Name: absolute quantification of aβ1-42 in csf using a mass spectrometric reference measurement procedure
Uploaded: 2017-03-21T14:00:00.000+00:00
Duration: 8 min 40 s
Description: Watch this Scientific Journal Video about Absolute Quantification of AÎ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

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.

NOT: Bu protokol, başlangıç ​​malzemesi olarak, her Aβ peptidi için 50 ug / ml bir konsantrasyonda en az 50 uL hacimde, gerektirir. Aβ peptidler, deiyonize su içinde% 20 asetonitril (ACN) ve% 4, konsantre amonyak çözeltisi (hac / hac) içinde çözülmüş ve 80 ° C &#39;de muhafaza edilmesi gerekir. Dikkat: güvenlik bilgisi için Tablo 1&#39;e bakınız. Çözümler 1. hazırlanması <ol><li> ACN 20 ml deiyonize su içinde konsantre edilmiş amonyak 4 mL (~% 25) seyreltilmesi ile% 20 ACN ve% 4, 100 ml deiyonize edilmiş su (hacim / hacim) konsantre amonyak çözeltisi hazırlayın. iyonu giderilmiş su ile 100 mL nihai hacim ayarlayın. her gün taze olun. </li><li> 50 mL&#39;lik bir son hacme kadar iyonu giderilmiş su içinde guanidin-hidroklorür 26.08 g çözülmesiyle 5 M guanidin-hidroklorid 50 mL hazırlayın. 20 ° C&#39;de saklayın ve taze Aylık&#39;ı yapmak. </li><li> (Deiyonize su içinde% 4 fosforik asit 200 mL Hazırlama V /h) deiyonize su içinde konsantre edilmiş fosforik asit (~% 85) 9.4 ml seyreltilmesiyle. iyonu giderilmiş su ile 200 mL nihai hacim ayarlayın. Buzdolabında saklayın ve taze haftalık yapmak. </li><li> ACN, 37.5 mL deiyonize su içinde konsantre edilmiş amonyak 5 mL (~% 25) seyreltilmesi ile deiyonize su içinde 50% 75 ACN mL ve% 10 konsantre edilmiş amonyak çözeltisi (hac / hac) hazırlayın. iyonu giderilmiş su ile 50 ml nihai hacim ayarlayın. her gün taze olun. </li><li> Rutin klinik analizden de tanımlanan artık örneklerden elde edilen kalibratör, insan CSF en az 2.5 mL çözülme. </li><li> 150 mM Na, 3.0 mM K, 1.4 mM Ca, 0.8 mM Mg, 1.0 mM p ve deiyonize su içinde 155 mM CI ihtiva eden suni CSF hazırlamak ve 4 mg / mL&#39;lik nihai bir konsantrasyona kadar sığır serum albümini ekleyin. Sadece 1 ml gelecekte kullanılmak üzere kısım bunu, ve mağaza, analiz başına gerekli, ama büyük hacimli hazırlamak olduğunu. </li></ol> Kalibratörlerimizden 2. Hazırlık <ol><li> 4 ug 0.5 mL hazırlayın / ml 10.5 mL&#39;lik bir mikrosantrifüj tüpü içinde% 20 ACN ve% 4 konsantre edilmiş amonyak 0.46 mL / 15 mL NAβ 142 50 ug 40 uL eklenmesiyle 5 NAβ 1-42 peptid. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> 2 mL mikrosantrifüj tüpü içinde% 20 ACN ve% 4 konsantre edilmiş amonyak 1.95 mL 4 ug / ml 15 NAβ 142 50 uL eklenmesiyle, 100 ng / ml 15 NAβ 1-42 peptid 2 mL hazırlayın. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> Tablo 2&#39;de belirtilen her bir çözelti miktarı karıştırılarak altı kalibratör solüsyonları (AF) hazırlayın. 0.5, 1.5, ve 2 mL mikrosantrifüj tüpleri kullanın. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> karşılık gelen kalibrasyon çözümleri ve 1 dakika boyunca bir vorteks karıştırıcıda Tablo 3. Karışımı uygun olarak insan CSF eklenerek 0.5 mL mikrosantrifüj tüplerinde (çift olarak) son kalibratörleri hazırlayın. </li></ol> Dahili standardın 3. hazırlanması <ol><li> 2 mL mikrosantrifüj tüpü içinde% 20 ACN ve% 4 konsantre edilmiş amonyak 1.968 mL / 13 mL CAβ 142 50 ug 32 uL eklenmesiyle / ml 13 CAβ 1-42 peptid 0.8 ug 2 mL hazırlayın. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> 5 ml mikrosantrifüj tüpü içinde% 20 ACN ve% 4 konsantre edilmiş amonyak 4.9 ml 0.8 ug / ml 0.1 ml ilave edilerek 16 ng / ml 13 CAβ 1-42 peptid 5 mL hazırlayın. 