What does one-dimensional gel electrophoresis-based western blotting data really mean in the reality of proteoforms?

One-dimensional gel electrophoresis-based western blotting (1DE-WB) is a routine technique for protein analysis. However, multiple immunoreactive bands are often found for a single protein target in 1DE-WB, yet only the band corresponding to the theoretical molecular weight of the canonical protein is considered relevant and additional immunoreactive bands are dismissed as false positives. However, what additional bands actually contain has not been established. Proteoforms are the final structural and functional entities of a gene product. Here, multiple immunopositive bands in the 1DE-WB analysis of growth hormone and prolactin in human pituitary tissue samples were identified by MS, 2DE-WB, or 2DE-WB combined with MS, and immunopositive bands were found to contain multiple proteoforms; a range of published 1DE-WB data indicate this is likely a common phenomenon. Thus, multiple 1DE-WB positive bands are likely to represent different proteoforms rather than non-specific reactions and MS, 2DE-WB, or 2DE-WB combined with MS are effective approaches to identify proteoforms and thus more thoroughly understand 1DE-WB data.