非常感谢 Tanveer Batth 和同事在建立预分馏法方面的建议。
这项工作得到了瑞典研究理事会、Wallström 和 Sjöblom 基金会、枪支和 Bertil Stohne 基金会 Stiftelse、马格努斯 Bergwall 基金会、Åhlén 基金会、Alzheimerfonden、Demensförbundet、Stiftelsen för 的资助。格姆拉斯坦 Tjänarinnor, 克努特和爱丽丝的基础, Frimurarestiftelsen, 和 Västra Götalandsregionen。
这个项目的主要资助对象是 Kaj Blennow, 亨利 Zetterberg 和约翰 Gobom。

下面所述的协议是在以前的研究中使用的一个精炼版本, 其中大量的内源性多肽在人类 CSF15中被识别出来。对原始协议的更新包括对脑脊液化学预处理的细微改动, 以及用于离线高 pH 反相高效 HPLC 预分馏的梯度优化。
道德考虑
所有关于瑞典病人和控制材料的研究都得到了伦理委员会的批准: St. 约兰·佩尔松 (文献 2005-554-31/3)。来自阿姆斯特丹痴呆症患者的脑脊液样本和在国立神经病学和神经外科医院采集的样本被用于研究, 得到所有参与的病人的书面同意, 并得到区域伦理委员会的批准。这里所用的材料主要是从为诊断目的而抽取的样品中提取的左脑脊液, 在被纳入我们的研究之前就已被识别。没有可能将样本追溯到任何个别捐助者或捐助国集团。
1. 人体脑脊液的提取:
<ol><li>使用标准的协议22, 通过腰椎穿刺提取脑脊液 (必须由训练有素的医生执行)。</li>
	<li>通过离心 2500 x g 去除细胞碎片和其他非水溶性物质20分钟。</li>
	<li>目测检查脑脊液样本中是否有可能显示穿刺出血导致的血液污染的变色。血液中蛋白质浓度的升高和蛋白酶的存在会对分析结果产生很大影响。</li>
</ol>2. 脑脊液预处理 (1.5 毫升样本量, 无量化):
<ol><li>室温 (RT) 解冻1.5 毫升脑脊液等分, 将内容转移到10毫升聚丙烯管, 并将80µL 1 米乙碳酸氢盐 (TEAB) 作为缓冲剂。</li>
	<li>添加0.65 毫升8米胍盐酸盐 (GdnHCl) (活性浓度 GdnHCl: 2.4 米) 和涡旋轻轻在 RT 为10分钟。 注: chaotropic 剂, GdnHCl, 都影响溶剂粘度和与多肽链的相互作用, 这导致蛋白质的展开是积极有利的23。因此, GdnHCl 在随后的过滤过程中溶解蛋白质聚合体并增加内源性肽的回收。</li>
	<li>添加60µL 200 毫米水三 (2-乙) 膦盐酸盐 (TCEP) (活性浓度 TCEP: 6 毫米) 和孵育55° c 1 小时, 以减少半胱氨酸 disulphides。</li>
	<li>添加35µL 400 毫米碘 (iaa) (活性浓度 iaa: 4 毫米) 和孵化在 RT 在黑暗中为30分钟到烷基化胱氨酸。在随后的样品制备过程中, 添加烷基基团可确保半胱氨酸残留物在任何时候都不能自发形成新的二硫化物桥。</li>
	<li>在分子过滤之前, 加入3.25 毫升的 de 电离水和涡旋来稀释样品, 导致样品总量为5.5 毫升。这一步的作用是稀释 GdnHCl 到低于1米, 因为较高的浓度已被观察到有时会导致从过滤器设备的聚合物物质浸出。</li>
</ol>3. 脑脊液的预治疗 (10 x 150 µL 样本量, 等压标签-定量):
<ol><li>将150µL 的脑脊液等分从10人的 RT 中解冻, 然后将内容转移到单个1.5 毫升低束缚微离心管, 并将8µL 1 M TEAB 作为缓冲剂。</li>
	<li>添加65µL 8 米 GdnHCl (活性浓度 GdnHCl: 2.4 米) 和涡旋轻轻在 RT 为10分钟。</li>
	<li>添加6µL 200 毫米的水 TCEP (活性浓度 TCEP: 6 毫米) 和孵化在55° c 为 1 h, 以减少半胱氨酸 disulphides。</li>
	<li>添加3.5 µL 400 毫米水性 iaa (活性浓度 iaa: 6 mm), 在 RT 和黑暗中孵育30分钟至烷基化胱氨酸。</li>
	<li>准备等压标签套件 (e. g, 串联质量标签10plex 等压标签试剂)。允许等压标记试剂瓶在打开前达到 RT, 以避免不必要的试剂水化, 加入41µL 的 HPLC 级乙腈 (AcN), 用温和的搅拌溶解5分钟。</li>
	<li>将30µL 等压标签试剂溶液转移到相应的样品中, 并在温和搅拌下孵育1小时。等压标签试剂包括一个琥珀酰亚胺-酯基团, 它与在多肽 N 总站和赖氨酸残留物中存在的主要氨基酸反应。</li>
	<li>加8µL 5% 羟胺 (活性浓度羟胺: 0.16%), 在 RT 处轻轻摇动20分钟, 以熄灭标签反应。由于分开地被标记的样品将被结合, 保证标签反应被淬火。通过添加大量的胺类以羟胺的形式, 其余的等压标签试剂可以作出反应, 从而使惰性。</li>
	<li>结合每一个10单独标签的样品在一个单一的15毫升聚丙烯管的内容。</li>
