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. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36+were detected. Tandem mass spectrometry was performed on β‐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 β‐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.
- Award ID(s):
- 1709075
- PAR ID:
- 10055377
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 8
- Issue:
- 9
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 6499 to 6507
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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