A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting

Proteomics and other
protein-based analysis methods such as Western
blotting all face the challenge of discriminating changes in the levels
of proteins of interest from inadvertent changes in the amount loaded
for analysis. Mass-spectrometry-based proteomics can now estimate
the relative and absolute amounts of thousands of proteins across
diverse biological systems. We reasoned that this new technology could
prove useful for selection of very stably expressed proteins that
could serve as better loading controls than those traditionally employed.
Large-scale proteomic analyses of SDS lysates of cultured cells and
tissues revealed deglycase DJ-1 as the protein with the lowest variability
in abundance among different cell types in human, mouse, and amphibian
cells. The protein constitutes 0.069 ± 0.017% of total cellular
protein and occurs at a specific concentration of 34.6 ± 8.7
pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily
detectable with several peptides, it can be helpful in normalization
of proteomic data sets. In addition, DJ-1 appears to be an advantageous
loading control for Western blot that is superior to those used commonly
used, allowing comparisons between tissues and cells originating from
evolutionarily distant vertebrate species. Notably, this is not possible
by the detection and quantitation of housekeeping proteins, which
are often used in the Western blot technique. The approach introduced
here can be applied to select the most appropriate loading controls
for MS-based proteomics or Western blotting in any biological system.