	<li>将6.4 毫升的 de 电离水添加到组合样品和涡流中, 以减少 AcN 浓度从12% 到 3%, 到 GdnHCl 浓度 GdnHCl 到1米以下, 这是由于较高浓度, 有时会导致聚合物物质的浸出。从过滤器设备。</li>
</ol>4. 分子量切断过滤
<ol><li>为了去除潜在的污染物, 条件分子过滤器通过装载10毫升水 1 M GdnHCl, 25 毫米 TEAB 并且离心为 15 min 在 2500 x g 和 RT, 放弃流经 (FT)。</li>
	<li>在过滤器上加载整个样本量 (5 毫升无标签的 csf (步骤 2.5) 或10毫升压标记的 csf (步骤 3.9)), 并在 30 x g 和 RT 上离心2500分钟, 在集合容器中留出流量 (FT)。过滤的结果是多肽和小蛋白质分离从更大的蛋白质和残余细胞残骸。这一步可以被看作是 peptidomic 等同于使用蛋白水解蛋白质来产生肽在蛋白质组实验。</li>
	<li>负载5毫升的25毫米 TEAB (水) 的过滤器和离心机为15分钟, 2500 x g 和 RT, 以增加恢复肽。</li>
	<li>从前两个步骤 (总共10毫升的非标签或15毫升压标签的样本) 的组合罚球, 并进行样品酸化。</li>
</ol>5. 固相萃取--盐析和样品清理
<ol><li>酸化在固相萃取 (SPE) 之前从步骤4.4 中筛选出的样品。酸化是为了改善溶质 (肽) 相互作用与固定相的固相的 SPE 墨盒。</li>
	<li>对于无标签的样品 (容量10毫升): 添加550µL 1% 乙酸酸 (TFA)-样品总体积: 10.55 毫升与 0.05% TFA。</li>
	<li>为等压标记的样品 (容量15毫升): 增加20毫升 0.1% TFA 到酸化和更低的 AcN 集中从3% 到 1%-样品的总容量:30 毫升与 0.066% TFA。 注: TFA 还添加了它作为离子配对试剂的功能, 它可以提高多肽的保留能力, 而不能够与 SPE 墨盒的包装材料保持足够强的疏水作用。</li>
	<li>如果 ph > 3, 滴定样品与20% 磷酸, 直到样品 ph 值是 < 3。</li>
	<li>通过添加1毫升的 84% AcN, 0.1% 甲酸 (FA), 并放弃 FT. 重复一次。在随后的步骤中, 需要对滤筒过滤器进行调理, 以去除不需要的物质, 否则洗与肽一起;进一步, 调节增加过滤器的渗透性。</li>
	<li>平衡的 SPE 墨盒加1毫升的 0.1% TFA, 丢弃 FT. 重复一次。确保过滤器在最后一个平衡步骤后不会运行干燥-在过滤器的顶部保持一个小体积。为了使过滤器能够通过去除从调理步骤中留下的疏水物质 (乙腈) 来准备滤池以保持多肽的平衡。</li>
	<li>加载整个样本量 (10.55 毫升为非标签样本或30毫升压标签的样品), 在几个部分, 如果必要的, 让 FT 运行成废物。确保墨盒在取样加载轮或最后一个取样卷加载后不会运行干燥, 请在过滤器顶部保留一小卷。</li>
	<li>通过1毫升的 0.1% TFA 的过滤器, 以消除盐和试剂和丢弃的 FT. 重复一次。确保墨盒在每次洗涤步骤后不会运行干燥-在墨盒顶部保持少量液体。</li>
	<li>放置1.5 毫升低束缚微离心管下的墨盒和洗的样品通过1毫升 84% AcN, 0.1% FA 超过墨盒。</li>
	<li>在真空离心机中, 在干燥、贮存于-80 ° c 或立即进行高 pH 分馏时, 将溶剂从脱盐试样中蒸发。</li>
</ol>6. 离线高 pH 反相 HPLC 样品分离
<ol><li>
制备水基高 pH (HpH) 流动相: HpH 缓冲器 A: 纯净水 HpH 缓冲器 B: 84% AcN HpH 缓冲器 C:25 mM NH4哦 HpH 加载缓冲区: 2.5 mM NH4哦, 2% AcN 注意: HpH 加载缓冲区用作传输解决方案和示例缓冲区</li>
	<li>16µL HpH 加载缓冲液中的样品再溶解20分钟</li>
	<li>负载15µL 的样品的 HPLC 系统与内部分数收集器的 96-深井板配置根据 Batth et al.20 , 但有少量更改。分级在100µL/分钟的流量超过 pH 稳定的分离柱 (C18 3.5 µm, 2.1 毫米 x 250 毫米) 和收集一小部分每分钟在线性 60 min 梯度。</li>
	<li>使用以下渐变时间点: t = 0 分钟, B = 1%, C = 10%;t = 4 分钟, B = 1%, C = 10%, 开始分数集合;t = 76 分钟, B = 70%, C = 10%;结束分数收集;t = 76.5 分钟, B = 85%, C = 10%;t = 80 分钟, B = 85%, C = 10%;t = 80.5 分钟, b = 1%, c = 10% 和 t = 90 min, b = 1%, c = 10%。</li>
	<li>在12口井中重复地收集分数, 从而在一个圆的模式中, 使分数间隔12分钟, 产生12分, 每个分数包含6串联的子分数。</li>
