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.

Hinweis: Dieses Protokoll erfordert Aliquots von mindestens 50 &amp; mgr; l, mit einer Konzentration von 50 ug / ml für jedes Aß-Peptid, als Ausgangsmaterial. Die Aß-Peptide sollten in 20% Acetonitril (ACN) und 4% konzentrierter Ammoniaklösung in entionisiertem Wasser (v / v) und gelagert bei 80 ° C aufgelöst werden. Achtung: Siehe Tabelle 1 für Sicherheitshinweise. 1. Herstellung der Lösungen <ol><li> Herstellung von 100 ml 20% ACN und 4% konzentrierter Ammoniaklösung in entionisiertem Wasser (v / v) mit 20 ml ACN verdünnt und 4 ml konzentrierter Ammoniak (~ 25%) in deionisiertem Wasser. Stellen Sie das Endvolumen auf 100 ml mit VE-Wasser. Machen Sie jeden Tag frisch. </li><li> Vorbereitung 50 ml 5 M Guanidin-Hydrochlorid durch Auflösen von 26,08 g Guanidin-Hydrochlorid in deionisiertem Wasser auf ein Endvolumen von 50 mL. Aufbewahren bei 20 ° C und frisch monatlich machen. </li><li> Bereiten 200 ml 4% ige Phosphorsäure in deionisiertem Wasser (v /v) von 9,4 ml konzentrierter Phosphorsäure verdünnen (~ 85%) in destilliertem Wasser. Stellen Sie das Endvolumen auf 200 ml mit VE-Wasser. Im Kühlschrank aufbewahren und wöchentlich frisch zu machen. </li><li> Vorbereitung 50 ml 75% ACN und 10% konzentrierter Ammoniaklösung (v / v) in entionisiertem Wasser mit 37,5 ml ACN verdünnt und 5 ml konzentriertem Ammoniak (~ 25%) in deionisiertem Wasser. Stellen Sie das Endvolumen auf 50 ml mit VE-Wasser. Machen Sie jeden Tag frisch. </li><li> mindestens 2,5 ml menschlichen CSF für die Kalibratoren auftauen, von de-identifizierte übrig gebliebenen Proben aus der klinischen Routine-Analyse erhalten. </li><li> Bereiten künstlichen CSF, enthaltend 150 mM Na, 3,0 mM K 1,4 mM Ca, 0,8 mM Mg, 1,0 mM P, und 155 mM Cl in deionisiertem Wasser und fügen Rinderserumalbumin in einer Endkonzentration von 4 mg / mL. Nur 1 ml pro Analyse benötigt, sondern bereiten ein großes Volumen, aliquoten es und Speicher für den späteren Gebrauch. </li></ol> 2. Herstellung der Kalibratoren <ol><li> Bereiten 0,5 ml 4 &amp; mgr; g / mL 15 NAβ 1-42 Peptid durch Zugabe von 40 &amp; mgr; l von 50 &amp; mgr; g / mL 15 NAβ 142 bis 0,46 ml 20% ACN und 4% konzentriertem Ammoniak in einer 0,5-ml - Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> Bereiten 2 ml 100 ng / ml 15 NAβ 1-42 Peptid durch Zugabe von 50 &amp; mgr; l der 4 &amp; mgr; g / mL 15 NAβ 142-1,95 ml 20% ACN und 4% konzentriertem Ammoniak in einem 2 ml Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> Bereiten Sie sechs Kalibratorlösungen (AF) durch die Volumina jeder Lösung in Tabelle 2 angegeben zu mischen. Verwenden Sie 0,5, 1,5 und 2 ml Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> Bereiten Sie die endgültige Kalibratoren (in zweifacher Ausfertigung) in 0,5 ml Mikrozentrifugenröhrchen durch die entsprechenden Kalibrierlösungen Hinzufügen und menschlichen CSF gemäß Tabelle 3. Mischen auf einem Vortex-Mischer für 1 min. </li></ol> 3. Vorbereitung des internen Standards <ol><li> Bereiten 2 ml von 0,8 &amp; mgr; g / mL 13 CAβ 1-42 Peptid durch Zugabe von 32 &amp; mgr; l von 50 &amp; mgr; g / mL 13 CAβ 142 bis 1,968 ml 20% ACN und 4% konzentriertem Ammoniak in einem 2 ml Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> Bereiten 5 ml 16 ng / ml 13 CAβ 1-42 Peptid durch Zugabe von 0,1 ml von 0,8 ug / ml bis 4,9 ml 20% ACN und 4% konzentriertem Ammoniak in einem 5 ml - Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li></ol> 4. Vorbereitung der Responsefaktor Probe HINWEIS: Die Responsefaktor (RF) Bestimmung wird durchgeführt , um die Konzentration des markierten Peptids zur Kalibrierung (15 NAβ 1-42) verwendet zu bestimmen. Dies erfordert , dass die Konzentration des nativen Aß 1-42 Peptid wurde unter Verwendung von Aminosäureanalyse (AAA) bestimmt. Somit wird das Volumen und die Konzentration von nativen Aß 1-42 Peptid Aliquotsmüssen die Anforderungen der AAA zu erfüllen. <ol><li> Bereiten 0,5 ml 4 &amp; mgr; g / mL nativen (unmarkiert) A &amp; bgr; 1-42 durch Zugabe von 40 ul von 50 ug / ml nativem Aß 1-42 bis 0,46 ml 20% ACN und 4% konzentriertem Ammoniak in einer 0,5-ml - Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> Bereiten Sie eine 2-ml 40 ng / ml Mischung aus einheimischen und 15 NAβ 1-42 durch Zugabe von 20 &amp; mgr; l von 4 ug / ml nativen Aß 1-42 und 20 &amp; mgr; l von 4 ug / ml 15 NAβ 1-42 bis 1,96 ml 20% ACN und 4% konzentriertem Ammoniak in einem 2 ml Mikrozentrifugenröhrchen. Mit Hilfe eines Vortex-Mischer für 1 min. </li><li> In 20 ul der 40 ng / ml Mischung auf 0,38 ml künstlichem CSF in einem 0,5 ml Mikrozentrifugenröhrchen. Bereiten Sie Duplikate und mischen auf einem Vortex-Mischer für 1 min. </li></ol> 5. Probenvorbereitung HINWEIS: auftauen die Proben auf einer Walze bei Raumtemperatur gemessen werden. <ol><li>In 0,18 ml jeder Kalibrator, Responsefaktor und unbekannten Probe (einschließlich der Qualitätskontrolle [QC] Proben, falls verwendet) zu einem 1 ml Protein 96-Deep-Well - Platte, nach 1 (eine vollständige Platte unter der Annahme verwendet wird ). Achten Sie darauf, um die Proben in oder in der Nähe, der Boden der Vertiefungen hinzuzufügen. </li><li> In 20 ul des internen Standards in jede Vertiefung (dh Kalibratoren, Responsefaktoren, QCs und Unbekannten); ist es entscheidend, den Tropfen an der Seite des Schachts nahe der Oberfläche der Probe zu lösen, ohne die Pipettenspitze eingetaucht. </li><li> In 0,2 ml 5 M Guanidin-Hydrochlorid in jede Vertiefung. </li><li> Platzieren Sie die Probenplatte auf einem Schüttler und mischen Sie die Proben für 45 Minuten bei 1100 Umdrehungen pro Minute. Die optimale Frequenz kann je nach Instrumentierung abweichen. Die Frequenz und Amplitude des Mischers so, dass die Lösungen gründlich gemischt und keine Tropfen des internen Standards oder CSF sind auf der Seite der Vertiefungen ungemischten gelassen. </li><li> In 0,2 ml 4% Phosphorsäurezu gut jeder. Vortex kurz mischen. </li></ol> 6. Festphasenextraktion HINWEIS: In jedem Waschen, Laden und Eluierungsschritte gelten die niedrigste mögliche Vakuum nach Zugabe der Lösung ist und