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.

Most human genes can produce multiple protein isoforms that should appear at multiple positions of polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS), but most published results of Western blotting show only one protein. We performed SDS-PAGE of proteins from several human cell lines, isolated the proteins at the 72-, 55-, 48-, 40-, and 26-kD positions, and used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although ACTB and GAPDH are 41.7-kD and 36-kD proteins, respectively, LC-MS/MS identified peptides of ACTB and GAPDH at all of these SDS-PAGE positions, making us wonder whether they produce some unknown protein isoforms. The NCBI (National Center for Biotechnology Information, USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one non-coding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatic analysis identified a 17.6-kD isoform encoded by the non-coding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with great similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to ACTB. These data collectively disqualify GAPDH and ACTB from serving as the reference genes for determination of the protein level in such techniques as Western blotting, a leading role these two genes have been playing for decades in the biomedical research.