Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry
Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry
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DOI：
10.1021/acs.analchem.7b02426
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发表时间：
2017-11-21
影响因子：
7.4
通讯作者：
Patrie, Steven M.
中科院分区：
文献类型：
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作者：
Plymire, Daniel A.;Wing, Casey E.;Patrie, Steven M.
髓鞘碱性蛋白（Myelin basic protein，MBP）在神经元髓鞘中起着重要的结构和功能作用。翻译的MBP表现出极端的微观异质性，许多选择性剪接变体（ASV）和翻译后修饰（PTM），据报道与中枢神经系统成熟，髓鞘稳定性，以及各种脱髓鞘和脱髓鞘疾病的病理生物学。传统的生物分析工具不能同时有效地检测ASV和PTM事件，这限制了对MBP微异质性在人类生理和疾病中的作用的理解。为了满足这一需求，我们报告了一个自上而下的蛋白质组学管道，该管道结合了表面多孔反相液相色谱（SPLC），傅里叶变换质谱（FTMS），数据独立采集（DIA）与分离器分离（NSD），并对齐数据处理资源，以快速表征小鼠组织内丰富的MBP蛋白质型。三层蛋白质型鉴定和表征工作流程解析了四种已知的MBP ASV和数百种差异修饰状态，这些状态来自于在类似于0.5 μ g材料上运行的单个90分钟SPLC-FTMS。这包括单独的14.1 kDa ASV的323种蛋白形式。我们还确定了两个新的ASV从一个替代的转录起始位点（ATSS）的MBP基因，以及一个从未有过的特征S-酰化事件连接棕榈酸，油酸和硬脂酸在C78的17.125 kDa的ASV。
Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de-and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease. To address this need, we report on a top-down proteomics pipeline that combines superficially porous reversed-phase liquid chromatography (SPLC), Fourier transform mass spectrometry (FTMS), data-independent acquisition (DIA) with nozzle-skimmer dissociation (NSD), and aligned data processing resources to rapidly characterize abundant MBP proteoforms within murine tissue. The three-tier proteoform identification and characterization workflow resolved four known MBP ASVs and hundreds of differentially modified states from a single 90 min SPLC-FTMS run on similar to 0.5 mu g of material. This included 323 proteoforms for the 14.1 kDa ASV alone. We also identified two novel ASVs from an alternative transcriptional start site (ATSS) of the MBP gene as well as a never before characterized S-acylation event linking palmitic acid, oleic acid, and stearic acid at C78 of the 17.125 kDa ASV.