	<li>在 RT 和 3000 rpm 之前, 用真空离心法除去样品中的溶剂, 直到干燥为止, 并在-80 ° c 之前将样品储存在 LC-质谱分析之前。</li>
</ol>7. LC-质谱
<ol><li>
准备水的流动阶段: 缓冲器 A: 0.1% FA 缓冲器 B: 0.1% FA, 84% AcN 装货缓冲: 0.05% TFA, 2% AcN 注: 装载缓冲器用作装载泵的传输方案、采样缓冲器和移动相位。</li>
	<li>通过增加6µL 加载缓冲器并在 RT 处晃动20分钟来重新溶解每个12馏分</li>
	<li>负载5µL 样品的纳米流高效液相色谱, 操作在陷井专栏配置 (陷井专栏:75 µm x 2 cm, C18, 100 Å孔大小, 3 µm 微粒大小; 分离专栏: C18, 75 µm x 500 毫米, 100 Å孔大小, 2 µm 微粒大小, 以 150 nL/分钟的流速进行多肽分离, 使用以下梯度: t = 0 min, B = 2%;t = 10 分钟, B = 2%;t = 11 分钟, B = 7%;t = 100 分钟, B = 26%;t = 170 分钟, B = 45%;t = 175 分钟, B = 80%;t = 181 分钟, b = 2%, t = 210 分钟, b = 2%。</li>
	<li>
在高分辨率混合质谱仪上, 通过纳米电喷雾界面进行高效液相色谱联用。在m/z范围 350-1400 的分辨率设置下, 以 MS 模式记录全扫描频谱 (2.0e5 AGC 目标) 12万。
	<ol>
<li>在数据相关的采集模式下操作质谱仪, 从前十最强峰值的m/z > 150, 并在1.0e4-1 的强度范围内选择 ms/毫秒谱. 0e5 用于片段离子分析。使用 1 m/z的四极隔离窗口隔离前体离子, 最大注射时间为100毫秒, RF 镜头在60%。</li>
		<li>将动态排除应用于十五年代的排除时间和± ppm 的m/z公差。在高碰撞能量分解 (HCD) 单元中执行碎片, 并在 orbitrap 的分辨率设置为 5万 (5.0e4 AGC 目标值) 记录 ms/ms 收购。</li>
	</ol>
</li>
</ol>8. 多肽识别
<ol><li>
为肽鉴定提交结果. 原始文件从质谱分析到蛋白质组学搜索引擎应用以下设置: 注: 在我们以前的研究中15, 三搜索引擎;吉祥物 v2.4, Sequest HT, 和峰值 v7.5 是并行使用, 这里指定的设置是为所有三搜索引擎, 除非另有规定。大多数可调节的设置是普遍的多肽/蛋白质识别和应该有相应的设置在任何给定的搜索引擎。 频谱选择器: 最小前体质量: 350 Da 最大.前体质量: 5000 Da 扫描类型: 完全 信号/噪声阈值: 1。5 序列数据库搜索 数据库: UniProt_SwissProt [version2015_11] 分类学: 智人 酶: 无 最大.错过分裂: 0 仪器 (仅吉祥物): ESI 陷阱 最小肽长度 (仅 SequestHT): 6 最大.多肽长度 (仅 SequestHT): 144 前体质量公差:15 ppm 碎片质量公差: 0.05 Da 静态修改: Carbamidomethyl (C);[如果标签]TMT10plex (N 期) 动态修改: 氧化 (M);[如果标签]TMT10plex (K) 多肽谱匹配 (PSM) 验证器 过滤 (仅限吉祥物和 Sequest) 或诱饵融合 (仅限峰值) 目标罗斯福: 0.01 基于: q 值的验证</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+human+cerebrospinal+fluid:+discovery+and+verification+of+biomarker+candidates+in+neurodegenerative+diseases+using+quantitative+proteomics.">Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics.</a> J Proteomics. 74 (4), 371-388 (2011).</li><li>H&#246;ltt&#228;, 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=A+single+dose+of+the+&#947;-secretase+inhibitor+semagacestat+alters+the+cerebrospinal+fluid+peptidome+in+humans.">A single dose of the &#947;-secretase inhibitor semagacestat alters the cerebrospinal fluid peptidome in humans.</a> Alzheimers Res Ther. 8 (1), 11 (2016).