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.

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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.

للتعرف على طريقة وصفها، بدلا من استخدام مصفوفة البديلة، استخدمنا نهج بديل تحليلها 13، 14، 15، 16، والتي تمكن المعايرة في السائل النخاعي البشري. ينطوي على نهج تحليلها البديل مختلفين معايير المسمى isotopically. واحد (15 NAβ 142) يستخدم لتوليد منحنى المعايرة في السائل النخاعي البشري، في حين يتم استخدام (13 CAβ 142) آخر كمعيار داخلي. ثم يتم استقراء مجهولة الذاتية Aβ 142 تركيزات من منحنى المعايرة شيدت باستخدام 15 NAβ 142/13 CAβ 142 نسبة من الذاتية Aβ 142/13 CAβ نسبة 142 المحسوبة. وقد تم استخدام المنهج تحليلها بديلة، حيث لا يوجد CSF خالية من تحليلها المتاحة، وانخفاض Aβ 142 الانتعاشوقد لوحظ عند استخدام الأصلي Aβ 142 في مصفوفة البديلات خلال تطوير الأسلوب. منذ 15 NAβ 142 و أصلي Aβ 142 قد تعطي استجابات مختلفة في مرض التصلب العصبي المتعدد، وتركيز 15 NAβ 142 يتم تعديل قياس عينة، وRF عينة السائل النخاعي الاصطناعية التي تحتوي على تركيزات متساوية من 15 NAβ 142 و أصلي Aβ 142، مع تركيز معروف بواسطة AAA.The عامل استجابة العزم قد تختلف بين الطيف الإعلام المختلفة بسبب الاختلافات المحتملة في نقاء النظائر من 15 الببتيد N-وصفت بين دفعات. وبالتالي، ينبغي تحديد عامل استجابة لكل يوم قياس. الخطوات الأكثر أهمية في هذا البروتوكول هي إعداد معايرة والعينات الترددات اللاسلكية. الببتيدات Aβ، خصوصا Aβ 1-42، هي مسعور جدا وتلتصق بسهولة إلى نصائح ماصة لأنابيب ND أسطح 8 و 17 و 18. لتقليل الخسائر من الببتيدات Aβ خلال pipetting ل، فمن المهم للغاية لتشبع نصائح ماصة قبل التسليم. ويفضل أن يتم تجاهل ثلاثة مجلدات من الحل الببتيد قبل تسليمها إلى الأنبوب الذي يحتوي الحل الجديد. اعتمادا على حجم وتركيز المحلول، وهذا ليس من الممكن دائما. نهج ثاني أفضل بالطبع إلى ماصة الحل الببتيد صعودا وهبوطا ثلاث مرات قبل التسليم. لنفس السبب، من المهم أن تستخدم الأحجام المناسبة للأنابيب، وتجنب كميات كبيرة باطلة. وتشير البيانات التي تم نشرها مسبقا لطريقة بان الانتعاش ضمن 100٪ (15٪) (15). كانت الأخطاء النسبية للمعايرة محسوبة إلى أقل من 15٪ من مجمل يحددها منحنى تدريج 19. واحد سالحد bvious من هذه التقنية هو أن يكون المنخفض للالإنتاجية مقارنة المناعية الآلي. ومع ذلك، فإن الغرض من الطريقة الموصوفة هي عالية الدقة وعدم الإنتاجية. ويمكن أيضا أن يتم توسيع هذه الطريقة لتشمل Aβ 1-38 وAβ 1- 40، والتي هي أقصر 19. الحد آخر من هذا الأسلوب هو أن المشغل سوف يحتاجون إلى تدريب قياس الطيف الكتلي واسعة النطاق قبل تشغيل التحليل على الصك. الكمي باستخدام المناعية يتوقف على التفاعل بين الجسم المضاد والمستضد. يمكن أن تتأثر هذا التفاعل من خلال وجود مكونات العينة التي قد تتداخل أو تتنافس مع التفاعل. وبالإضافة إلى ذلك، يمكن أيضا التفاعل تتأثر التشكل للمستضد. وهذه الآثار هي من الصعب السيطرة عليها ويعتقد أن السبب الرئيسي كان من الصعب التوفيق بين النتائج بين منصات المناعية وبين المختبرات. بيكويستند تقدير ause مع MS على عد مباشرة الجزيئات المستهدفة بالنسبة إلى مستقر، ومعيار المسمى النظير، الكمي مطلق ويتأثر عموما من قبل هذه الآثار المصفوفة. وبالإضافة إلى ذلك، يجب أن تكون معتمدة من القياسات بروتين التشخيص من قبل المناعية عن طريق سلسلة متصلة من إجراءات القياس العليا والمواد، من التحقق من صحة LC-MS / MS والمعايير الداخلية مستقرة المسمى النظير لخطط إدارة المبردات وإدارة علاقات العملاء، وبالتالي تحسين القابلية للمقارنة و موثوقية النتائج 20 و 21. في الختام، فإن الشرطة العسكرية الملكية وصفه لقياس Aβ 142 في السائل النخاعي هو خطوة هامة في تطوير إدارة علاقات العملاء التي من شأنها أن تساعد على إرساء العام تركيز النهائي لAβ 142 في السائل النخاعي. بالضبط قطع العرضية مهمة للغاية لتشخيص بدقة م في وقت مبكر وذات أهمية قصوى عندما أنواع جديدة من الأدوية المعدلة للمرض تصل إلى العيادة.

