Absolute Quantification of Protein Concentration through Electrospray-Differential Mobility Analysis

M. Li, J. Tan, M.J. Tarlov, M.R. Zachariah
University of Mayland College Park, US

Keywords: protein concentration, analytical approach development, electrospray-differential mobility analysis, BSA

Summary:

Accurate and reliable protein quantification is essential over a wide range of enzymatic and biopharmaceutical researches. The most commonly adopted quantification assays employed are ultraviolet-visible (UV) absorbance and dye-bindings. However, they suffer from accuracy limitations. On the other hand, more accurate method, amino acid analysis, is laborious and time consuming especially for large protein quantification. In this study, we introduced a fast and accurate analytical approach to measure absolute protein concentration for which a systematic and robust protocol was developed. This methodology was based on statistical analysis of droplet entrapped non-specific oligomer formation in electrospray process. Absolute number concentration of proteins in solution was directly related to observed dimer to monomer ratio. The instrument we used was Electrospray-Differential Mobility Analyzer (ES-DMA) which differentiated monomers, dimers from higher order oligomers based on electric mobility and Condensation Particle Counter (CPC) which was used to count observed protein particles (Fig. 1). NIST standard reference material (SRM 927, Bovine Serum Albumin (BSA)) was used to validate our protocol. Through systematic studies (Fig. 2 and Fig. 3), we showed that our method was pretty accurate and robust with a measured BSA concentration of 65.8g±1.6g/L compared with NIST certified value of 65.41g±0.82g/L