</li><li>Cao, Z., Tang, H. Y., Wang, H., Liu, Q., Speicher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Comparison+of+Fractionation+Methods+for+In-depth+Analysis+of+Plasma+Proteomes.">Systematic Comparison of Fractionation Methods for In-depth Analysis of Plasma Proteomes.</a> J Proteome Res. 11 (6), 3090-3100 (2012).</li><li>Chiu, C. W., Chang, C. L., Chen, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+peptide+fractionation+strategies+used+in+proteome+analysis.">Evaluation of peptide fractionation strategies used in proteome analysis.</a> J Sep Sci. 35 (23), 3293-3301 (2012).</li><li>Gilar, M., Olivova, P., Daly, A. E., Gebler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonality+of+separation+in+two-dimensional+liquid+chromatography.">Orthogonality of separation in two-dimensional liquid chromatography.</a> Anal Chem. 77 (19), 6426-6434 (2005).</li><li>Batth, T. S., Francavilla, C., Olsen, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-line+high-pH+reversed-phase+fractionation+for+in-depth+phosphoproteomics.">Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.</a> J Proteome Res. 13 (12), 6176-6186 (2014).</li><li>Yang, F., Shen, Y., Camp, D. G., Smith, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pH+reversed-phase+chromatography+with+fraction+concatenation+for+2D+proteomic+analysis.">High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis.</a> Expert Rev Proteomics. 9 (2), 129-134 (2012).</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>Moglich, A., Krieger, F., Kiefhaber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+the+effect+of+urea+and+guanidinium+chloride+on+the+dynamics+of+unfolded+polypeptide+chains.">Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains.</a> J Mol Biol. 345 (1), 153-162 (2005).</li><li>Ho&#776;ltta&#776;, 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=An+Integrated+Workflow+for+Multiplex+CSF+Proteomics+and+Peptidomics+Identification+of+Candidate+Cerebrospinal+Fluid+Biomarkers+of+Alzheimer&#8217;s+Disease.">An Integrated Workflow for Multiplex CSF Proteomics and Peptidomics Identification of Candidate Cerebrospinal Fluid Biomarkers of Alzheimer&#8217;s Disease.</a> J Proteome Res. 14 (2), 654-663 (2014).</li></ol>

