Quantitative Top-Down Proteomics: A Review

Top-down proteomics approaches have emerged as powerful strategies for investigating the effects of proteoform modifications, such as amino acid substitutions, truncation, and post-translational modifications (PTMs), on protein function and activity in biological systems. In addition to proteoform structure, proteoform abundance also influences cellular biochemistry by regulating signaling pathways and altering protein-protein interaction dynamics. Thus, accurate measurement of proteoform abundance is critical for examining cellular phenotypes and disease states. The application of quantitative proteomics strategies to top-down proteomics, however, has been challenging due to difficulties in extracting and solubilizing intact proteins, the wide dynamic range in proteoform abundance, limited sensitivity for low‑abundance species, and the need for advanced separations and robust deconvolution algorithms. Recent advances in top-down proteomics strategies, including methods to improve proteoform mass spectrometry data collection, sample preparation, and data analysis, have laid the groundwork for quantitative top-down proteomics. Here, we provide a comprehensive review of current quantitative top-down proteomics strategies and examine the applications, advantages, and challenges, including label-free, metabolic labeling, chemical tag labeling, and targeted quantification strategies.