Abstract
Housekeeping proteins are essential endogenous controls for normalization as they are expected to be stably expressed. However, the stability of the expression level of housekeeping proteins needs to be assessed considering various experimental conditions. Our study evaluated the degree of variability of 7 commonly used housekeeping proteins with regard to their potential utility as normalizers in 56 pairs of matched colorectal adenocarcinoma (CRC) tissue samples and 6 pairs of hepatocellular carcinoma (HCC) tissue samples using multiple reaction monitoring (MRM) and Western blot analyses. A comprehensive experimental design and strict statistical analysis revealed that the expression levels of these 7 housekeeping proteins were not as stable as expected and they all exhibited upregulations to varying degrees in both the CRC and the HCC tissue samples. Consequently, we verified that using the amount of total protein instead of that of an individual protein can serve as a preferable control for studies of protein expression that require normalization.
Highlights
The endogenous control is a widely used concept in gene expression studies, as it is the foundation of accurate quantification
Our study evaluated the degree of variability of 7 commonly used housekeeping proteins with regard to their potential utility as normalizers in 56 pairs of matched colorectal adenocarcinoma (CRC) tissue samples and 6 pairs of hepatocellular carcinoma (HCC) tissue samples using multiple reaction monitoring (MRM) and Western blot analyses
MRM quantitative proteomics revealed the elevated expression of housekeeping proteins in CRC tumor tissues
Summary
The endogenous control is a widely used concept in gene expression studies, as it is the foundation of accurate quantification. Housekeeping genes, which mainly function in cellular maintenance, are commonly used as endogenous controls as they are often considered to be adequately and stably expressed [1]. Cells are exposed to rapidly changing microenvironments as a result of various changes in metabolic conditions. To survive these stresses, cells must adopt various strategies to increase their adaptability to the rapidly changing microenvironments [2]. Cells must adopt various strategies to increase their adaptability to the rapidly changing microenvironments [2] These subtle adjustments may affect both the active genes, which are sensitive to local environments, and the housekeeping genes, which maintain vital functions
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