P1-261: DEVELOPMENT OF A UNIFIED -OMIC PREPARATION METHODOLOGY: AN ALTERNATIVE, NON-IP APPROACH TO TARGETED LC-MS/MS DETECTION OF SECRETED AMYLOID-ß PEPTIDES IN CEREBROSPINAL FLUID AND IPSC-DERIVED FAMILIAL ALZHEIMER'S DISEASE MODELS
P1-261: DEVELOPMENT OF A UNIFIED -OMIC PREPARATION METHODOLOGY: AN ALTERNATIVE, NON-IP APPROACH TO TARGETED LC-MS/MS DETECTION OF SECRETED AMYLOID-ß PEPTIDES IN CEREBROSPINAL FLUID AND IPSC-DERIVED FAMILIAL ALZHEIMER'S DISEASE MODELS
复制标题
P1-261：统一组学制备方法的开发：一种替代的非 IP 方法，用于靶向 LC-MS/MS 检测脑脊液中分泌的淀粉样肽和 IPSC 衍生的家族性阿尔茨海默病模型
DOI：
10.1016/j.jalz.2019.06.816
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Leckey C
中科院分区：
文献类型：
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作者：
Leckey C
背景在组学研究中，液-质联用(LC-MS)提供了一个平台来验证多个生物标记物，并开发出可以更容易地转化为临床病理实验室的极其特异的测试。在阿尔茨海默病中，针对淀粉样蛋白(Aβ，Aβ)多肽的靶向LC-MS分析目前依赖于样品制备过程中的免疫沉淀(IP)进行浓缩。在这项研究中，我们提出了一种替代的非IP制备方法，允许从患者脑脊液和家族性AD IPSC衍生模型的细胞培养液中浓缩和检测生物体液中的多肽，特别是短和长Aβ物种。方法我们开发了一种样品制备方法，该方法利用固相萃取和不同的化学洗脱条件从同一样品中分离出蛋白质、代谢物、多肽和脂类。特别是，这种方法是作为从生物体液中分离和浓缩Aβ肽的IP的替代制备而发展起来的。我们还在三重四极杆质谱仪(Xevo TQ-XS)上建立了短肽(Aβ1-14/1-15/1-16/1-17)和长肽(Aβx-37/x-38/x-40)的靶向LC-MS/MS分析方法，用于检测脑脊液和N2B27细胞培养上清液中分泌的Aβ多肽。简写为A-β肽、A-β1-15和A-β1-17在750μL脑脊液中检测到。对于较长的Aβ多肽，在750βL脑脊液中也检测到Aβx-37和Aμx-38。为提高灵敏度和检测其他Aβ多肽以及其他AD生物标志物，进一步的技术优化正在进行中。结论该方法可以提供一种新的、快速和有针对性的方法来测量生物体液中的Aβ多肽，特别是较少研究的脑脊液中的短Aβ物种。在使用稳定同位素示踪剂的研究中，使用LC-MS/MS作为这种方法中的分析平台，可以测量不同的生物标志物及其在健康和疾病中的动力学特征。
BackgroundIn-omics research, liquid chromatography-mass spectrometry (LC-MS) offers a platform to validate multiple biomarkers and develop extremely specific tests that can be more easily translated into a clinical pathology laboratory. In Alzheimer's disease, targeted LC-MS assays for Amyloid-β (Aβ) peptides currently rely on immunoprecipitation (IP) during sample preparation for enrichment. In this study, we propose an alternative non-IP preparation method to allow for enrichment and detection of peptides from biofluids, in particular short and long Aβ species, from patient CSF and cell media from familial AD iPSC-derived models.MethodsWe have developed a sample preparation method utilising solid phase extraction (SPE) and varying chemical elution conditions to fractionate proteins, metabolites, peptides and lipids from the same sample. In particular, this method was developed as an alternative preparation to IP isolation and enrichment of Aβ peptides from biofluids. We have also developed targeted LC-MS/MS assays (MRMs) for short (Aβ 1-14/1-15/1-16/1-17) and long (Aβ x-37/x-38/x-40) peptides on a triple quadrupole mass spectrometer (Xevo TQ-XS) to detect secreted Aβ peptides in CSF and N2B27 cell media from iPSC-derived cortical neurons.ResultsPreliminary results show that both short and long Aβ peptides can be selectively enriched from C18 columns under mild elution conditions (20-30% ACN, 0.1% TFA) and with high recovery. For short Aβ peptides, Aβ 1-15 and Aβ 1-17 were detectable in 750μl of CSF. For longer Aβ peptides, Aβ x-37 and Aβ x-38 were also detectable in 750μl of CSF. Further technical optimisation for increased sensitivity and detection of other Aβ peptides, as well as other AD biomarkers, is on-going.ConclusionsThis approach could offer a new, rapid and targeted approach to the measurement of Aβ peptides in biofluids, particularly the lesser-studied short Aβ species in CSF. In studies utilising stable isotope tracers, the use of LC-MS/MS as the analytical platform in this approach could allow for measurement of different-omic biomarkers and their kinetic profiles in health and disease.