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.

주 :이 프로토콜은 출발 물질로서 각각 Aβ 펩타이드를 50㎍ / ㎖의 농도에서 50 μL의 분취 량을 필요로한다. Aβ 펩타이드의 탈 이온수 중 20 % 아세토 니트릴 (ACN) 및 4 % 진한 암모니아 용액 (V / V)에 용해시키고, 80 ℃에서 보관해야한다. 주의 : 안전 정보는 표 1을 참조하십시오. 솔루션 1. 준비 <ol><li> ACN 20㎖의 탈 이온수에서 농축 암모니아 4 ㎖ (~ 25 %)을 희석하여 20 % ACN과 4 % 100 ㎖를 탈 이온수 (v / v)로 농축 암모니아 용액을 제조 하였다. 탈 이온수 100 ㎖에 최종 볼륨을 조정합니다. 신선한 매일 확인합니다. </li><li> 50 ㎖의 최종 부피의 탈 이온수에 구아니딘 염산염 26.08 g을 용해하여 5 M 구아니딘 히드로 클로라이드 50 ㎖를 준비한다. 20 ° C에서 보관하고 신선한 매월합니다. </li><li> (탈 이온수 4 % 인산 200㎖를 준비 V /V)의 탈 이온수에서 농축 인산 (~ 85 %) 9.4 mL로 희석. 탈 이온수 200 ㎖에 최종 볼륨을 조정합니다. 냉장고에 저장하고 신선한 주간을합니다. </li><li> ACN 37.5 mL의 탈 이온수에서 농축 암모니아 5 ㎖ (~ 25 %)를 희석하여, 탈 이온수 50 75 % ACN ㎖의 10 % 진한 암모니아 용액 (V / V)를 준비한다. 탈 이온수로 50 ㎖에 최종 볼륨을 조정합니다. 신선한 매일 확인합니다. </li><li> 일상적인 임상 분석 드 식별 남은 샘플에서 얻은 교정기, 인간 CSF의 적어도 2.5 mL로 녹여. </li><li> 150 mM의 나트륨, 3.0 mM의 K, 1.4 mM의 CA, 0.8mm의 마그네슘, 1.0 mM의 P, 및 탈 이온수 155 mM의 CL을 포함하는 인공 CSF를 준비하여 4 mg / ml의 최종 농도로 우 혈청 알부민을 추가한다. 단 1 mL의 향후 사용을 위해 분취 량을, 저장, 분석 당 필요하지만, 많은 양을 준비한다. </li></ol> 구경 2. 준비 <ol><li> 4 μg의 0.5 mL의 준비 / mL의 10.5 ㎖의 마이크로 원심 튜브에 20 % ACN과 4 % 진한 암모니아 용액을 0.46 / ㎖ 15 142 NAβ 50 μg의 40 μL를 첨가하여 5 NAβ 1-42 펩티드. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 2 ML의 microcentrifuge 관에서 20 % ACN과 4 % 진한 암모니아 1.95 mL의 4 μg의 / ㎖ 15 NAβ 142 50 μL를 첨가하여 100 NG / ㎖ 15 NAβ 1-42 펩티드이 용액을 준비한다. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 표 2에 나타낸 각각의 용액의 양을 혼합하여 용액 여섯 교정기 (AF)를 준비한다. 0.5, 1.5, 2 ML의의 microcentrifuge 튜브를 사용합니다. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 해당 교정 솔루션 및 1 분을위한 와류 믹서 표 3. 