在作者之间没有任何竞争的利益, 财务或其他, 报告。

采用高效液相色谱法预分馏步骤, 采用分子量超滤法回收内源性多肽11降低了相对样本复杂度, 从而使5倍大要研究的样本量。这反过来增加了每个分数中所含的肽子集的浓度, 从而提高了检测低丰肽的几率。
通过对三种蛋白质组软件并行使用的内源肽进行识别, 可以扩大已知的脑脊液 endopeptidome 超过10倍。共16395内源性多肽在一个合并脑脊液标本的初步试验中?...

脑脊液中的生物标志物目前正转化为神经退行性疾病的研究。在阿尔茨海默氏病, 最常见的神经退行性疾病, 影响超过6000万人的全球1,2, 一个生物标记三重组成的肽淀粉样β, 微管稳定蛋白 tau, 和磷酸化 tau 形态, 能检测出高灵敏度和特异性的疾病, 并已纳入诊断研究标准3。在其他神经退行性疾病, 如帕金森氏病和多发性硬化症, 蛋白质组研究发现了许多生物标记候选, 其中一些目前正在评估临床研究4,5 ,6。
除了蛋白质, CSF 还含有丰富的内源性多肽7,8,9,1011.这些肽构成了许多脑源性蛋白质的裂解产物, 同时也代表了一种潜在的重要的疾病生物标志物来源。为了增加人类脑脊液中已识别的内源性多肽的库存, 并在临床研究中进行 csf endopeptidomic 分析, 开发了用于样品制备和 LC-MS 分析的方法 (在图 1中包含了一个简短的协议方案).该方法在最近的一项研究中的应用, 导致了来自几个非特定诊断个体的汇集脑脊液样本中的近16400内源性脑脊液肽的鉴定, 扩大已知 csf endopeptidome 十倍13。该方法可选择与等压标签法结合使用, 以量化。
样品制备
脑脊液中蛋白质质量的主要来源是血浆成分 (如白蛋白和免疫球蛋白) 通过血脑屏障14,15。它们的高丰度阻碍了对低丰度脑源样件的检测。内源性肽可以很容易地与高丰度的蛋白质分离, 从而允许大量的脑脊液肽提取物用于 LC-MS 分析, 从而能够检测到低丰度的肽。
在这里提出的协议中, 分子量的分子过滤是用来分离脑脊液肽的蛋白质部分;已在以前的几项研究中使用的方法8、9、10、11、12、16。过滤步骤之后是一个离线反相高效液相色谱预分馏步骤, 在高 pH 移动相位梯度。通过串联两个反相 HPLC 步骤, 以 pH 值为主要区别, 两个步骤的选择性差异主要是由于不同的肽电荷状态而导致的多肽潴留。在酸性条件下, 高 pH 肽预分馏在 LC-MS 的应用在增加肽识别17,18, 甚至是在复杂生物的优势样品比较正交分离模式19, 如强 cat 离子交换 (SCX) 和 RP20。为了缩短分析时间, 使用了串联方案, 将每12个th分数 (例如、分数1、13、25、37和 49) 汇集在一起, 由于反相高效色谱法的高分辨力, 仍然很大程度上避免了不同的肽的共洗LC-MS 步骤20,21中的分数。
肽鉴定
peptidomic 研究中的肽鉴定与蛋白质组学研究不同, 因为在数据库搜索中不能指定酶解理, 因此, 识别率通常低于11。最近的一项研究13表明, Sequest 和吉祥物获得的内源性多肽的识别率在使用自适应评分法修改相应软件程序的默认评分算法时得到了显著改善算法过滤, 表明内源肽的最佳评分算法与胰肽13不同。在这项研究中, 基于自动肽从头测序使用软件峰值 (BSI) 被发现是互补的两个片段离子指纹为基础的搜索引擎, 导致了显着更大的一套识别肽.

<table border="0" cellpadding="0" cellspacing="0" width="1564">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">&#160;1 M Triethylammonium bicarbonate</td>
			<td style="width:339px;">Fluka, Sigma-Aldrich</td>
			<td style="width:339px;">17902-100ML</td>
			<td style="width:339px;">TEAB</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">&#160;8 M Guanidinium hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G7294-100ML</td>
			<td>GdnHCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Pierce</td>
			<td>20490</td>
			<td>TCEP</td>
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		<tr height="38">
			<td height="38" style="height:38px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td>I1149-5G</td>
			<td>IAA</td>
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		<tr height="38">
			<td height="38" style="height:38px;">Hydroxylamine 50% (w/w)</td>
			<td>Sigma-Aldrich</td>
			<td>457804-50ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acetonitrile, Far UV, HPLC gradient grade</td>
			<td>Sigma-Aldrich</td>
			<td>271004-2L</td>
			<td>AcN</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Formic acid</td>
			<td>Fluka, Sigma-Aldrich&#160;</td>
			<td>56302-1mL-F</td>
			<td>FA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Triflouroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508-10AMP</td>
			<td>TFA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>30501-1L-1M</td>
			<td>NH4OH</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td>MWCO-filter</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sep-Pak C18, 100 mg</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td>SPE-column</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Resprep 12-port SPE Manifold&#160;</td>
			<td>Restek</td>
			<td>26077</td>
			<td>Vacuum manifold</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">TMT10plex Isobaric Label Reagent Set</td>
			<td>Thermo Fisher Scientific</td>
			<td>90110</td>
			<td>TMT10plex</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">UltiMate 3000 RSLCnano LC System</td>
			<td>Dionex</td>
			<td>5200.0356</td>
			<td>Online sample separation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ultimate 3000 RPLC Rapid Separation Binary System</td>
			<td>Dionex</td>
			<td>IQLAAAGABHFAPBMBEZ</td>
			<td>Offline high-pH fractionation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Orbitrap Fusion Tribrid mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td style="width:339px;">IQLAAEGAAPFADBMBCX</td>
			<td>Mass spectrometer for sample analysis</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Proteome Discoverer 2.0&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGABSFAKJMAUH</td>
			<td>Proteomics search platform</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Mascot v2.4</td>
			<td>Matrix Science</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sequest HT</td>
			<td>Thermo</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">PEAKS v7.5&#160;</td>
			<td>Bioinformatic Solutions Inc.)</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap 100, 75 &#181;m x 2 cm, C18, 100 &#197; pore size, 3 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164535</td>
			<td>Trap column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap C18, 75 &#181;m x 500 mm, 100&#197; pore size,&#160;2 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164942</td>
			<td>Separation Column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Savant SpeedVac High Capacity Concentrators</td>
			<td>Thermo Fisher Scientific</td>
			<td>SC210A-230</td>
			<td>SpeedVac/Vacuum concentrator</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">XBridge Peptide BEH C18 Column, 130&#197;, 3.5 &#181;m, 2.1 mm X 250 mm</td>
			<td>Waters</td>
			<td>186003566</td>
			<td>Separation Column (micro HPLC)</td>
		</tr>
	</tbody>
</table>

