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Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry
S D Shi1, M E Hemling, S A Carr
1Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Analytical Chemistry
|February 24, 2001
Summary
Electron capture dissociation (ECD) minimizes phosphorylation loss in proteins, enabling accurate site-specific analysis. This method offers complementary data to collisionally activated dissociation (CAD) for identifying post-translational modifications.
Area of Science:
- Mass spectrometry
- Proteomics
- Biochemistry
Background:
- Electron capture dissociation (ECD) is effective for peptide and protein ion dissociation.
- ECD preserves post-translational modifications like glycosylation and carboxylation.
- Collisionally activated dissociation (CAD) can lead to loss of phosphorylation during analysis.
Purpose of the Study:
- To extend ECD's capability to analyze phosphorylation in peptides and proteins.
- To demonstrate ECD's advantage over CAD in preserving phosphorylation.
- To enable site-specific characterization of phosphorylation on intact proteins.
Main Methods:
- Activated ion ECD was used for dissociation of multiply charged ions.
- Analysis of phosphorylated peptides and bovine beta-casein.
- Comparison of ECD and CAD fragmentation patterns.
Main Results:
- Minimal loss of phosphorylation was observed in ECD spectra.
- ECD and CAD provided complementary backbone cleavages for modification site identification.
- ECD identified a phosphorylation site at Ser-15 and localized others in bovine beta-casein.
Conclusions:
- ECD is a valuable method for analyzing phosphorylation without significant loss of modification.
- ECD facilitates site-specific characterization of phosphorylation on intact proteins.
- This technique advances the understanding of post-translational modifications in proteomics.