Identification of Phosphorylation Sites by ECD FT-ICR/MS

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(Last Updated On: April 4, 2020)
MARCKS analogue test peptides
MARCKS analogue test peptides. Image: Woodling et al. 2007

ECD FT-ICR/MS is a type of mass spectroscopy that is equipped with Electron Capture Dissociation as a source of ionization and Fourier Transform-Ion Cyclotron Resonance as a mass filter/analyzer. To identify the sites of post-translational modifications of a given protein, different analytical tools can be employed such as SDS-PAGE, acid hydrolysis, X-ray crystallography, etc but all of them are somewhat difficult and time-consuming.


Using ECD FT-ICR/MS, it is easy to determine the site of phosphorylation of proteins. Researchers have used ECD FT-ICR/MS to determine the phosphorylation sites of the Phosphorylation Site Domain of the Myristoylated Alanine-Rich C Kinase (MARCKS) Protein.

MARCKS protein is an acidic membrane protein consisting of 309 amino acid residues. It plays an important role in the regulation of brain development, cellular adhesion, and cell migration. It also promotes tumor suppression and helps in neurosecretion. It binds to the calmodulin (Ca binding protein), membrane phospholipids, and actin filaments.

MARCKS protein contains three domains, phosphorylation site domain (PSD), myristoylated N-terminal domain and MARCKS homology domain. The PSD domain of the MARCKS protein contains 25 amino acid residues including 5 serine residues. In previous studies, researchers had used FAB-MS and the result of which reveals that serine residue-2 within the PSD is first phosphorylated followed by the serine residue-1.

For this study, researchers synthesized 7 test peptides with 13 amino acids containing 3 serine residues. Three peptides were singly phosphorylated and three were doubly phosphorylated while one was non-phosphorylated (as control). These test peptides were homologous to the PSD of the MARCKS protein. Test peptides were phosphorylated by rat brain PKC and purified by HPLC while molecular weights were confirmed by MALDI-TOF-MS.

They performed ECD FT-ICR/MS to all the 7 test peptides and analyzed their fragment ions. They found fragment ions resulting from cleavage of N-and C-sides of the modified serine residue of the MARCKS analogue-2 revealing that Ser-3 is the site of modification. A similar scenario was observed for the MARCKS analogue-3 and 4 representing the phosphorylation of Ser-7 and Ser-10 residues respectively.

ECD FT-ICR-MS of MARCKS analogue 1 and 2
ECD FT-ICR-MS of MARCKS analogue 1 and 2. Image: Woodling et al. 2007

In the same way, researchers found the presence of fragment ions z11 and c3 of the MARCKS analogue-5 that reveal the phosphorylation of Ser-3 while fragment ions z7 and c7 reveals the phosphorylation of Ser-7. However, there was no evidence for the phosphorylation of Ser-10. A similar scenario was seen in the case of MARCKS analogue-6 and 7.

ECD-FT-ICR mass spectra for the MARCKS analogue 5 and synthetic PSD segment identical to that of the MARCKS protein
ECD-FT-ICR mass spectra for the MARCKS analog 5 and synthetic PSD segment identical to that of the MARCKS protein. Image: Woodling et al. 2007

From the reports mentioned above, it is clear that electron capture dissociation can be used to confirm the known structure of the phosphorylated peptides. Therefore, analyzing the structure of the peptide with unknown sites of phosphorylation can no longer be problematic.

Then after researchers analyzed the peptides with unknown sites of phosphorylation based on the test peptides by comparing the predicted electron capture dissociation fragments of two of the mono-(or di-) phosphorylated test peptides with the actual experimental data of the fragments of electron capture dissociation obtained from the third mono-(or di-) phosphorylated test peptide.

Such as, researchers compared the experimental data of fragment ions obtained for each of the singly phosphorylated test peptides with predicted data of other two singly phosphorylated test peptides to determine how many experimental fragment ions are similar to that of the predicted fragment ions. For example, a comparison of the experimental peak list for MARCKS analogue-3 with those predicted data for the peptide-2 and 4.

Similarly, researchers compared the experimentally obtained peak lists of the doubly phosphorylated MARCKS analogue-6 with those of the predicted data for doubly phosphorylated MARCKS analogue-5 and 7. Most of the predicted fragment ions were similar to that of the experimentally obtained fragment ions indicating consistency in the experimentally generated electron capture dissociation fragment ions. Thus, incorrect sites of phosphorylation can’t be assigned if the wrong predicted peak list is compared with experimental data.

Using the fragmentation guidelines of test peptides, researchers tried to determine the phosphorylation sites of PSD of the MARCKS protein. For this purpose, researchers synthesized a synthetic PSD segment containing 25 amino acids with 5 Ser residues and subjected to the ECD FT-ICR/MS. The synthetic PSD segment was identical to the PSD segment of the MARCKS protein and was phosphorylated in vitro. However, the extent of phosphorylation was unknown before mass spectrometric analysis.

Since the PSD segment contains 5 Ser residues that may be phosphorylated leading to the production of fragment ions of identical mass. It may be possible to obtain five different peptide sequences for this polypeptide. Therefore, researchers used the same approach as for the test peptides. They compared the experimentally obtained data of fragment ions with that of the predicted data of the PSD segment phosphorylated at each of the five Ser residues.

The presence of fragment ions c8, c9, c10, and c11 indicates that some portion of the PSD is phosphorylated at the Ser-8 while fragment ions z15, z16, and z17 suggested the phosphorylation of any one of Ser-12, Ser-16, Ser-19 or Ser-23. Whereas, the absence of experimental data for fragment ions with masses corresponding to the predicted fragment ions z3, z4, z5 or z6 from the PSD segment phosphorylated at Ser-23 strongly suggests that none of the singly phosphorylated PSD polypeptides is modified at Ser-23.

From the result above, it is clear that phosphorylation of the PSD segment results in the single phosphorylation of at least two different serine residues. Some of the phosphorylation takes place at Ser-8 while some at either Ser-19 or Ser-23 (most probably Ser-19). This result is not in agreement with those of the previously obtained data using the FAV-MS that predicted the first phosphorylation of Ser-12.

This is because the previous study carried out by FAB-MS was involved in the PSD domain containing only 21 amino acid residues with only four Ser residues unlike in this study where the researchers had used PSD domain containing 25 amino acid residues with 5 Ser residues.

In conclusion, ECD FT-ICR/MS was successfully utilized for the determination of the site of phosphorylation for a series of singly and doubly phosphorylated peptides (MARCKS analogs) based on the extremely efficient fragmentation. Researchers applied the same approach to the PSD domain identical to that of the MARCKS protein, containing 5 Ser residues. They found that the most common sites of phosphorylation were Ser-8 and Ser-19.

Reference: American Society for Mass Spectrometry

Article DOI: 10.1016/j.jasms.2007.09.010

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