Data Independent Acquisition Mass Spectrometry Method for Improved Neuropeptidomic Coverage in Crustacean Neural Tissue Extracts
Data Independent Acquisition Mass Spectrometry Method for Improved Neuropeptidomic Coverage in Crustacean Neural Tissue Extracts
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DOI：
10.1021/acs.analchem.8b05734
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发表时间：
2019-04-16
影响因子：
7.4
通讯作者：
Li, Lingjun
中科院分区：
文献类型：
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作者：
DeLaney, Kellen;Li, Lingjun
神经肽是神经和神经内分泌系统中一类重要的信号分子，但由于其在体内浓度低，存在许多干扰伪影，因此研究具有挑战性。通常，复杂组织提取物中神经肽的质谱分析的限制不是由于神经肽低于检测限，而是由于在液相色谱洗脱时间期间未选择用于串联质谱的离子，因此未被鉴定。在这项研究中，数据独立采集（DIA）的方法，以提高覆盖率的神经组织中的神经肽从模式生物C。北极光确定了最佳质荷比范围和隔离窗口，并随后用于检测更多的神经肽提取物从大脑和心包器官比传统的数据依赖的采集方法。DIA方法导致在大脑中检测到几乎两倍的神经肽，在心包器官中检测到大约1.5倍的神经肽。还探索了技术和生物学重现性，发现与原始方法相比有所改善，3次重复注射中有3次检测到56%的神经肽，3次生物学重复中有3次检测到62%的神经肽。此外，68个推定的新的神经肽进行了检测和鉴定的从头测序。并对该方法的定量准确性进行了探讨。预计开发的方法是有用的，以获得更深的剖析的神经肽，特别是那些在低丰度，在各种样品类型。
Neuropeptides are an important class of signaling molecules in the nervous and neuroendocrine system, but they are challenging to study due to their low concentration in vivo in the presence of numerous interfering artifacts. Often the limitation of mass spectrometry analyses of neuropeptides in complex tissue extracts is not due to neuropeptides being below the detection limit but due to ions not being selected for tandem mass spectrometry during the liquid chromatography elution time and therefore not being identified. In this study, a data independent acquisition (DIA) method was developed to improve the coverage of neuropeptides in neural tissue from the model organism C. borealis. The optimal mass-to-charge ratio range and isolation window were determined and subsequently used to detect more neuropeptides in extracts from the brain and pericardial organs than the conventional data dependent acquisition method. The DIA method led to the detection of almost twice as many neuropeptides in the brain and approximately 1.5-fold more neuropeptides in the pericardial organs. The technical and biological reproducibility were also explored and found to be improved over the original method, with 56% of neuropeptides detected in 3 out of 3 replicate injections and 62% in 3 out of 3 biological replicates. Furthermore, 68 putative novel neuropeptides were detected and identified with de novo sequencing. The quantitative accuracy of the method was also explored. The developed method is anticipated to be useful for gaining a deeper profiling of neuropeptides, especially those in low abundance, in a variety of sample types.