About three-fourths of mouse proteins unexpectedly appear at a low position of SDS-PAGE, often as additional isoforms, questioning whether all protein isoforms have been eliminated in gene-knockout cells or organisms.

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high-throughput approach to identify these isoforms. Using an oversimplified top-down LC-MS/MS strategy, we detected, around the 26-kD position of SDS-PAGE, proteins produced from 782 genes in a Cdk4-/- mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one-fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS-PAGE. These 213 proteins are considered as the wild type (WT). The remaining three-fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two-thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger-group or a smaller-group, respectively, for their appearance at a higher or lower position of SDS-PAGE. For instance, at this 26-kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS-PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4-/- cell line, thus wondering whether some of other gene-knockout cells or organisms show similar incompleteness of the knockout.