sample preparation for endopeptidomic analysis in human cerebrospinal fluid

本协议描述了一种用于识别人脑脊液中内源性多肽的方法。为此, 本文提出了一种基于分子量分子过滤和质谱分析的方法, 并结合离线高 pH 反相高效液相色谱预分馏步骤。
脑脊液分泌是去除中枢神经系统细胞分子的主要途径。因此, 中枢神经系统中的许多过程都反映在脑脊液中, 使它成为一种有价值的诊断液。CSF 有一个复杂的组成, 包含的蛋白质的浓度范围为 8-9 的数量级。除了蛋白质, 以前的研究还表明存在大量的内源性多肽。虽然不像蛋白质那样广泛研究, 但它们也可能作为生物标志物而具有潜在的兴趣。
内源性肽通过分子过滤与脑脊液蛋白含量分离。通过去除样品中的大多数蛋白质含量, 可以增加研究的样品体积, 从而也能提高内源性多肽的总量。在 LC-质谱分析之前, 通过在碱性 pH 值上采用反相 (RP) HPLC 预分馏步骤, 解决了过滤肽混合物的复杂性。分馏与一个简单的串联方案结合在一起, 其中60个分数被汇集到 12, 分析时间消耗量可以从而减少, 当仍然主要避免 co 洗脱。
通过使用三种不同的肽/蛋白质识别软件程序, 并结合结果进行了自动肽鉴定。不同的方案是互补的, 而不是可比的, 少于15% 的标识重叠之间的三。

本文介绍的方法已在三项研究中应用和评价前的样品前分馏 (表 1)。第一项研究使用离线 LC 在 MALDI 靶板上发现脑脊液分数, 并导致730已确定内源性肽11。在以下两项研究中, 使用了等压标签。主要在病例/对照研究中, 对 CSF endopeptidome 和蛋白质组中的潜在生物标志物进行鉴定和描述, 同时24, 并在第二项研究中使用等压标记用于监测治疗效果体内γ-分泌抑制?...

Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

提出了一种人脑脊液内源性多肽的质谱分析方法。通过采用分子量切断过滤、色谱预分馏、质谱分析以及随后的多肽识别策略组合, 就可以将已知的脑脊液 peptidome 扩大近十倍以前的研究。

人脑脊液 Endopeptidomic 分析的样品制备

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

sample-preparation-for-endopeptidomic-analysis-human-cerebrospinal

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Sahlgrenska Academy at University of Gothenburg. 提出了一种人脑脊液内源性多肽的质谱分析方法。通过采用分子量切断过滤、色谱预分馏、质谱分析以及随后的多肽识别策略组合, 就可以将已知的脑脊液 peptidome 扩大近十倍以前的研究。

视频: Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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.

一种改进的麻醉小鼠脑脊液采集方法

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

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

恒河猴脑脊液中Aβ和Tau水平重复经颅磁刺激的试点研究

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...