The Proteoform Landscape of Current Top-Down Proteomics

Top-down proteomics (TDP) aims for the proteome-wide characterization of intact proteoforms, i.e., the distinct molecular forms of proteins, which are the functional entities in biological systems. Over recent years, TDP has rapidly evolved, enabling increasingly comprehensive proteoform identifications across diverse samples. However, a systematic assessment of TDP's experimental capabilities and biases is lacking. Here, we analyzed proteoform identifications reported by 359 TDP datasets from 69 studies published over the past decade. Despite substantial differences in sample type and experimental design, highly consistent trends were observed across studies, including a strong bias toward abundant, low-molecular-weight proteoforms and frequently detected modifications. This analysis provides a reference for state-of-the-art TDP experiments and defines capabilities and limitations of current in-depth proteoform identification. Furthermore, it establishes a realistic baseline for laboratories entering the field, highlights the need for future analytical developments, and provides a benchmark for evaluating future advances.