Mass Spectrometric Analysis of Phosphorylation Modification in 14-3-3ε Protein

In the present study, 14-3-3ε protein was over-expressed in HEK293 cells, which was genetically attached with 6 × His tag. The protein was purified and enriched by nickel-charged resin and gel electrophoresis, and then was subject to tryptic digestion. The phosphorylated peptide fragments within 14-3-3ε protein were enriched by TiO2 affinity chromatography, followed by mass spectrometry analysis. Totally twelve phosphorylation sites along the primary structure of 14-3-3ε protein were identified, most of which had not been reported previously. The results indicated that bio-mass spectrometry coupled with manual interpretation was quite feasible to identify the phosphorylation modification in 14-3-3ε protein over-expressed in HEK293 cells.