1 dakika boyunca vorteks mikserde karıştırılır. </li></ol> Yanıt Faktörü Numunenin 4. hazırlanması Not: yanıt faktörü (RF) belirlenmesi kalibrasyon (15 NAβ 1-42) için kullanılan işaretli peptidin konsantrasyonunu belirlemek için gerçekleştirilir. Bu yerli Aβ 1-42 peptid konsantrasyonu amino asit analizi (AAA) ile tespit edilmiştir gerektirir. Bu durumda, ses ve yerli Aβ 1-42 peptid alikotları konsantrasyonuAAA gereklerini yerine getirmek gerekir. <ol><li> 0.5 mL&#39;lik bir mikrosantrifüj tüpü içinde% 20 ACN ve% 4 konsantre edilmiş amonyak 0.46 mL / mL nativ Aβ 1-42 50 ug 40 uL eklenmesiyle (etiketlenmemiş) 4 ug / mL nativ Aβ 1-42 0.5 mL hazırlayın. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> % 20 1.96 mi, 4 ug / mL nativ Aβ 1-42 ve 4 ug 20 ul / ml 15 NAβ 1-42 20 uL eklenmesiyle yerli 15 NAβ 1-42 bir 2 mL 40 ng / ml karışımı hazırlayın ACN ve% 4 2 mL mikrosantrifüj tüpü içinde amonyak konsantre edildi. 1 dakika boyunca vorteks mikserde karıştırılır. </li><li> 0.5 mL mikrosantrifüj tüpü içinde suni CSF 0.38 mL 40 ng / ml karışımı 20 uL ekleyin. çiftleri hazırlayın ve 1 dakika boyunca bir vorteks karıştırıcıda karıştırılır. </li></ol> 5. Numune Hazırlama NOT: örnekleri çözülme rulo üzerinde oda sıcaklığında ölçülecek. <ol><li>Şekil 1&#39;e göre, bir 1 ml protein 96 derin oyuklu plakaya her kalibratör, yanıt faktörü ve (kullanıldığı takdirde kalite kontrol [QC] örnekleri dahil) bilinmeyen numunenin 0.18 mL ekleyin (tam bir plaka varsayılarak kullanılır). örnekleri eklemeyi unutmayın, ya da kuyu dibine, yakın olun. </li><li> Her iyi (yani, kalibratörler, tepki faktörleri, QCS ve bilinmeyenler) iç standardın 20 uL ekleyin; o pipet batış olmadan numunenin yüzeyine iyice yakın tarafında damla serbest bırakmak için önemlidir. </li><li> Her bir oyuğa 0.2 ml 5 M guanidin-hidroklorür ekleyin. </li><li> bir mikro çalkalayıcı örnek plakasını ve 1100 rpm&#39;de 45 dakika süre ile numuneler karıştırın. Optimal frekans enstrümantasyon bağlı olarak farklılık olabilir. Mikserin frekans ve genliği ayarlayın çözümler iyice karıştırılır ve iç standart veya CSF hiçbir damla kuyuların tarafında karışmamış bırakılır böylece. </li><li> % 4 fosforik asit 0.2 mL eklemeher oyuğa. Vortex kısaca karıştırın. </li></ol> 6. Katı Faz Ekstraksiyon Not: Tüm yıkama, yükleme ve elüsyon adımlarda, çözelti ilave edildikten sonra mümkün olan en düşük vakum uygulanır ve yük veya çözelti elüt edilmesi için gerektiği şekilde yavaş yavaş artar. Her yükleme ve elüsyon adım arasındaki vakum devre dışı bırakın. <ol><li> karma modunda atıklar için bir rezervuar tepsi koyun, ekstraksiyon plakası manifold bölmesi katyon-değişim, 96-çukurlu bir katı faz ekstraksiyonu (SPE) plaka. </li><li> Her bir oyuğa 0.2 ml metanol eklenerek SPE sorbent hale getirin. </li><li> her kuyuya% 4 fosforik asit 0.2 ml ilave edilerek sorbent dengelenmesi. </li><li> SPE plakasına derin kuyu plaka tüm örnekleri (her kuyuda yaklaşık 0.62 ml) aktarın. SPE plakasına derin kuyu plaka örnekleri aktarırken sekiz kanallı pipet kullanın; Numuneler, bir intern içeren, çünkü tüm örneklerin tamamı veya eşit hacimlerde aktarmak için önemli değildirdeğişimleri telafi edecek al standardı. </li><li> Numuneler her kuyuya% 4 fosforik asit 0.2 ml ilave edilerek üzerinden geçtikten sonra sorbent yıkayın. </li><li> yıkayıcı çözücü sorbentten elüt sonra, bir toplama plakası ve tüpler haznesi tepsisini değiştirin. </li><li> iki kez% 75 ACN /% 10 konsantre amonyak 50 uL eklenmesiyle sorbentten örnek Zehir, ve bu çözelti, sorbent geçmesi için çok düşük bir vakum gerektirdiğini not edin. her ilave arasında vakum devre dışı bırakmak için unutmayın. <ol><li> İSTEĞE BAĞLI: toplama plakası veya tüpleri Seal ve -80 ° C&#39;de onları dondurmak. 