ABSTRACT Individual cells have distinctive molecular characteristics, including biologically relevant proteoforms. Although single‐cell (SC) molecular omics offer unprecedented insights into cellular heterogeneity and function, the characterization of proteoforms in SCs remains an uncharted territory. Mass spectrometry (MS)‐based proteomics is a key technology for analyzing proteins and their proteoforms in SCs. Top‐down proteomics (TDP) is the MS technique of choice to characterize proteoforms from the bulk of cellular extracts. TDP has also shown proficiency in characterizing these biomolecules in SC. Recent advances in sample processing, separation techniques, MS instrumentation, and computational approaches have evolved the application of TDP to SC analysis, overcoming longstanding technical barriers and opening new avenues for large‐scale studies. This review discusses the importance of TDP for profiling proteoforms within SC and provides examples of the biological relevance of these biomolecules. Furthermore, this review describes recent advances in the TDP for SC research while providing a detailed overview of MS‐based methods for analysis of proteoforms in individual cells. The potential and limitations of these MS‐based approaches are discussed, along with perspectives about the future direction of the SC‐TDP field.
Melby et al. (2026) studied this question.