schrittweise nach Bedarf erhöhen, um die Lösung zu laden oder zu eluieren. Deaktivieren Sie das Vakuum zwischen jedem Lade- und Elutionsschritt. <ol><li> Setzen Sie ein Vorratsfach für Abfall unter einem gemischten Modus, Kationenaustausch, 96-Well Festphasenextraktion (SPE) Platte in der Extraktionsplatte Verteilerkammer. </li><li> Erhaltung der SPE Sorbens durch Zugabe von 0,2 ml Methanol zu jeder Vertiefung. </li><li> Äquilibrieren das Sorbens durch Zugabe von 0,2 ml 4% ige Phosphorsäure zu jeder Vertiefung. </li><li> Übertragen Sie alle Proben (etwa 0,62 ml in jeder Vertiefung) aus dem Deep-Well-Platte an der SPE-Platte. Verwenden Sie einen Acht-Kanal-Pipette, wenn die Proben aus dem Deep-Well-Platte an der SPE-Platte zu übertragen; es ist nicht entscheidend, um die gesamte oder gleiche Volumina aller Proben zu übertragen, da die Proben einen Praktikanten enthaltenal-Standard, der für Variationen kompensieren. </li><li> Waschen des Sorbens, nachdem die Proben durch Zugabe von 0,2 ml 4% -iger Phosphorsäure in jede Vertiefung durchlaufen haben. </li><li> Nach dem Waschlösungsmittels aus dem Sorbens eluiert wurde, mit einer Sammelplatte oder Rohre, die die Vorratsfach ersetzen. </li><li> Eluieren der Probe aus dem Sorbens durch zweimalige Zugabe von 50 &amp; mgr; l von 75% ACN / 10% konzentriertem Ammoniak, und beachten Sie, dass diese Lösung eine sehr niedrige Vakuum erfordert durch das Sorbens zu passieren. Denken Sie daran, das Vakuum zwischen jeder Zugabe zu deaktivieren. <ol><li> Optional: Verschließen Sie die Sammelplatte oder Rohre und sie bei -80 ° C einfrieren. Entfernen Sie die Dichtung von der Sammelplatte oder Rohre vor 6.8.2 zu Schritt fortfahren. </li><li> Trocknen Sie die Eluate durch Verwendung von Vakuum-Zentrifugation (ohne Anwendung von Wärme); Dies kann von einem bis zu mehreren Stunden dauern, abhängig von der Vakuumzentrifuge. </li><li> Verschließen Sie die Behälter und frieren sie bei -80 ° C. </li></ol></li></ol> 7. Flüssige Chromatographie <ol><li> Vorbereitung der mobilen Phase A (5% ACN und 0,3% konzentriertem Ammoniak in entionisiertem Wasser [v / v]), B (4% deionisiertes Wasser und 0,1% konzentriertem Ammoniak in ACN [v / v]) und Nadelspülung (50% ACN und 4% konzentriertem Ammoniak in entsalztem Wasser [v / v]). <ol><li> Für 500 ml der mobilen Phase A, 25 ml ACN und 1,5 ml konzentrierter Ammoniak zu entionisiertem Wasser. Stellen Sie die endgültige Volumen auf 500 ml mit VE-Wasser. </li><li> Für 500 ml der mobilen Phase B, 500 &amp; mgr; l konzentrierter Ammoniak und 25 ml VE-Wasser zu ACN. Stellen Sie die endgültige Volumen auf 500 ml mit ACN. </li><li> Bereiten 250 ml Nadelspülung durch Zugabe von 120 ml ACN und 10 ml konzentriertem Ammoniak zu entionisiertem Wasser. Stellen Sie das Endvolumen auf 250 ml mit entionisiertem Wasser. </li><li> Setzen Sie den mobilen Phasen A und B und Nadelwaschflaschen öffnet in einem Ultraschallbad für 20 Minuten vor dem Gebrauch mit dem LC-System </li></ol></li><li> Man löst jede Probe mit 25 &amp; mgr; l von 20% ACN und 