مرض الزهايمر (م) هو الشكل الأكثر شيوعا من الخرف ويصيب حوالي 35 مليون شخص في جميع أنحاء العالم 1. بصمات عصبية مرضية من مرض يعتقد على نطاق واسع تكمن في جوهر م المرضية هي التشابك الليفي العصبي بين الخلايا من hyperphosphorylated بروتين تاو 2 ولويحات خارج الخلية التي تتكون من (Aβ) الببتيد 3 المجمعة اميلويد بيتا. وتماشيا مع هذا، وقد تم مؤخرا شملت تقييم أمراض الترسبات في الجسم الحي من المؤشرات الحيوية في المعايير البحوث التشخيصية لمدة 4 م. لقياس السائل النخاعي من Aβ 1-42، هي عدة المناعية المتاحة وتستخدم في العديد من المختبرات السريرية 5. تركيز Aβ 1-42 في السائل النخاعي هو أقل ما يقرب من 50٪ في المرضى ميلادي مما كانت عليه في كبار السن الطبيعي معرفيا، مما يعكس ترسب الببتيد في لويحات رعين 6 و 7. ويتم تحليل هذه المؤشرات الحيوية بشكل رئيسي باستخدام المناعية (أي التقنيات المعتمدة الضد)، ولكن قد تتأثر هذه المقايسات من الآثار مصفوفة 8. استخدام المناعية على منصات التكنولوجيا المختلفة وعدم وجود فحص التقييس 9، 10 يجعل إدخال العالمية تركيزات قطع الصعبة 11 و 12. ومن شأن RMP التحقق من صحة تحليلي تسمح معايرة موحدة للمنصات فحص مختلفة، مما أدى مثالي في المقارنة أفضل عبر منصات التحليلية وفي تحسين السيطرة على العوامل التي تساهم في تقلب قياس العام. الكمي المطلق للAβ 1-42 باستخدام ضعت طريقة LC-MS / MS يتغلب على العديد من القضايا المرتبطة ميتاليك القائم على الأجسام المضادةiques. سيتم استخدام طريقة، على النحو الوارد في الشرطة العسكرية الملكية من قبل اللجنة المشتركة للتتبع في طب المختبرات (JCTLM قاعدة بيانات تحديد عدد C11RMP9)، لتحديد تركيز المطلق للAβ 1-42 في المراجع معتمد (CRM) لتنسيق CSF Aβ 1 قياسات -42 عبر تقنيات ومناهج تحليلية. وينبغي أن يكون سير العمل وصفه ذات الصلة لتطوير أساليب مرشح مرجعية لالببتيدات والبروتينات داخل مناطق أخرى من الأدوية.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;Elution Plate</td>
			<td>Waters</td>
			<td>186001830BA&#160;</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 &#38; 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&#252;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.

