Comparative study on protein quantitation by digital PCR with G2-EPSPS as an example

Abstract

Protein quantification is an important issue for pharmaceutical development, clinical diagnosis, and food safety. Generally, protein concentration is relatively quantified with an external standard by high performance liquid chromatography, mass spectrometry, immunoassay and so on. However, as a molecular biological method, PCR is commonly used for nucleic acid analysis. With the proximate ligand assay approach, quantitative PCR (qPCR) could be also used for protein quantitation. Digital PCR (dPCR) is recently proposed for absolutely quantitation of nucleic acid with the advantage of high accuracy and less interference without external standard. With the aid of proximate ligand assay approach, dPCR could be also potentially used for both relative and absolute protein quantitation. However, less work has been reported for the method performance of PCR based protein quantitation. In this paper, we have developed both relative and absolute quantitation methods by qPCR and dPCR with G2-EPSPS protein as an example. Traditional ELISA method was also developed as comparison. Isotope dilution mass spectrometry (IDMS), which is primary method for protein quantitation based on its primary sequence, was used to assign the accurate concentration of G2-EPSPS for comparison. The method performance was thoroughly evaluated. For relative quantitation, it revealed that the dPCR showing better repeatability and reproducibility compared with qPCR and ELISA methods, especially for higher level sample analysis. And qPCR showed similar repeatability and reproducibility as ELISA method. It is interesting that the absolute concentration obtained by dPCR was much lower than that obtained by IDMS. One possible reason was that only those binding to both mAbs simultaneously could be quantified by dPCR, therefore, it is kind of “active concentration”. Comparatively, the concentration obtained by IDMS was just based on the primary sequence of protein. The study demonstrated the potential advantage of dPCR used for relative protein quantitation and the risk of lower concentration obtained by dPCR when it was used for absolute protein quantitation. However, the dPCR could be a potential primary method for active protein concentration measurement. This study could benefit the correct development and usage of protein quantitation methods based on dPCR. It is also anticipated that the dPCR based protein quantitation could be widely used in the future.