4% KonzenAmmoniaklösung ted und legen Sie sie auf einem Schüttler für 20 min. Zentrifuge nach unten den Proben und legen Sie sie in den Autosampler (halten bei 7 ° C). </li><li> Injizieren 20 ul der Probe auf einem 1 x 250 mm 2 Polystyrol-Divinylbenzol (Reversed Phase) monolithische Säule bei 50 ° C gehalten. <ol><li> Verwenden , um die LC - Gradienten in Tabelle 4 gezeigt mit einer Durchflussrate von 0,3 mL / min. Umleiten die ersten beiden und die letzten fünf Minuten zu verschwenden (post-Säule) unter Verwendung eines Umleitungsventil, um die Verunreinigung des Massenspektrometers zu reduzieren. </li></ol></li></ol> 8. massenspektrometrische Analyse HINWEIS: Diese Parameter für einen Quadrupol-Orbitrap Hybrid-Massenspektrometer mit aheated Elektrospray-Ionisierung Quelle ausgestattet verwendet wurden. <ol><li> Die Parameter für den Ionenquelle gemäß Tabelle 5. </li><li> Stellen Sie das MS - Gerät die 4+ Ladungszustände von nativen Aß 1-42 (1,129.48 Massen zu isolierenLadungs-Verhältnis [m / z]), 15 NAβ 1-42 (1.143,00 m / z) und 13 CAβ 1-42 (1,179.50 m / z) in der Quadrupol - Massenanalysator mit einer Isolationsbreite von 2,5 m / z. </li><li> Fragment, das die isolierten Peptide in der Kollisionszelle mit einer normalisierten Kollisionsenergie (NCE) von 17,0. Diese müssen möglicherweise für jedes Instrument, selbst des gleichen Typs abgestimmt werden (und insbesondere dann, wenn andere Arten von Instrumenten, wie beispielsweise einem Triple-Quadrupol-MS). </li><li> Notieren Sie sich die Fragmentspektren mit einer Auflösung von 17.500, mit einer automatischen Verstärkungsregelung Ziel von 2 x 10 5 Kosten und einem maximalen Einspritzzeit von 250 ms. </li></ol> 9. Datenverarbeitung <ol><li> Verwenden , um die Summe der Produkt - Ionen (mit einem Massentoleranz von ± 250 Millimasseneinheiten [MMU]) in Tabelle 6 die chromatographischen Bereiche für jedes Peptid berechnet. Beachten Sie, dass die Ionenarten und Zahlen werden nur für die native Aß gezeigt <sub&gt; 1-42 Produkt - Ionen, da sie die gleichen für beide 15 NAβ 1-42 und 13 CAβ 1-42. </li><li> Bestimmen Sie die durchschnittliche Reaktionsfaktor der beiden Reaktionsfaktor Proben durch die Fläche unter der Kurve dividiert (chromatographischen Peak) von 15 NAβ 1-42 mit der Fläche unter der Kurve von nativen Aß 1-42. </li><li> Stellen Sie die Konzentration des 15 NAβ 1-42 für die Kalibrierung verwendet , indem er mit dem Reaktionsfaktor in Schritt 9.2 berechnet multipliziert wird . </li><li> Einer Eichkurve , die durch die Flächenverhältnisse von 15 NAβ 1-42 bis 13 CAβ 1-42 von den beiden Sätzen von Kalibratoren gegen die Konzentration aufgetragen ist . </li><li> Berechnen der Steigung und Achsenabschnitt der Kalibrierungskurve lineare Regression verwendet wird. </li><li> Berechnen Sie den Flächenverhältnis von nativen Aß 1-42 auf den internen Standard (13 CAβ 1-42) für unbekannten Proben. </li><li> Extrapolation der Konzentration von unbekannten Proben aus der Kalibrierungskurve unter Verwendung der Steigung und Achsenabschnitt in Schritt 9.5. </li></ol>

