Abstract
Previously, we have demonstrated the effect of salt bridges on the electron capture dissociation mass spectrometry behavior of synthetic model phosphopeptides and applied an ion mobility spectrometry/molecular modeling approach to rationalize the findings in terms of peptide ion structure. Here, we develop and apply the approach to a biologically derived phosphopeptide. Specifically, we have investigated variants of a 15-mer phosphopeptide VVGARRSsWRVVSSI (s denotes phosphorylated Ser) derived from Akt1 substrate 14-3-3-ζ, which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. ECD fragmentation patterns for the eight phosphopeptide variants show greater sequence coverage with successive R → L substitutions. Peptides with two or more basic residues had regions with no sequence coverage, while full sequence coverage was observed for peptides with one or no basic residues. For three of the peptide variants, low-abundance fragments were observed between the phosphoserine and a basic residue, possibly due to the presence of multiple conformers with and without noncovalent interactions between these residues. For the five variants whose dissociation behavior suggested the presence of intramolecular noncovalent interactions, we employed ion mobility spectrometry and molecular modeling to probe the nature of these interactions. Our workflow allowed us to propose candidate structures whose noncovalent interactions were consistent with the ECD data for all of the peptides modeled. Additionally, the AMBER parameter sets created for and validated by this work are presented and made available online ( http://www.biosciences-labs.bham.ac.uk/cooper/datasets.php ).
Highlights
Electron capture dissociation (ECD)[1] is a fragmentation technique typically employed in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers and, more recently, in other mass spectrometers by the addition of a linear ECD cell.[2,3] ECD makes use of low-energy electrons to irradiate multiply charged analyte ions and induce fragmentation
We have shown that the ECD behavior of the doubly charged ions of eight peptides derived from 14-3-3-ζ results in a reduced sequence fragment coverage when there are two or more arginine residues present
For the five peptides (Pep-01−Pep-05) for which reduced sequence coverage was observed, we employed an ion mobility spectrometry/molecular modeling workflow to probe the structures of their [M+2H]2+ ions further
Summary
Electron capture dissociation (ECD)[1] is a fragmentation technique typically employed in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers and, more recently, in other mass spectrometers by the addition of a linear ECD cell.[2,3] ECD makes use of low-energy electrons to irradiate multiply charged analyte ions and induce fragmentation.
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