State‐of‐the‐art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome

Abstract

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β-tubulin and β-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

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“…There has been an extensive debate on which house-keeping proteins (HKPs) can be used as steady-level controls in molecular biology experiments (40). Recent research has shown that classic HKPs may actually change their expression under specific conditions (41). For example in our dataset, GAPDH, a popular HKP, exhibits stable levels for most treatments, but could not be a good control for studies with bortezomib and few other compounds (Fig.…”

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“…However, stable expression under treatment conditions has not been studied for most of these proteins. Indeed, in practice, a number of these HKPs show inconsistent expression levels under different treatments (Vigelsø, Dybboe et al 2014;Janes 2015;Lee, Jo et al 2016). For example, GAPDH, one of the most extensively used HKPs showed variable expression in our compound panel especially for bortezomib ( Figure 31A).…”

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“…2d). Finally, we observed an association between the intensity of peptides from a housekeeping protein and the known proportion of human tissue in each sample (TAR DNA-binding protein 43 27 ; Fig. 2e).…”