Influence of N‑terminal Residue Composition on the Structure of Proline-Containing b₂⁺ Ions

To probe the structural implications of the proline residue on its characteristic peptide fragmentation patterns, in particular its unusual cleavage at its C-terminus in formation of a b₂ ion in XxxProZzz sequences, the structures of a series of proline-containing b₂⁺ ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy and fragment ion hydrogen–deuterium exchange (HDX). Five different Xxx-Pro b₂⁺ ions were studied, with glycine, alanine, isoleucine, valine, or histidine in the N-terminal position. The residues selected feature different sizes, chain lengths, and gas phase basicities to explore whether the structure of the N-terminal residue influences the Xxx-Pro b₂⁺ ion structure. In proteins, the proline side chain-to-backbone attachment causes its peptide bonds to be in the cis conformation more than any other amino acid, although trans is still favored over cis. However, HP is the only b₂⁺ ion studied here that forms the diketopiperazine exclusively. The GP, AP, IP, and VP b₂⁺ ions formed from protonated tripeptide precursors predominantly featured oxazolone structures with small diketopiperazine contributions. In contrast to the b₂⁺ ions generated from tripeptides, synthetic cyclic dipeptides VP and HP were confirmed to have exclusive diketopiperazine structures

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein–ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same <i>m</i>/<i>z</i> but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR