Analyzing a mixture of protein isoforms (proteoforms) with different combinations of PTMs, by bottom-up is usually not successful, as most of the information is lost upon trypsin digestion (Figure). On the contrary, in a top-down analysis, each proteoform is analyzed and fragmented individually, providing extra-information on the primary structure of the isoforms. Although early top-down analyses used collision induced dissociation (CID), alternative dissociation techniques based on electron transfer (ETD) or capture (ECD), are now providing complementary fragmentation profiles, significantly increasing protein sequence coverages. Furthermore, ETD and ECD, unlike CID, do not lead to the loss of certain labile PTMs such as phosphorylations, explaining their more and more common use in proteomics.
Figure: The combination of post-translational modifications (green symbols), mutations (colored squares) and alternative splicing (dotted squares), can sometimes produce numerous protein isoforms of proteoforms. After digestion (bottom-up, left), it is impossible to know the origin of the trypsic peptides. In top-down (right), each proteoform is selected and fragmented individually.