6.8.2 adıma geçmeden önce toplama plakası veya tüplerden contayı sökün. </li><li> (Isı uygulamadan) vakum santrifüj kullanılarak eluatları Kuru; Bu vakum santrifüj bağlı olarak birkaç saat birinden alabilir. </li><li> kapları mühür ve -80 ° C&#39;de onları dondurmak. </li></ol></li></ol> 7. Sıvı Chromatografisi <ol><li> Ve iğne yıkama (% 50 ACN (ACN [hacim / hacim]% 4, deiyonize su ve% 0.1 konsantre edilmiş amonyak), B (iyonu giderilmiş su [hacim / hacim]% 5 ACN ile% 0.3 konsantre edilmiş amonyak), mobil faz A Hazırlama ve% 4, deiyonize su içinde amonyak konsantre edildi: [hacim / hacim]). <ol><li> Mobil faz A, 500 ml ACN 25 mL deiyonize su konsantre amonyak 1.5 ml. iyonu giderilmiş su ile 500 mL nihai hacim ayarlayın. </li><li> mobil faz B, 500 ml konsantre amonyak 500 uL ACN iyondan arındırılmış 25 ml su ilave edin. ACN ile 500 mL son ses seviyesini ayarlayın. </li><li> ACN, 120 ml deiyonize su, konsantre amonyak 10 ml ilave iğne yıkama 250 mL hazırlayın. deiyonize su ile 250 mL son ses seviyesini ayarlayın. </li><li> LC sistemi kullanmadan önce 20 dakika süreyle sonikasyon banyosunda açık mobil fazlar A ve B iğne yıkama şişeleri koymak </li></ol></li><li> 25 uL konsantrasyon% ACN% 20 ve 4 ile her bir örnek çözülürted amonyak çözeltisi, 20 dakika süre ile bir çalkalama üzerine yerleştirin ve. örneklerin aşağı santrifüj ve alıcısı içinde koyun (7 ° C&#39;de tutmak). </li><li> 50 ° C &#39;de muhafaza edilen bir 1 x 250 ila 2 mm polistiren-divinilbenzen (ters faz) monolitik kolon üzerinde örneğin 20 ul enjekte edilir. <ol><li> 0.3 mL / dakika bir akış oranı ile Tablo 4&#39;te gösterilmiştir LC gradyanı kullanın. kütle spektrometresi kirlenmesini azaltmak için yönlendirme vanası kullanılarak (post-sütun) atık ilk iki ve son beş dakika yönlendir. </li></ol></li></ol> 8. Kitle spektrometrik analiz NOT: Bu parametreler aheated elektrosprey iyonizasyon kaynağı ile teçhiz edilmiş bir dört kutuplu-Orbitrap hibrid kütle spektrometresi kullanıldı. <ol><li> Tablo 5&#39;e uygun iyon kaynağı parametrelerini ayarlar. </li><li> (Yerli Aβ 1-42 4+ şarj durumlarını izole etmek için 1,129.48 kütle cinsinden MS cihazı ayarlayın2,5 m / z yalıtım genişliği kuadruple kitle analizatöründe için şarj oranı [m / z]), 1-42 15 NAβ (1,143.00 m / z), ve 13 CAβ 1-42 (1,179.50 m / z). </li><li> 17.0 normalleştirilmiş çarpışma enerjisi (NCE) ile çarpışma hücresinde izole peptidler parçalara. (Örneğin, bir üçlü dört MS gibi araçlarla diğer türleri, kullanımı ve özellikle de) da aynı türde Bu, her enstrüman için ayarlanmış olması gerekebilir. </li><li> 2 x 10 5 ücretleri otomatik kazanç kontrol hedefine ve 250 ms maksimum enjeksiyon süresi ile 17.500 çözünürlüğe sahip fragmanı spektrumları kaydedin. </li></ol> 9. Veri İşleme <ol><li> Her peptit için kromatografik alanları hesaplamak için Tablo 6&#39;da (± 250 mili kütle birimlik [mmu] kütle toleransla) Ürün iyonlarının toplamı kullanın. iyon türleri ve sayıları yalnızca yerel Aβ için gösterilen unutmayın <sub&gt; 1-42 ürün iyonları, bu 15 NAβ 1-42 ve 13 CAβ 1-42 için aynı olduğundan. </li><li> Yerel Aβ 1-42 eğri altında kalan alan 15 NAβ 1-42 eğrisi (kromatografik tepe) altındaki alanı bölünmesiyle iki farklı yanıt faktörü numunelerin ortalama yanıt faktörünü belirleyin. </li><li> Adım 9.2 hesaplanan tepki faktörü ile çarpılarak kalibrasyon için kullanılan 15 NAβ 1-42 konsantrasyonu ayarlayın. </li><li> Konsantrasyonuna karşı kalibratör iki grup 13 CAβ 1-42 kadar NAβ 1-42, 15 alan oranları işaretlenmesiyle bir kalibrasyon eğrisi oluşturmak. </li><li> lineer regresyon kullanılarak kalibrasyon eğrisinin eğimini ve kesişme hesaplayın. </li><li> Un iç standart (CAβ 1-42 13) yerli Aβ 1-42 alanı oranını hesaplamakBilinen örnekler. </li><li> Adım 9.5 &#39;de elde edilen eğim ve kesişme kullanarak kalibrasyon eğrisinden bilinmeyen örneklerinin konsantrasyonunu tahmin. </li></ol>

