Top-Down Proteomics: Why and When?

Manifold biological processes at all levels of transcription and translation can lead to the formation of a high number of different protein species (i.e., proteoforms), which outnumber the sequences encoded in the genome by far. Due to the large number of protein molecules formed in this way, which span an enormous range of different physicochemical properties, proteoforms are the functional drivers of all biological processes, creating the need for powerful analytical approaches to decipher this language of life. While bottom-up proteomics has become the most widely used approach, providing features such as high sensitivity, depth of analysis, and throughput, it has its limitations when it comes to identifying, quantifying, and characterizing proteoforms. In particular, the major bottleneck is to assign peptide-level information to the original proteoforms. In contrast, top-down proteomics (TDP) targets the direct analysis of intact proteoforms. Despite being characterized by a number of technological challenges, the TDP community has established numerous protocols that allow easy implementation in any proteomics laboratory. In this viewpoint, we compare both approaches, argue that it is worth embedding TDP experiments, and show fields of research in which TDP can be successfully implemented to perform integrative multi-level proteoformics.