Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS‐PAGE), is a technique commonly used to detect specific proteins. SDS‐PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography – mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non‐malignant HEK293 and cancerous MDA‐MB231 (MB231) cells separated using SDS‐PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS‐PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer‐therapy studies.