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

Authors: Fagerquist, Clifton - Keith

Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/28/2020
Publication Date: 9/6/2020
Citation: Fagerquist, C.K. 2020. 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. International Journal of Mass Spectrometry. 457:116433. https://doi.org/10.1016/j.ijms.2020.116433.
DOI: https://doi.org/10.1016/j.ijms.2020.116433

Interpretive Summary: The ability to identify proteins and protein toxins expressed by foodborne pathogens by mass spectrometry rests on the analysis of the fragment ions generated by cleavage of their polypeptide backbone. These sequence-specific fragment ions are sufficiently unique such that the correct protein sequence can be identified/distinguished from hundreds of thousands incorrect sequences. The intensity of fragment ions has not received much attention or utility as a metric of the identification process. The B-subunit sequences of two Shiga toxin 2 subtypes have been studied with respect to the effect of amino acid substitutions on the efficiency of protein backbone fragmentation by the aspartic acid effect (AAE) fragmentation mechanism. It was found that asparagine (N) residues, which were not previously associated with the AAE mechanism, can fragment the protein backbone by this mechanism although at a rate that is ten times less efficient than that of an aspartic acid (D) residue. As D versus N residues differ in mass by only 1 Da, the relative intensity of the fragment ions generated by these two residues may be a better metric to deduce/identify the residue responsible for protein fragmentation than simply relying on fragment ion mass-to-charge (m/z) assuming it contains the amino acid substitution in its sequence.

Technical Abstract: 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 residues. In addition to polypeptide backbone cleavage (PBC) on the C-terminal side of D and glutamic acid residues by the aspartic acid effect (AAE) fragmentation mechanism, we also observe cleavage on the C-terminal side of N-residues. We estimate 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 are of equal length, the difference in PBC efficiency is likely due to differences in the gas phase acidities (Hacid = 0.4 eV) of their side-chain functional groups: [-(C=O)OH] for D versus [-(C=O)NH2] for N. We propose 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, if there are no other residue substitutions between two protein sequences, the b-fragments would also differ by 1 Da, and the complementary y-type fragment would have exactly the same mass. In consequence, the relative abundance of fragment ions from a D versus N residue might be a better indicator of which residue was responsible for PBC than simply relying upon fragment ion m/z.