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	<li>Querfurth, H. W., LaFerla, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> N Engl J Med. 362 (4), 329-344 (2010).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+neuropathology+of+Alzheimer's+disease.">Evolution of the neuropathology of Alzheimer's disease.</a> Acta Neurol Scand Suppl. 165, 3-12 (1996).</li><li>Blennow, K., de Leon, M. J., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> Lancet. 368 (9533), 387-403 (2006).</li><li>Dubois, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+research+diagnostic+criteria+for+Alzheimer's+disease:+the+IWG-2+criteria.">Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria.</a> Lancet Neurol. 13 (6), 614-629 (2014).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat Rev Neurol. 6 (3), 131-144 (2010).</li><li>Buchhave, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+levels+of+beta-amyloid+1-42,+but+not+of+tau,+are+fully+changed+already+5+to+10+years+before+the+onset+of+Alzheimer+dementia.">Cerebrospinal fluid levels of beta-amyloid 1-42, but not of tau, are fully changed already 5 to 10 years before the onset of Alzheimer dementia.</a> Arch Gen Psychiatry. 69 (1), 98-106 (2012).</li><li>Olsson, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+and+blood+biomarkers+for+the+diagnosis+of+Alzheimer's+disease:+a+systematic+review+and+meta-analysis.">CSF and blood biomarkers for the diagnosis of Alzheimer's disease: a systematic review and meta-analysis.</a> The Lancet Neurology. 15 (7), 673-684 (2016).</li><li>Bjerke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confounding+factors+influencing+amyloid+Beta+concentration+in+cerebrospinal+fluid.">Confounding factors influencing amyloid Beta concentration in cerebrospinal fluid.</a> Int J Alzheimers Dis. , (2010).</li><li>Dumurgier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersite+variability+of+CSF+Alzheimer's+disease+biomarkers+in+clinical+setting.">Intersite variability of CSF Alzheimer's disease biomarkers in clinical setting.</a> Alzheimers Dement. 9 (4), 406-413 (2013).</li><li>Mattsson, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+biomarker+variability+in+the+Alzheimer's+Association+quality+control+program.">CSF biomarker variability in the Alzheimer's Association quality control program.</a> Alzheimers Dement. 9 (3), 251-261 (2013).</li><li>Kang, J. H., Korecka, M., Toledo, J. B., Trojanowski, J. Q., Shaw, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Utility+and+Analytical+Challenges+in+Measurement+of+Cerebrospinal+Fluid+Amyloid-beta1-42+and+tau+Proteins+as+Alzheimer+Disease+Biomarkers.">Clinical Utility and Analytical Challenges in Measurement of Cerebrospinal Fluid Amyloid-beta1-42 and tau Proteins as Alzheimer Disease Biomarkers.</a> Clin Chem. 59 (6), 903-916 (2013).</li><li>Mattsson, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+measurement+procedures+for+Alzheimer's+disease+cerebrospinal+fluid+biomarkers:+definitions+and+approaches+with+focus+on+amyloid+beta42.">Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid beta42.</a> Biomark Med. 6 (4), 409-417 (2012).</li><li>Ahmadkhaniha, R., Shafiee, A., Rastkari, N., Kobarfard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+of+endogenous+androgenic+steroids+in+cattle's+meat+by+gas+chromatography+mass+spectrometry+using+a+surrogate+analyte+approach.">Accurate quantification of endogenous androgenic steroids in cattle's meat by gas chromatography mass spectrometry using a surrogate analyte approach.</a> Anal Chim Acta. 631 (1), 80-86 (2009).</li><li>Jemal, M., Schuster, A., Whigan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography/tandem+mass+spectrometry+methods+for+quantitation+of+mevalonic+acid+in+human+plasma+and+urine:+method+validation,+demonstration+of+using+a+surrogate+analyte,+and+demonstration+of+unacceptable+matrix+effect+in+spite+of+use+of+a+stable+isotope+analog+internal+standard.">Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard.</a> Rapid Commun Mass Spectrom. 17 (15), 1723-1734 (2003).</li><li>Leinenbach, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+candidate+reference+measurement+procedure+for+quantification+of+amyloid-beta+in+cerebrospinal+fluid.">Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-beta in cerebrospinal fluid.</a> Clin Chem. 60 (7), 987-994 (2014).</li><li>Li, W., Cohen, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+endogenous+analytes+in+biofluid+without+a+true+blank+matrix.">Quantitation of endogenous analytes in biofluid without a true blank matrix.</a> Anal Chem. 75 (21), 5854-5859 (2003).</li><li>Perret-Liaudet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+collection+tubes:+a+critical+issue+for+Alzheimer+disease+diagnosis.">Cerebrospinal fluid collection tubes: a critical issue for Alzheimer disease diagnosis.</a> Clin Chem. 58 (4), 787-789 (2012).</li><li>Lewczuk, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52 (2), 332-334 (2006).</li><li>Pannee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+measurement+procedure+for+CSF+amyloid+beta+(A&#946;)1-42+and+the+CSF+A&#946;1-42+/A&#946;1-40+ratio+-+a+cross-validation+study+against+amyloid+PET.">Reference measurement procedure for CSF amyloid beta (A&#946;)1-42 and the CSF A&#946;1-42 /A&#946;1-40 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceability+to+a+common+standard+for+protein+measurements+by+immunoassay+for+in-vitro+diagnostic+purposes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceability+in+laboratory+medicine.">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.