믹스에 따라 인간의 CSF를 추가하여 0.5 ML의의 microcentrifuge 튜브 (중복)에 최종 교정기를 준비합니다. </li></ol> 내부 표준 3. 준비 <ol><li> 2 ML의 microcentrifuge 관에서 20 % ACN 4 % 농축 암모니아 1.968 mL의 / mL의 13 CAβ (142) 50 μg의 32 μL를 추가하여 / mL의 13 CAβ 1-42 펩타이드 0.8 μg의 2 용액을 준비합니다. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 5 ML의 microcentrifuge 관에서 20 % ACN과 4 % 진한 암모니아 용액 4.9 μg의 0.8 / mL의 0.1 mL를 첨가하여 16 NG / ㎖ 13 CAβ 1-42 펩티드 5 ㎖를 준비한다. 1 분 동안 와동 혼합기에서 혼합한다. </li></ol> 응답 인자 샘플 4. 준비 참고 : 응답 팩터 (RF) 결정은 교정 (15 NAβ 1-42)에 사용되는 표지 된 펩타이드의 농도를 결정하기 위해 수행된다. 이것은 천연 Aβ 1-42 펩티드의 농도는 아미노산 분석 (AAA)를 사용하여 측정 된 것을 필요로한다. 따라서, 체적 고유 Aβ 1-42 펩티드 분취 농도AAA의 요구 사항을 충족해야합니다. <ol><li> 0.5 ML의 microcentrifuge 관에서 20 % ACN 4 % 농축 암모니아 0.46 mL의 / mL의 기본 Aβ 1-42 50 μg의 40 μL를 추가하여 (레이블이 지정되지 않은) 4 μg의 / ML의 기본 Aβ 1-42 0.5 mL로 준비합니다. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 20 %의 1.96 mL의 4 μg의의 / mL의 기본 Aβ 1-42 및 4μg 20 μL / mL의 15 NAβ 1-42을 20 μL를 추가하여 기본 15 NAβ 1-42의 2 mL의 40 NG / ML의 믹스를 준비 ACN과 4 %는 2 ML의 microcentrifuge 관 암모니아를 농축시켰다. 1 분 동안 와동 혼합기에서 혼합한다. </li><li> 0.5 ML의 microcentrifuge 관에서 인공 CSF의 0.38 mL의 40 NG / ML의 믹스의 20 μL를 추가합니다. 중복을 준비하고 1 분 동안 와류 믹서에 혼합한다. </li></ol> 5. 샘플 준비 주의 : 샘플을 해동은 롤러 실온에서 측정한다. <ol><li>그림 1 항에있어서, 1 mL의 단백질 96 깊은 웰 플레이트에 각 교정기, 응답 계수, 및 (사용 된 경우 품질 관리 [QC] 샘플 포함) 미지 시료의 0.18 mL를 넣고 (전체 판을 가정하는 데 사용됩니다). 에 샘플을 추가해야하거나, 우물의 바닥에 근접합니다. </li><li> 각 웰 (즉, 교정기, 응답 인자, QCS, 그리고 미지수)에 내부 표준의 20 μL를 추가; 이는 피펫 팁을 침지하지 않고 시료 표면에 충분히 가까운 측의 방울을 방출하는 것이 중요하다. </li><li> 각 웰에 0.2 ㎖의 5 M 구아니딘 염산염을 추가합니다. </li><li> 마이크로 통에 샘플 플레이트를 배치하고 1,100 rpm에서 45 분 동안 샘플을 섞는다. 