确定蛋白质表达和翻译后修饰(PTM)的变化对于阐明细胞信号转导和疾病机制至关重要。常规的基于抗体的方法具有固有的问题,例如高质量抗体的有限可用性和批次间的差异。基于自上而下的质谱(MS)的蛋白质组学已成为表征和定量蛋白质修饰的最强大的方法。然而,仍然缺乏同时确定蛋白质表达和PTM变化的可靠方法。在这里,我们开发了一个简单而强大的基于自上而下的液相色谱(LC)/MS的靶向蛋白质组学平台,用于高通量和高可重复性地同时定量蛋白质表达和PTM.我们使用这种方法来分析来自不同物种的各种肌肉类型的肌节亚蛋白质组,这成功地揭示了骨骼肌异质性和心脏发育变化的肌节蛋白同工型表达和PTM。这个有针对性的自上而下的蛋白质组学平台为复杂混合物中同时准确定量肌节蛋白表达和PTM提供了一个极好的“抗体无关”替代方案,这通常适用于不同的物种和各种组织类型。
Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent \'antibody-independent\' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.