Tarif edilen yöntem için, bunun yerine bir yedek matris kullanılarak, insan CSF kalibrasyon sağlar taşıyıcı analit yaklaşım 13, 14, 15, 16, kullanılır. vekil analit yaklaşım iki farklı izotopik olarak etiketlenmiş standartları içerir. Başka bir (13 CAβ 142), dahili standart olarak kullanılır ise bir (15 NAβ 142), insan CSF kali...

Alzheimer hastalığı (AD), demansın en yaygın biçimidir ve dünya çapında 1 ila yaklaşık 35 milyon insanı etkiler. Yaygın AD patogenezinde özünde yalan inanılan hastalığın nöropatolojik işaretlerinden toplu amiloid-beta (Aβ) peptitler 3 oluşan hiperfosforile tau proteininin 2 hücre içi nörofibrillerin ve hücre dışı plaklar vardır. Buna paralel olarak, biyomarkerların tarafından in vivo plak patoloji değerlendirmesi son zamanlarda AD 4 araştırma tanı kriterleri dahil edilmiştir. Aβ 1-42 BOS ölçümleri için, çok sayıda bağışıklık mevcuttur ve çok sayıda klinik laboratuarlarda 5 kullanılır. BOS Aβ 1-42 konsantrasyonu br plaklarda peptidin birikme yansıtan bilişsel normal yaşlılarda daha AD hastalarında yaklaşık olarak% 50 daha düşüktürain 6, 7. Bu biyolojik esas olarak immün (örneğin, antikor bazlı teknikleri) kullanılarak analiz edilir, ancak bu deneyler, matriks etkileri 8 etkilenebilir. Farklı teknoloji platformları üzerinde immunoassay ve tahlil standardizasyon 9 eksikliği kullanımı 10 11, 12 zorlu küresel kesme konsantrasyonlarının giriş yapar. Analitik doğrulanmış RMP ideal analitik platformlarda daha iyi karşılaştırılabilirlik ve genel ölçüm değişkenliği katkıda bulunan faktörlerden daha iyi kontrol sonuçlanan farklı tahlil platformları üniforma kalibrasyonunu imkan verecek. Geliştirilmiş LC-MS / MS yöntemi kullanılarak Aβ 1-42 mutlak ölçümü antikor bazlı techn ile bağlantılı sorunların üstesinden geliriques. Laboratuvar Tıp İzlenebilirlik için Ortak Komitesi (JCTLM veritabanı kimlik numarası C11RMP9) tarafından bir RPM olarak listelenen yöntem, BOS Aβ 1 uyumlaştırmak için bir Sertifikalı Referans Malzeme (CRM) içinde Aβ 1-42 mutlak konsantrasyonunu belirlemek için kullanılır teknikleri ve analitik platformlarda -42 ölçümler. açıklanan iş akışı Tıbbın diğer alanlarında peptidler ve proteinler için aday başvuru yöntemlerinin geliştirilmesi için alaka olmalıdır.

