Keeping It Native: Non‐Denaturing Approaches to Top‐Down Protein Mass Spectrometry

Top‐down proteomics and top‐down mass spectrometry approaches provide direct characterization of proteoforms; the specific molecular forms of proteins, including post‐translational modifications, truncations, alternative splicing, and sequence variants. To date, most top‐down studies have been performed under denaturing conditions, losing critical information about protein folding and noncovalent protein‐protein and protein‐ligand interactions. However, recent efforts to apply non‐denaturing, or native, ionization to top‐down mass spectrometry and top‐down proteomics have shown substantial promise for characterizing these higher‐order and more transient interactions. This review covers progress over the last 35 years in non‐denaturing top‐down approaches and addresses the inherent strengths and challenges of the technique. We cover three major areas of applications: (1) Ligand binding and drug discovery, (2) Structural biology, and (3) Proteomics aspects and bioinformatics.