Top-down mass spectrometry of intact phosphorylated ?-casein: Correlation between the precursor charge state and internal fragments.

ABSTRACT: Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision-induced dissociation top-down approach for characterizing phosphoproteins on a quadrupole time-of-flight mass spectrometer. ?-casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact ?-casein ions with charged states up to 36+ were detected. Tandem mass spectrometry was performed on ?-casein ions of different charge states (12+ , and 15+ to 28+ ) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N-terminal amide bond adjacent to proline residues (Xxx-Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx-Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of ?-casein further complicated the spectra. Our results suggest that when interpreting top-down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.

SUBMITTER: Chen J 

PROVIDER: S-EPMC6779312 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature