Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed‐phase liquid chromatographic system

ABSTRACT Although the efficient separation of intact protein mixtures is extremely difficult, reversed‐phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high‐performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile‐phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5‐fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins. Copyright © 2014 John Wiley & Sons, Ltd.