Strong cation-exchange combined with mass spectrometry reveals the glycoform heterogeneity of sialylated glycoproteins

Sialylation is an important modification of proteins, related to protein life and bioactivity. However, the evaluation of sialylation is only based on the average molecular composition by peptide mapping and glycan profiling because sialylated proteins are usually too heterogeneous to obtain good quality mass spectra by conventional intact mass analysis methods. In this study, a simple strong cation exchange-mass spectroscopy (SCX-MS) method was developed for intact mass analysis of sialylated glycoproteins. The developed SCX-MS method provided good separation for sialylated glycoproteins and had an inherent characteristic of native MS. Thus, the intact mass analysis of highly heterogeneous glycoprotein, which cannot be obtained by reversed-phase liquid chromatography (RPLC)-MS and size exclusion chromatography (SEC)-MS methods, can be well analyzed using the current SCX-MS method. First, the method was developed and optimized using the etanercept monomer. Conditions including MS parameters, flow rate, and gradient were investigated. Then, the developed method was used to analyze a new recombinant vaccine, protein 1. Similar to the etanercept monomer, the intact molecular information of protein 1, which cannot be obtained by RPLC-MS and SEC-MS, can be achieved using SCX-MS. Combined with information obtained on peptide mapping and glycan profiles obtained by LC-MS, the new vaccine was well characterized. Finally, the SCX-MS method was used to quickly evaluate the batch-to-batch reproducibility of protein 1. It was much faster than peptide mapping and glycan profiling methods and can provide information complementary to these strategies. It should be useful for many applications where speed and comprehensive characterization are required, such as recombinant sialylated vaccines and fusion proteins.