Structural analysis of high affinity divalent phosphopeptide hybrids of spleen tyrosine kinase

A set of synthetic phosphorylated peptidomimetic inhibitors of spleen tyrosine kinase (Syk), targeted towards its two tandem Src homology-2 (SH2) domains, was studied by nano-electrospray tandem mass spectrometry in both positive and negative ionisation mode. The design of the peptidomimetic compounds was based on the replacement of the intervening amino acid sequence of a Syk-binding di-phosphopeptide by non-peptide spacers based on either ethylene glycol or amino-propynyl-benzoate. Collision-induced dissociation (CID) spectra of the protonated molecular ions [M+H]⁺ allowed full characterisation of the peptide hybrids. Preferred cleavage at the amide bond N-terminal to the adjacent polyethylene glycol (PEG) and the propynyl-benzoate (PrB) linkers was observed. In general, it thus appears that preferred sequential amino acid fragmentation takes place from the N-terminus up to the linker molecule followed by subsequent internal fragmentation starting at the C-terminus. Additionally, tandem CID spectra of the doubly de-protonated molecular ions [M-2H]^2- of every compound showed the m/z 79/97 phosphate-specific ions plus a remarkably intense ion at m/z 297. The mechanism proposed for the m/z 297-ion occurrence goes through a five-membered ring formation giving an N-terminal pyroGlu structure as derived from MSⁿ spectra.