Spectroscopic Identification of Cyclic Imide b₂-Ions from Peptides Containing Gln and Asn Residues

Abstract

In mass-spectrometry based peptide sequencing, formation of b- and y-type fragments by cleavage of the amide C-N bond constitutes the main dissociation pathway of protonated peptides under low-energy collision induced dissociation (CID). The structure of the b2 fragment ion from peptides containing glutamine (Gln) and asparagine (Asn) residues is investigated here by infrared ion spectroscopy using the free electron laser FELIX. The spectra are compared with theoretical spectra calculated using density functional theory for different possible isomeric structures as well as to experimental spectra of synthesized model systems. The spectra unambiguously show that the b2-ions do not possess the common oxazolone structure, nor do they possess the alternative diketopiperazine structure. Instead, cyclic imide structures are formed through nucleophilic attack by the amide nitrogen atom of the Gln and Asn side chains. The alternative pathway involving nucleophilic attack from the side-chain amide oxygen atom leading to cyclic isoimide structures, which had been suggested by several authors, can clearly be excluded based on the present IR spectra. This mechanism is perhaps surprising as the amide oxygen atom is considered to be the better nucleophile; however, computations show that the products formed via attack by the amide nitrogen are considerably lower in energy. Hence, b2-ions with Asn or Gln in the second position form structures with a five-membered succinimide or a six-membered glutarimide ring, respectively. b2-Ions formed from peptides with Asn in the first position are spectroscopically shown to possess the classical oxazolone structure.

Deamidation reactions of protonated asparagine and glutamine investigated by ion spectroscopy

“…A variety of studies have addressed the deamidation reactions of protonated Gln-and Asn-containing proteins and peptides in a mass spectrometer, where the reactions are initiated by collisional activation, occur in the gas phase, and can be analyzed in terms of the mass-to-charge ratio of the resulting fragments. [1,3,6,[12][13][14][15][16][17][18][19]31,32] In order to gain detailed mechanistic insight into these reactions, the deamidation of protonated Asn and Gln has been studied using a variety of methods including energy-dependent collision-induced dissociation, deuterium exchange, and threshold collision-induced dissociation (TCID) combined with theoretical investigations at various levels of theory.…”

Effect of the Asn side chain on the dissociation of deprotonated peptides elucidated by IRMPD spectroscopy

“…In light of the present findings for Asn-containing peptides, the behavior of the glutamine (Gln) residue is of particular interest as it also contains an amide moiety, though linked to the backbone by a longer alkyl chain. For protonated peptides containing Asn and Gln, similar involvement of the side-chain amide in the reactions forming bions was recently established using ion spectroscopy [42]. The role of Gln in the dissociation of deprotonated peptides is currently under study in our group.…”

“…As established for protonated peptides, the side chain of Asn and Gln may become involved in the rearrangement reactions forming b 2 -type fragments [38][39][40][41][42]. It is therefore of interest to investigate what the effect of these residues is in the dissociation of deprotonated peptides.…”

Gas-phase conformations of small polyprolines and their fragment ions by IRMPD spectroscopy

“…Infrared multiple photon dissociation (IRMPD) spectroscopy is a technique that provides direct conformational insight for gas-phase ions and has been applied in the past to identify the conformation of peptide fragment ions [15,[35][36][37][38]. Here, we use IRMPD spectroscopy in compliment with computational methods to examine the conformations of a series of small polyprolines (3-6 residues) and the influence of the cis/trans amide bond conformation on their fragmentation pathways.…”