<html> يتم استخدام الإعداد لوحة في الشكل رقم 1 لوحة كاملة من العينات. إذا ليتم تحليلها أقل عينات غير معروفة، وتدريج الثاني، ينبغي أن توضع مجموعات الترددات اللاسلكية، ومراقبة الجودة بعد نهاية الشوط الأول من العينات غير معروف. كما رأينا في الشكل 2، ومعايرة قريبة من خط الانحدار، مع الانحرافات المعيارية منخفضة. هذا الأسلوب لديه مستوى أدنى من تقدير حجم 150 غ / مل ومستوى أعلى من تقدير من 4000 جزء من الغرام / مل. الانحراف المعياري المتبقي من معايرة ينبغي بالطبع أن يكون في أدنى مستوى ممكن. إذا المعايرة غير الخطية، يجب التخلص المدى (اعتمادا على شدة)، والانحراف هو على الأرجح بسبب تقنية pipetting لغير صحيحة و / أو أخطاء في التخفيف من معايرة. يجب أن يكون معامل الاختلاف (CV) من مكررات أقل من 20٪، ولكن يفضل أن يكون أقل من 10٪. <html> ogether.within الصفحات = &quot;1&quot;&gt; والأم 15 N- و 13 CAβ 1-42 أزل من العمود LC في وقت واحد (لأنها تختلف فقط على مستوى النظائر)، مع ما يقرب من قمم متماثلة ودون المخلفات كبير (الشكل 3 ). يجب أن يتم تنفيذ لا يقل عن عشرة قياسات لكل الذروة الكروماتوغرافي ويمكن تعديلها مع الوقت حقن الأقصى في طريقة الصك. ويمكن قياس كل الببتيدات ثلاثة في وقت واحد لجميع القياسات (المعايرة، RF، والمجاهيل) خلال تحليل MS. ومع ذلك، إذا حساسية الطريقة هي دون المستوى الأمثل، وينبغي قياس الببتيدات فقط التي تهم كل حقنة (أي، فقط قياس 15 N- و 13 CAβ 1-42 للمعايرة، وأصلي و15 NAβ 1-42 لعينات الترددات اللاسلكية، وأصلي و13 CAβ 1-42 للعينات غير معروفة). الرقم 1 &quot;SRC =&quot; / ملفات / ftp_upload / 55386 / 55386fig1.jpg &quot;/&gt; الشكل 1: تخطيط من SPE وفي آبار عميقة لوحات. تخطيط نموذجي من معايرة (AF)، عامل استجابة عينة (RF)، وعينات مراقبة الجودة (QC)، والمجاهيل. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55386/55386fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/55386/55386fig2.jpg" /> الشكل 2: منحنى المعايرة. منحنى المعايرة شيدت باستخدام 15 NAβ 1-42 في 172، 572، 1144، 2287، 3431، و 4574 جزء من الغرام / مل (معدلة باستخدام عامل الاستجابة) و 13 CAβ 1-42 كمعيار الداخلي في السائل النخاعي البشري (ن = 2) . يتم رسم نسبة مساحة 15 NAβ 1-42 / 13 CAβ 1-42 (العمودي)ضد تركيز (المحور السيني). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55386/55386fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/55386/55386fig3.jpg" /> الشكل 3: المخطط الاستشرابي. اللوني من 0.500 نانوغرام / مل مواليد (الذاتية) Aβ 1-42 (لوحة العليا) و 1.6 نانوغرام / مل 13 CAβ 1-42 (اللوحة السفلية) في السائل النخاعي البشري. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55386/55386fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (4)" src="/files/ftp_upload/55386/55386fig4.jpg" /> الشكل 4: الكمي للمجهول Aβ 1-42 في عينات مجهولة &lt;./ قوي&gt; يتم احتساب نسبة الذروة منطقة بقسمة الأم Aβ 1-42 الذروة الكروماتوغرافي المنطقة مع المعيار الداخلي (13 CAβ 1-42) ذروة منطقة الكروماتوغرافي. ومن استقراء تركيز الأصلي Aβ 1-42 في عينة من منحنى المعايرة. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55386/55386fig4large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (4)" src="/files/ftp_upload/55386/55386table1.jpg" /> الجدول 1: معلومات السلامة. معلومات السلامة للمواد الكيميائية المستخدمة لهذا البروتوكول. