Enhancing top-down proteomics of brain tissue with FAIMS
Enhancing top-down proteomics of brain tissue with FAIMS
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DOI：
10.1101/2021.01.18.427216
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发表时间：
2021-01
期刊：
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通讯作者：
J. Fulcher;Aman Makaju;Ronald J. Moore;Mowei Zhou;D. Bennett;P. D. De Jager;W. Qian;L. Paša-Tolić;V. Petyuk
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作者：
J. Fulcher;Aman Makaju;Ronald J. Moore;Mowei Zhou;D. Bennett;P. D. De Jager;W. Qian;L. Paša-Tolić;V. Petyuk
阿尔茨海默病和帕金森病的蛋白质组学研究为神经退行性疾病提供了有价值的见解。到目前为止，这些研究主要局限于自下而上的方法，阻碍了人们表征蛋白质“完整”状态的程度。自上而下的蛋白质组学（TDP）克服了这一限制，然而，它通常仅限于观察最丰富的蛋白质形式和相对较小的尺寸。因此，离线分馏技术通常用于降低样品复杂性，限制吞吐量。更高通量的替代方法是在线分馏，如气相高场不对称波形离子迁移谱法（FAIMS）。利用来自阿尔茨海默病脑组织的高复杂性样本，我们描述了在TDP中添加FAIMS如何有效地提高蛋白质组覆盖的深度。例如，在−50补偿电压（CV）下实施FAIMS，观察到的非冗余蛋白质形态的平均数量（1,833±17,n = 3）比没有（754±35）增加了一倍多。我们还发现FAIMS可以影响变形形态及其电荷包膜的传输，这取决于它们的大小。重要的是，FAIMS能够鉴定完整的β淀粉样蛋白（Aβ），包括与阿尔茨海默病密切相关的易聚集的Aβ1-42变体。
Proteomic investigations of Alzheimer’s and Parkinson’s disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein’s “intact” state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimer’s disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at −50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 ± 17, n = 3), compared to without (754 ± 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ1-42 variant which is strongly linked to Alzheimer’s disease.