최적의 주파수는 기기에 따라 다를 수 있습니다. 혼합기의 주파수 및 진폭을 설정 용액 철저히 혼합하고, 내부 표준 또는 CSF의 어떠한 방울을 웰의 측면에 섞이지 남지 않도록. </li><li> 4 % 인산 0.2 mL를 넣고또한 각. 소용돌이 간략하게 섞는다. </li></ol> 6. 고체 단계 추출 참고 : 모든 세척,로드 및 용출 단계에서는 솔루션을 추가 한 후 가장 낮은 진공을 적용하고로드하거나 솔루션을 용출 필요에 따라 점차적으로 증가한다. 각각의로드 및 용출 단계 사이의 진공을 해제합니다. <ol><li> 혼합 모드에서 폐기물 저장소 트레이를 넣고 추출 플레이트 매니 폴드 챔버 내에서 양이온 교환, 96 웰 고체상 추출 (SPE) 판. </li><li> 각 웰에 0.2 ㎖의 메탄올을 첨가하여 SPE 흡착제를 조정. </li><li> 각 웰에 4 % 인산 0.2 mL를 첨가하여 흡착 평형. </li><li> 특수 목적 판에 깊은 웰 플레이트에서 모든 샘플 (각 웰 약 0.62 ㎖)에 전송합니다. 특수 목적 판에 깊은 웰 플레이트에서 샘플을 전송할 때 8 채널 피펫을 사용하여; 샘플이 인턴 포함 이래로, 모든 시료의 전체 또는 같은 부피를 전송하는 것이 중요하지 않다변화를 보상합니다 알 표준입니다. </li><li> 샘플을 각 웰에 4 % 인산 0.2 mL를 첨가하여 통과 한 후 흡착제를 세척한다. </li><li> 세정 용매 흡착제에서 용출 한 후, 컬렉션 플레이트 또는 튜브 저수지 트레이를 교체합니다. </li><li> 배 75 % ACN / 10 % 농축 암모니아 50 μL를 첨가하여 흡착제로부터 시료를 용출하고,이 용액이 흡착제를 통과하는 매우 낮은 진공을 요구주의. 각 추가 사이의 진공을 해제해야합니다. <ol><li> 선택 사항 : 컬렉션 플레이트 또는 튜브를 밀봉하고 -80 ° C에서 그들을 고정. 6.8.2 단계로 진행하기 전에 수집 판 또는 튜브에서 씰을 제거합니다. </li><li> (열을 가하지 않고) 진공 원심 분리를 이용하여 용출액을 건조; 이것은 진공 원심 분리기에 따라 몇 시간이 하나로부터 취할 수있다. </li><li> 용기를 밀봉하고 -80 ° C에서 그들을 고정. </li></ol></li></ol> 7. 액체 크로마토 그래피에그래피로 <ol><li> , 바늘 세척 (50 % ACN (ACN [V / V]에서 4 % 탈 이온수와 0.1 % 농축 암모니아), B (탈 이온수 [V / V] 5 % ACN 0.3 % 농축 암모니아) 모바일 A 상을 준비 4 %가 탈 물에 암모니아를 집중 [V / V]). <ol><li> 이동상 A의 500 ㎖를 들어, ACN 25 mL의 탈 이온수에 집중 암모니아 1.5 mL를 넣어. 탈 이온수 500 ㎖에 최종 볼륨을 조정합니다. </li><li> 이동상 B의 500 ㎖를 들어, 진한 암모니아 500 μL와 ACN을 증류수 25 mL를 첨가. ACN 500 mL의에 최종 볼륨을 조정합니다. </li><li> ACN 120 mL의 탈 이온수에 집중 암모니아 10 ㎖를 추가하여 바늘 세척 250 mL로 준비합니다. 