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> حل تدريج </td><td> حجم 100 نانوغرام / مل 15 NAβ 1-42 حل (مل) </tد&gt; <td> حجم 20٪ إيه سي و 4٪ الأمونيا (مل) </td><td> الحجم النهائي (مل) </td><td> النهائي 15 NAβ تركيز 1-42 (نانوغرام / مل) </td></tr><tr><td> ا </td><td> 0.20 </td><td> 0.30 </td><td> 0.50 </td><td> 40.00 </td></tr><tr><td> ب </td><td> 0.15 </td><td> 0.35 </td><td> 0.50 </td><td> 30.00 </td></tr><tr><td> C </td><td> 0.20 </td><td> 0.80 </td><td> 1.00 </td><td> 20.00 </td></tr><tr><td> د </td><td> 0.10 </td><td> 0.90 </td><td> 1.00 </td><td> 10.00 </td></tr><tr><td> E </td><td> 0.05 </td><td> 0.95 </td><td> 1.00 </td><td> 5.00 </td></tr><tr><td> F </td><td> 0.03 </td><td> 1.97 </td><td> 2.00 </td><td> 1.50 </td></tr></tbody></table> الجدول 2: حلول المعاير. حلول تدريج أعدت في 20٪ إيه سي و 4٪ تتركز الأمونياتستخدم لمعايرة ارتفاعه CSF. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> تدريج </td><td> حجم محلول المعاير المقابلة (مل) </td><td> حجم السائل النخاعي البشري (مل) </td><td> الحجم النهائي (مل) </td><td> 15N-Aβ1-42 النهائي تركيز (نانوغرام / مل) </td></tr><tr><td> ا </td><td> 0.02 (A) </td><td> 0.18 </td><td> 0.20 </td><td> 4.00 </td></tr><tr><td> ب </td><td> 0.02 (B) </td><td> 0.18 </td><td> 0.20 </td><td> 3.00 </td></tr><tr><td> C </td><td> 0.02 (C) </td><td> 0.18 </td><td> 0.20 </td><td> 2.00 </td></tr><tr><td> د </td><td> 0.02 (D) </td><td> 0.18 </td><td> 0.20 </td><td> 1.00 </td></tr><tr><td> E </td><td> 0.02 (E) </td><td> 0.18 </td><td> 0.20 </td><td> 0.50 </td></tr><tr><td> F </td><td> 0.02 (F) </td><td>0.18 </td><td> 0.20 </td><td> 0.15 </td></tr></tbody></table> الجدول 3: أجهزة القياس. معايرة أعد CSF البشري. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> الوقت (دقيقة) </td><td> الطور المتحرك٪ B </td></tr><tr><td> 0 </td><td> 5 </td></tr><tr><td> 1 </td><td> 5 </td></tr><tr><td> 6 </td><td> 20 </td></tr><tr><td> 7 </td><td> 90 </td></tr><tr><td> 9 </td><td> 90 </td></tr><tr><td> 10 </td><td> 5 </td></tr><tr><td> 15 </td><td> 5 </td></tr></tbody></table> الجدول 4: LC التدرج. التدرج LC المستخدمة مع معدل تدفق ثابت من 300 ميكرولتر / دقيقة. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> معامل </td><td> القيمة </td></tr><tr><td> الغاز غمد </td><td> 50 </td></tr><tr><td> الغاز المساعد </td><td> 6 </td></tr><tr><td> رذاذ الجهد </td><td> 4.4 كيلو فولت </td></tr><tr><td> S-عدسة RF </td><td> 61 </td></tr><tr><td> درجة حرارة سخان </td><td> +190 ° C </td></tr><tr><td> درجة الحرارة الشعرية </td><td> +350 ° C </td></tr></tbody></table> الجدول 5: ضبط مصدر ايون. المعلمات لمصدر أيون التي سيتم تحديدها في البرنامج أداة ضبط. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> السلائف أيون </td><td> أيونات المنتج </td></tr><tr><td> Aβ1-42 الأصلية (م / ض 1129.58، 4+) </td><td> 915.19 (b334 +)، 943.21 (b344 +)، 975.98 (b354 +)، 1000،74 (b364 +)، 1029،51 (b384 +)، 1054.03 (b394 +)، 1078،79 (B404 +)، 1107،06 (b414 +)، 1163،23 (b313 +)، 1200،25 (b323 +)، 1257،29 (b343 +)، 1300،96 (b353 +)، 1333،66 (B363 +)، 1372،00 (b383 +)، 1405،02 (b393 +) </td></tr><tr><td> 15N-Aβ1-42 (م / ض 1143.00، 4+) </td><td> 926.41، 954.68، 987.95، 1012.71، 1041.22، 1066.99، 1091.75، 1120.28، 1177.18، 1215.55، 1272.58، 1316.92، 1349.94، 1388.63، 1422.31 </td></tr><tr><td> 13C-Aβ1-42 (م / ض 1179.50، 4+) </td><td> 955.33، 985.11، 1019.37، 1045.14، 1074.65، 1100.67، 1126.69، 1156.40، 1253.43، 1313.14، 1358.50، 1393.19، 1432.21، 1466.90 </td></tr></tbody></table> الجدول 6: الأيونات المستخدمة لتقدير. يتم عزل الدول تهمة 4+ من أيونات السلائف في محلل كتلة رباعي، مع عرض العزلة من 2.5 م / ض. وتستخدم أيونات المنتج (مع التسامح الشامل لل± 250 MMU) لحساب المناطق الكروماتوغرافي عن كل الببتيد. أنواع أيون والأرقام ONLذ تعرض لالأم Aβ 1-42 أيونات المنتج، لأنها هي نفسها من أجل كل 15 NAβ 1-42 و 13 CAβ 1-42.