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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.

Für das Verfahren beschrieben, stattdessen eine Ersatzmatrix zu verwenden, haben wir den Surrogat - Analyt - Ansatz 13, 14, 15, 16, die Kalibrierung im menschlichen CSF ermöglicht. Der Ersatz Analyt Ansatz beinhaltet zwei verschiedene isotopenmarkierten Standards. Eine (15 NAβ 142) wird verwendet , um die Kalibrierungskurve in menschlichen CSF zu erzeugen, während ein...

Alzheimer-Krankheit (AD) ist die häufigste Form der Demenz und betrifft etwa 35 Millionen Menschen weltweit 1. Die neuropathologischen Kennzeichen der Krankheit weit verbreitet in den Mittelpunkt der AD Pathogenese zu liegen glaubte , sind intrazelluläre Neurofibrillen von hyperphosphorylated Tau - Protein 2 und extrazellulären Plaques , bestehend aus aggregierten Amyloid-beta (Aß) Peptide 3. Im Einklang mit dieser Situation hat die Beurteilung der Plaque Pathologie in vivo durch Biomarker vor kurzem in den Forschungs diagnostischen Kriterien für AD 4 aufgenommen. Für CSF Messungen von Aß 1-42, sind mehrere Immunoassays zur Verfügung und werden in vielen klinischen Laboratorien 5 verwendet. Die Konzentration von A &amp; bgr; 1-42 in CSF beträgt ungefähr 50% niedriger in AD - Patienten als in der kognitiv normalen älteren Personen, was die Abscheidung des Peptids in Plaques im br6 ain, 7. Diese Biomarker sind hauptsächlich unter Verwendung von Immunoassays analysiert (dh Antikörper-basierte Techniken), aber diese Assays können durch Matrixeffekte 8 beeinflusst werden. Die Verwendung von Immuntests auf verschiedenen Technologieplattformen und den Mangel an Standardisierung Assay 9, 10 aufgrund der Einführung von globalen abgeschnittene Konzentrationen schwer 11, 12. Eine analytisch validierte RMP würde die einheitliche Kalibrierung von verschiedenen Assay-Plattformen ermöglichen, idealerweise in eine bessere Vergleichbarkeit zwischen den Analyseplattformen zurückgehen und in eine bessere Kontrolle der Faktoren, die die Gesamtmess Variabilität beitragen. Die absolute Quantifizierung von Aß 1-42 des entwickelten LC-MS / MS - Verfahren überwindet viele der Probleme im Zusammenhang mit dem Antikörper-basierten techniques. Das Verfahren, aufgeführt als RMP durch den Gemischten Ausschuss für Rückverfolgbarkeit in der Labormedizin (JCTLM Datenbank - Identifizierungsnummer C11RMP9), werden verwendet , um die absolute Konzentration von Aß 1-42 in zertifiziertes Referenzmaterial (CRM) , um zu bestimmen CSF Aß 1 zu harmonisieren -42 Messungen über Techniken und Analyseplattformen. Die beschriebene Workflow sollte für die Entwicklung der Kandidatenreferenzverfahren für Peptide und Proteine ​​in anderen Bereichen der Medizin von Bedeutung sein.

<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.

Die Platte Setup in Abbildung 1 für eine volle Platte der Proben verwendet. Wenn weniger unbekannte Proben zu analysieren, sollte der zweite Kalibrator, RF, und QC-Sets nach der ersten Hälfte der unbekannten Proben platziert werden. Wie in Figur 2 zu sehen ist , sind die Kalibratoren nahe der Regressionslinie mit geringen Standardabweichungen. Diese Methode hat einen geringeren Grad an Quant...

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.

Absolute Quantifizierung von Aß<sub&gt; 1-42</sub&gt; In CSF eine massenspektrometrische Referenzmessverfahren verwenden

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

7.5K Aufrufe.  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.

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

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.

Analyse der β-Amyloid-induzierte Anomalien auf Fibrin Gerinnsel Struktur von Spektroskopie und Rasterelektronenmikroskopie

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

Forschung

Biochemie

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Eine Technik für Serial Sammlung von Liquor aus der Cisterna Magna in Maus

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biologie

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.

Ein hoher Durchsatz, Multiplexed und Proteom-CSF-Assay Gezielte Quantifizieren Neurodegenerative Biomarkers und Apolipoprotein E Isoformen-Status

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

Medizin

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.

Probenvorbereitung für die Endopeptidomic Analyse im menschlichen Liquor cerebrospinalis

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

Neurowissenschaften

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.

Ein verbessertes Verfahren zur Sammlung von Liquor cerebrospinalis aus narkotisierten Mäuse

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.

Visualisierung von Amyloid-β-Einlagen in das menschliche Gehirn mit Matrix-unterstützte Laser Desorption/Ionisierung bildgebende Massenspektrometrie

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.

Quantitative Messung von intrathecally Synthetisierten Proteinen bei Mäusen

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.

Voll- versus subregionale Quantifizierung der Amyloid-Beta-Belastung von Hirnschnitten der Maus

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.

Biochemische Reinigung und proteomische Charakterisierung von Amyloid-Fibrillenkernen aus dem Gehirn

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

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Millisekunden-Wasserstoff/Deuterium-Austausch-Massenspektrometrie zur Untersuchung der Alpha-Synuclein-Strukturdynamik unter physiologischen Bedingungen

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...

Absolute Quantifizierung von Aß

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