Polypeptide backbone cleavage on the C-terminal side of asparagine residues of metastable protein ions analyzed by MALDI-TOF-TOF-MS/MS and post-source decay

The gas phase dissociation of B-subunits of Shiga toxin 2 (Stx2) subtypes expressed by pathogenic Escherichia coli have been analyzed using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry and post-source decay (PSD). Two Stx2 subtypes (a and c) are distinguished by two amino acid substitutions involving aspartic acid (D), asparagine (N) and alanine (A) residues. In addition to polypeptide backbone cleavage (PBC) on the C-terminal side of D and glutamic acid (E) residues by the aspartic acid effect (AAE) fragmentation mechanism, PBC is also observed on the C-terminal side of N-residues. It is estimated that N-residues are at least an order-of-magnitude less efficient than D-residues at PBC. As the side-chains of D and N residues are of equal length, the difference in PBC efficiency is likely due to differences in the gas phase acidities (Delta Delta H-acid approximate to 0.4 eV) of their respective side-chain functional groups: [-(C=O)OH] for D versus [-(C=O)NH2] for N. It is proposed that the AAE mechanism for N-residues results in the formation of a b-type fragment with a 2,5-pyrrolidinedione (or succinimide) end-group at its C-terminus similar to previous proposals that predict formation of a b-type fragment with a 2,5-furanedione (or succinic anhydride) end-group for D-residues. As a D <-> N substitution differs in mass by only 1 Da, the b-fragment would also differ by 1 Da (if there are no other residue substitutions) and the complementary y-type fragment would have exactly the same mass. In consequence, the relative abundance of fragment ions caused by a D versus N residue might be a better indicator as to which residue was responsible for PBC rather than relying solely upon fragment ion m/z for this determination. Published by Elsevier B.V.