></table>

absolute quantification of a&#946;1-42 in csf using a mass spectrometric reference measurement procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

図1のプレート設定は、サンプルの完全版のために使用されます。少数の未知の試料を分析する場合は、第二校正器、RF、およびQCセットは未知サンプルの前半の後に配置する必要があります。 図2に見られるように、校正器は、低い標準偏差を用いて、回帰直線に近いです。この方法は、150 pg / ml...

Watch this Scientific Journal Video about Absolute Quantification of AÎ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Aβの絶対定量<sub&gt; 1-42</sub&gt; CSFに質量分析リファレンス測定手順を使用して

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

absolute-quantification-a1-42-csf-using-mass-spectrometric-reference

Research

JoVE Journal

Medicine

7.6K ビュー.  The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital. A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

ビデオ: Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

ヒト皮質組織におけるAβ多量体のSDS-PAGE/Immunoblotの検出は、抗原エピトープの検索を使用してホモジネート

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

神経科学

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

生化学

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

Β アミロイド誘起分光と電子顕微鏡によるフィブリン血栓構造異常の解析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

神経変性のバイオマーカーおよびアポリポタンパク質Eアイソフォームの状態を定量化するために高スループット、多重化およびターゲットプロテオームCSFアッセイ

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

医学

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

ひと脳脊髄液中の Endopeptidomic の分析のための試料作製

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

麻酔下のマウスから脳脊髄液のコレクションのための改良法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

マトリックス支援レーザー脱離イオン化質量顕微鏡を用いたひと脳のアミロイド β の堆積物の可視化

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

マウスにおけるインテコール内合成タンパク質の定量測定

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

マウス脳切片におけるアミロイドβ負荷の完全領域対小領域定量化

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Aβの絶対定量

要約

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