Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis

To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S‐transferase and β‐lactoglobulin. On the other hand, a low‐molecular‐weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high‐ and low‐molecular‐weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations. Copyright © 2013 John Wiley & Sons, Ltd.