탈 이온수 250 mL의에 최종 볼륨을 조정합니다. </li><li> 액정 시스템을 사용하기 전에 20 분 동안 초음파 욕조에서 열린 이동상 A와 B와 바늘 세척 병을 넣어 </li></ol></li><li> 25 μL 집중하고 % ACN 20 %의 4 각 샘플을 녹여테드 암모니아 용액을 20 분 동안 진탕에 배치합니다. 샘플 아래 원심 분리기 및 자동 시료 주입기에 배치 (7 ° C로 유지). </li><li> 50 ° C로 유지 된 1 × 250mm이 폴리스티렌 - 디 비닐 벤젠 (역상) 모노리스 컬럼 시료 20 μL를 주입한다. <ol><li> 0.3 mL / 분의 유속으로 표 4에 나타낸 LC 구배를 사용한다. 질량 분광계 오염을 감소시키기위한 전환 밸브를 사용하여 (이후 열) 낭비 처음 두 개의 지난 5 분 전환. </li></ol></li></ol> 8. 질량 분석 주의 : 이러한 파라미터 aheated 전기 분무 이온화 소스를 구비 한 하이브리드 orbitrap 극자 질량 분광계를 사용 하였다. <ol><li> 표 5에 따라 이온 소스의 매개 변수를 설정합니다. </li><li> (네이티브 Aβ 1-42의 4+ 충전 상태를 분리 1,129.48 질량을 MS 악기를 설정2.5 m / z의 분리 폭을 갖는 사중 극 질량 분석 장치에 충전 비율 [의 m / z), 1-42 15 NAβ (1,143.00 m / z), 13 CAβ 1-42 (1,179.50 m / z). </li><li> 17.0의 충돌 에너지 정규화 (NCE)에 충돌 셀의 격리 펩타이드 조각. (예를 들면, 삼중 사중 MS와 같은 악기의 다른 형태를 사용하여, 특히 경우)에도 동일한 형태의 이것은 각 기기 조율 할 필요가 있습니다. </li><li> 2 × 5 요금 자동 이득 제어 대상 및 250 ms의 최대 분사 시간과, 17.500의 해상도 단편 스펙트럼을 기록한다. </li></ol> 9. 데이터 처리 <ol><li> 각 펩티드에 대한 크로마토 그래피 영역을 계산하기 표 6 (± 250 밀리 질량 부 [MMU]의 질량 공차) 생성물 이온의 합을 사용한다. 이온의 종류와 숫자가 네이티브 Aβ에 대해 표시되어 있습니다 <suB&gt; 1-42 산물 이온들은 NAβ 15 1-42 13 CAβ 1-42 모두 동일하기 때문이다. </li><li> 천연 Aβ 1-42의 곡선 아래 영역 (15) NAβ 1-42 곡선 (크로마토 그래피 피크) 아래의 영역을 분할하여 두 응답 인자 샘플들의 평균 응답 인자를 결정한다. </li><li> 단계 9.2에서 계산 된 반응 인자를 곱하여 보정에 사용 된 15 NAβ 1-42의 농도를 조정한다. </li><li> 농도 대 구경 두 세트에서 13 CAβ 1-42에 NAβ 1-42 (15)의 면적비를 플로팅하여 교정 곡선을 구축. </li><li> 선형 회귀를 사용하여 보정 곡선의 기울기 및 절편을 계산한다. </li><li> 해제에 대한 내부 표준 (13 CAβ 1-42) 네이티브 Aβ 1-42의 면적 비율을 계산알려진 샘플. </li><li> 단계 9.5에서 얻어진 기울기 및 절편을 이용하여 검량선으로부터 미지 시료의 농도를 추정. </li></ol>

