Novel Strategies to Address the Challenges in Top-Down Proteomics

Top-down mass spectrometry (MS)-based proteomics is a powerful technology for comprehensively characterizing proteoforms to decipher post-translational modifications (PTMs) together with genetic variations and alternative splicing isoforms toward a proteome-wide understanding of protein functions. In the past decade, top-down proteomics has experienced rapid growth benefiting from groundbreaking technological advances, which have begun to reveal the potential of top-down proteomics for understanding basic biological functions, unraveling disease mechanisms, and discovering new biomarkers. However, many challenges remain to be comprehensively addressed. In this Account & Perspective, we discuss the major challenges currently facing the top-down proteomics field, particularly in protein solubility, proteome dynamic range, proteome complexity, data analysis, proteoform-function relationship, and analytical throughput for precision medicine. We specifically review the major technology developments addressing these challenges with an emphasis on our research group's efforts, induding the development of top-down MS-compatible surfactants for protein solubilization, functionalized nanoparticles for the enrichment of low-abundance proteoforms, strategies for multidimensional chromatography separation of proteins, and a new comprehensive user-friendly software package for top-down proteomics. We have also made efforts to connect proteoforms with biological functions and provide our visions on what the future holds for top-down proteomics.

Automated summary

Top-down proteomics is a powerful technology for comprehensively characterizing proteoforms and understanding protein functions, disease mechanisms, and biomarkers. However, challenges in protein solubility, data analysis, and proteome complexity remain. Recent technological developments include the development of new surfactants, nanoparticles for enriching proteoforms, and strategies for multi-dimensional chromatography separation of proteins.