<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.

Şekil 1 &#39;de plaka kurulumu numune tam levha kullanılır. Daha az bilinmeyen numuneler analiz edilecek ise, ikinci kalibratör, RF, ve QC setleri bilinmeyen numunelerin ilk yarısından sonra konulmalıdır. Şekil 2&#39;de görüldüğü gibi, kalibratörler, düşük ve standart sapmalar, regresyon çizgisine yakın bulunmaktadır. Bu yöntem, 150 pg / ml ve 4,000 pg / ml miktarının b...

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 mutlak Niceleme<sub&gt; 1-42</sub&gt; BOS bir kitle Spektrometrik Referans Ölçüm Prosedürü kullanma

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.5K Görüntüleme.  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

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-amiloid-indüklenen anormallikler Fibrin pıhtısı yapısına spektroskopisi ve elektron mikroskobu tarama Analizi

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

Biyokimya

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.

Cisterna Magna Fare Beyin Omurilik Sıvısı Seri Toplama Tekniği

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

Biyoloji

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.

Bir Yüksek Verimli, Çoklanmış ve Hedefli Proteomik BOS Deneyi nörodejeneratif Biyomarkerleri ve Apolipoprotein E İzoformları Durum belirlememize

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

Tıp

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.

İnsan serebrospinal sıvı Endopeptidomic analiz için numune hazırlama

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

Nörobilim

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.

Beyin omurilik sıvısı imzalat fareler gelen topluluğu için geliştirilmiş bir yöntem

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.

Amiloid β mevduat matris yardımlı lazer desorpsiyon/iyonlaşma kütle spektrometresi Imaging ile insan beynindeki görselleştirme

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.

Farelerde İntrathecally Sentezlenmiş Proteinlerin Kantitatif Ölçümü

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.

Fare Beyin Bölümlerinde Amiloid-Beta Yükünün Tam ve Alt Bölgesel Nicelleştirilmesi

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.

Beyinden Amiloid Fibril Çekirdeklerinin Biyokimyasal Saflaştırılması ve Proteomik Karakterizasyonu

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.

Fizyolojik Koşullar Altında Alfa-Sinüklein Yapısal Dinamiğinin İncelenmesi için Milisaniye Hidrojen / Döteryum Değişim Kütle Spektrometresi

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

Aβ mutlak Niceleme

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