<ol>
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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.

상술 한 방법 대신 대용 행렬을 이용하여, 우리는 인간 CSF의 교정을 가능하게 대리 분석 방법 13, 14, 15, 16을 사용 하였다. 대리 분석 방법은 두 가지 동위 원소 표지 된 표준을 포함한다. 또 (13 CAβ 142) 내부 표준으로 사용되는 동안 온 (15 NAβ 142), 인간 CSF의 보정 곡선을 ...

알츠하이머 병 (AD)은 치매의 가장 흔한 형태이며, 전 세계적으로 한 약 35 만명에 영향을 미친다. 널리 AD의 발병 기전의 핵심 거짓말을 믿고 병의 신경 병리학 적 특징은 집계 아밀로이드 - 베타 (Aβ) 펩타이드 3 구성된 hyperphosphorylated 타우 단백질이 세포 내 신경 섬유 엉킴 및 세포 외 플라크 있습니다. 이에 맞춰, 바이오 마커에 의해 생체 내에서 플라크 병리의 평가는 최근에 AD 4에 대한 연구 진단 기준에 포함되었습니다. Aβ 1-42의 CSF 측정을 위해 여러 가지 면역 사용할 수 있으며 많은 임상 실험실 (5)에 사용됩니다. CSF에서 Aβ 1-42의 농도는 BR에 플라크의 펩티드의 침착을 반영인지 정상 노인보다 AD 환자의 약 50 % 낮다아인 (6, 7). 이러한 생체는 주로 면역 (예를 들어, 항체 - 기반 기술)을 사용하여 분석하지만, 이러한 분석은 매트릭스의 효과 (8)에 의해 영향을받을 수있다. 다른 기술 플랫폼의 면역 분석 및 표준화 9의 부재의 사용은 10 11 12 어려운 글로벌 컷오프 농도로 도입한다. 해석 적 검증 RMP는 이상적으로 분석 플랫폼에서 더 나은 비교와 전체 측정 변동에 기여하는 요인의 더 나은 제어의 결과로, 다른 분석 플랫폼의 균일 보정을 허용합니다. 현상 된 LC-MS / MS 방법을 사용하여 Aβ 1-42의 절대 정량 항체 기반 TECHN와 관련된 많은 문제들을 극복iques. 실험 의약 추적을위한 합동위원회 (JCTLM 데이터베이스 식별 번호 C11RMP9)에 의해 RMP로 나열있어서, CSF Aβ 한 조화하는 인증 표준 물질 (CRM)에서 Aβ 1-42의 절대 농도를 결정하는 데 사용될 기술 및 분석 플랫폼에서 -42 측정. 설명 된 워크 플로는 의학의 다른 영역에서 펩타이드 및 단백질 후보 참조 방법의 개발을위한 관련이 있어야한다.

<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.

그림 1의 판 설치 샘플의 전체 판에 사용됩니다. 이하 미지 시료를 분석 할 경우, 제 교정기는 RF 및 QC 세트는 미지 시료의 전반부 뒤에 배치한다. 도 2에 도시 된 바와 같이, 구경이 낮은 표준 편차와, 회귀 직선에 가깝다. 이 방법은 150 pg / ml 인 4,000 pg / ml 인의 정량의 상위 레벨의 정량의 낮은 수준을 ?...

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.

Aβ의 절대 정량<sub&gt; 1-42</sub&gt; CSF의 질량 분석 참조 측정 절차를 사용하여

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

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7.5K 조회수.  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.

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

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

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

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

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

인간의 두피 조직에서 Aβ Multimers의 SDS-PAGE/Immunoblot 감지는 항원 에피토프 검색을 사용 Homogenates

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

연구

신경과학

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

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

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

맞춤형 HPLC 정화 프로토콜 즉, 올리고머 형성 할 수있는 수율 고순도 아밀로이드 베타 42과 아밀로이드 베타 펩티드 (40),

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

생화학

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.

분광학 그리고 스캐닝 전자 현미경 검사 법에 의해 섬유 소 혈전 구조에 β 아 밀 로이드 유도 비정상의 분석

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

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

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

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

높은 처리량, 멀티 플렉스 및 대상 단백체 CSF 분석은 퇴행성 신경 바이오 마커 및 아포 지단백 E 이소 상태를 정량화하기

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

의학

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.

인간의 중추 신 경계에서 Endopeptidomic 분석을 위한 시료 준비

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

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

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

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

마 취 쥐에서 척수의 컬렉션에 대 한 개선된 방법

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.

매트릭스 보조 레이저 탈 착/이온화 질량 분석 이미징와 인간의 뇌에서 아 밀 로이드 β 예금의 시각화

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.

쥐의 경내 합성 단백질의 정량적 측정

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.

마우스 뇌 절편에 대한 아밀로이드-베타 부하의 전체 대 하위 지역 정량화

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.

뇌에서 아밀로이드 피브릴 코어의 생화학 적 정제 및 프로테오믹 특성화

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

Aβ의 절대 정량

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