Watch this Scientific Journal Video about Absolute Quantification of AÃŽÂ²1-42 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.

الكمي المطلق للAβ<sub&gt; 1-42</sub&gt; في السائل الدماغي الشوكي عن طريق مرجع إجراء قياس كتلة الطيف

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

Nanyang Technological University

Research

JoVE Journal

Medicine

7.5K Views.  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.

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

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

التصوير بالرنين المغناطيسي الكمي من الإرواء الرئوي باستخدام معايرة وصفها سبين الشرياني

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

تجزئة والقياس من وحدات تخزين الدهون في الفئران نماذج السمنة عن طريق الأشعة السينية التصوير المقطعي

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

فوق الجافية قياس الضغط داخل الجمجمة في الفئران باستخدام محول ضغط الألياف الضوئية

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

الكمي لسرطان الثدي الغزو خلية باستخدام ثلاثي الأبعاد (3D) نموذج

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

الكمي للمناعة tacrolimus على وبقع الدم الجافة عن طريق LC-MS / MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

الكمي قداس الطيفية من التنميط Proteomes خلايا السرطان المستمدة من السائل والأورام الصلبة

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

تحليل شبكة الثقبة البيضوية الكهربائي تسجيلات في الفص الصدغي الصرع المرضى المقاوم للأدوية

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

التقاط الكمي المكانية للمخدرات في الآفات السل الرئوي بالليزر اللوني السائل ميكروديسيكشن الكتلي (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

We present a protocol to determine the levels of overall macular pigment, lutein, and zeaxanthin optical density in the central and parafoveal regions of the retina. The protocol includes a novel adjustable track system used to measure macular pigment optical density in the foveal eccentricity.

قياس الكاروتينات في Perifovea باستخدام مقياس انعكاس الصباغ البقعي

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

استخدام تطبيق اختبار Hirschberg الآلي لتقييم محاذاة العين

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

الكمي المطلق للAβ

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