Abstract
We have previously shown that the presence of phosphorylation can inhibit detection of electron capture dissociation (ECD) fragments of doubly charged peptide ions. The presence of non-covalent interactions, in the form of salt-bridges or ionic hydrogen bonds, prevents the separation of fragments following backbone cleavage. Here, we show the electron capture dissociation mass spectrometry of a suite of model peptides designed to investigate the relationship between phosphoserine and arginine position, namely AApSAnRAmKA (n=0–6, m=6–0), the presence of lysine residues (AApSAAKAARAKA) and AAApSARAAAAKAAAK, and the presence of proline A(A/P)ApSARAAA(A/P)KAAAK. The latter are analogous to the peptides studied previously. The results show that the presence of phosphoserine and basic amino acid residues alone does not inhibit ECD fragmentation, even when the number of basic amino acid residues is greater than the precursor charge state. Neither did the presence of proline in the peptide sequence suppress ECD backbone cleavage. Nevertheless, the presence and relative position of the phosphorylated residue do alter the observed backbone fragmentation abundance. In addition, the presence of phosphorylation appears to inhibit cleavage within the arginine side-chain regardless of the relative position of the arginine residue. The results suggest that ECD fragmentation behaviour is dependent on the three-dimensional structure of a peptide rather than its sequence.
Highlights
Electron capture dissociation is a tandem mass spectrometry technique which has proved highly useful in the analysis of protein post-translational modifications (PTMs) [1]
We have previously shown that the presence of phosphorylation can inhibit detection of electron capture dissociation (ECD) fragments of doubly charged peptide ions
The results show that the presence of phosphoserine and basic amino acid residues alone does not inhibit ECD fragmentation, even when the number of basic amino acid residues is greater than the precursor charge state
Summary
Electron capture dissociation is a tandem mass spectrometry technique which has proved highly useful in the analysis of protein post-translational modifications (PTMs) [1]. Capture of electrons by multiply charged peptide ions results in cleavage of N-C␣ bonds in the peptide backbone producing c and z fragment ions. We present the ECD mass spectrometry of doubly charged ions from a suite of model phosphopeptides, see Table 1. The results suggest that the presence of phosphoserine and basic amino acid residue alone are insufficient to inhibit detection of ECD fragments, even when the number of basic amino acid residues exceeds the charge state of the precursor ion. Loss of CH5N3 from the arginine side chain was reduced as a result of phosphorylation but was independent of the position of the arginine relative to the phosphoserine. Results from triplicate analyses were used to calculate the standard deviation of the normalized relative intensity (Ii) of each fragment ion (Eq (1) in supplementary material).
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