Desalting of Proteins Using MassPREP On-Line Desalting Cartridges Prior to Mass Spectrometry

In this application note we show a rapid, on-line desalting procedure with cycle times less than five minutes. 

LC-MS is a powerful tool used for the characterization of proteins. Reversed-phase HPLC is the most common mode of chromatography coupled with mass spectrometry. This separation technique can also be an effective method for the desalting of proteins. Pharmaceutical biotherapeutics and other proteins are often prepared or stored in salt containing buffer solutions such as PBS (phosphate buffered saline). These buffer solutions contain salts that form adducts with proteins, complicating data interpretation. These salts also suppress ionization during ESI-MS analysis. Therefore, separation of proteins from non-volatile salts is an important step prior to introduction into the mass spectrometer. Here we show a rapid, on-line desalting procedure with cycle times less than five minutes. The MassPREP On-Line Desalting Cartridge is a 2.1 x 10 mm device packed with polymer sorbent and was evaluated using acidic, (bovine serum albumin), basic, (cytochrome c), and large globular, (monoclonal antibody) protein samples. Parameters such as column carry over, loading, and lifetime were investigated. The results of this study show no sample carryover and excellent lifetime for repeated sample analysis under the conditions tested. 

Sample Preparation

Bovine serum albumin (BSA), cytochrome c, and monoclonal IgG, were obtained from Sigma Aldrich Inc. St. Louis Mo. All proteins were dissolved in phosphate buffered saline (PBS) at a concentration on 1µg/µL. 

Diveter valve, HPLC System, and Mass Spectrometer were all controlled by Waters MicroMass MassLynx Software.

Waters MassPREP On-Line Desalting Cartridges are an effective tool for the successful desalting of acidic, basic, and large globular proteins. The desalted proteins yielded abundant ESI-MS signal suitable for deconvolution and protein characterization. Studies determining loading capacity and carryover showed that up to 10 μg of monoclonal IgG was succesfully loaded with no breakthrough and no detected carryover in a subsequent analysis. The same results were obtained for BSA and cytochrome c for injected masses of up to 5 μg. Further, no loss of performance was observed after 100 consecutive injections were performed. The overall MassPREP desalting strategy provides an easy, fast (<5 minutes), and reproducible approach for the effective desalting of proteins stored in physiological salt buffers prior to high resolution structural analysis.