Fast top-down intact protein characterization with capillary zone electrophoresis-electrospray ionization tandem mass...

Capillary zone electrophoresis (CZE)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome c, myoglobin, bovine serum albumin (BSA), and β-casein) was used as the sample. The CZE-ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome c) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE-ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified.