A Comparative Study of qPCR, Western Blot and Mass Spectrometry for the Estimation of Protein Concentrations

It is of great biological and clinical value within proteomics to be able to accurately quantify proteins in a complex sample. However, this is a challenge due to the large dynamic range of protein concentrations, demonstrating the need for advance quantification techniques. The mass spectrometryand affinity-based techniques with their respective reagents need to be both sensitive and specific, which has to be ensured by validation of these reagents. In addition, mRNA abundances have been used to predict protein abundances for non-detectable proteins. However, the RNA-protein level correlation has been questioned. In this study, quantitative real-time polymerase chain reaction, western blot and mass spectrometry have been compared for the quantification of RBM3, ADAT2, ANLN and VIM in a siRNA knockdown system using the U251 cell line. Via the comparison, the antibodies and the QPrESTs commercialized by Atlas Antibodies were validated and a mass spectrometry assay developed. In general, it was found that the methods correlated well in their concentration estimations, further demonstrating the reproducibility of the antibodies and the QPrESTs. However, these results need to be verified with a larger cohort of replicates and the quantitative protocol needs to be optimized. Next step would be to analyze the methods in an induced protein expression system.