Influence of amino acid side chains on apparent selective opening of cyclic b₅ ions

Abstract

In this study, the possible influence of acidic, basic, and amide side chains on the opening of a putative macrocyclic b ion (b 5 ϩ ) intermediate was investigated. Collision induced dissociation (CID) of b 5 ions was studied using a group of hexapeptides in which amino acids with the side chains of interest occupied internal sequence positions. Further experiments were performed with permuted isomers of glutamine (Q) containing peptides to probe for sequence scrambling and whether the specific sequence site of the residues influences opening of the macrocycle. Overall, the trend for (apparent) preferential/selective opening of the cyclic b 5 ϩ , presumably due to the side chain, followed by the loss of the amino acid with active side group is: [3], is dependent, in part, on product ion distributions generated by CID. For bioinformatics approaches in particular, prediction of fragmentation patterns often employs rules that are rudimentary and simple. This may lead to invalid assignments of peptide and protein identity [3,4]. Certainly, a better understanding of fundamental gas-phase peptide fragmentation chemistry and physics would potentially lead to enhanced and more accurate bioinformatics-based MS/MS sequencing.Using low-energy collision induced dissociation (CID), fragmentation of protonated peptides traditionally involves charge (proton) mediated reactions, with induced cleavage of amide bonds leading to the generation of b, y, and a ions [5,6]. Development of the mobile proton model [7,8] of peptide fragmentation, and related amide bond cleavage pathways [9 -14], has been focused on the energetics and kinetics of proton mobilization. The more recent pathways in competition (PIC) fragmentation model [14] uses the mobile proton model as a foundation for understanding, but takes into account the structures and reactivity of key reactive configurations and primary fragments as well as transition states and their energies.There is a great deal of evidence that N-terminal b n type fragment ions have structures that include, at least in part, C-terminal oxazolone rings [9,15], and retain much of the primary sequence of the precursor peptide ion. However, more recent experiments [16 -19] strongly suggest that a macro-cyclic b ion isomer, or intermediate, can arise through cyclization of the linear, oxazolone-terminated b ions; this macrocyclic species can then open at different amide bonds to regain a linear, oxazolone terminated structure. One problematic outcome of such a cyclization and reopening process is the associated scrambling of the original primary sequence. This type of pathway is referred to as b-type scrambling of peptide fragment ions [16]. Above and beyond the "head to tail" type formation of the macrocycle, there exist several other possible processes that could play a significant role in the scrambling of sequence, the majority of which involve opening of the cyclic b ion. For example, peptides that contain acidic, basic, and amide side amino acids feature side chains that can serve as nucleo...

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

“…Upon collisional activation of an ornithine-containing peptide, the amine of the ornithine residue cyclizes via nucleophilic attack at the adjacent carbonyl group, resulting in a characteristic and preferential cleavage C-terminal to the carbonyl group. This phenomenon has been observed before, as lysine and many of its homologues have exhibited similar characteristics as a nucleophile previously [42][43][44][45].…”

Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

“…The mechanism of deletion of the internal lysine residue has been investigated by multi stage CID, chemical, and isotopic modification of the peptides and energy resolved CID techniques. Our results suggest that this selective deletion of the lysine residue from the interior of the peptide may not follow the sequence scrambling fragmentation pathway as reported earlier [21,[27][28][29][30][31][32]. A novel gas-phase peptide ion degradation pathway has been shown to be responsible for the specific loss of the internal lysine residues in the peptide.…”

“…Recent studies [21,[27][28][29][30][31][32] have shown that the b n -ions and their analogues of a peptide may undergo head-totail cyclization, forming a protonated cyclic intermediate through the nucleophilic attack by the N-terminal α-amino group on the carbonyl carbon of the C-terminal oxazolone ring. The authors showed that this cyclic protonated peptide intermediate [21,[27][28][29][30][31][32] undergoes ring opening at different amide bonds to form a series of the linear oxazolone terminated b n -ions of scrambled sequences. The cyclization and subsequent ring-opening process, was thus proposed to lead to scrambling of the original primary sequence of the b n -ions, which upon collisional activation could form the daughter ions corresponding to the neutral loss of an internal residue from the peptide.…”

“…There are a few such nondirect sequence ions have been identified recently, such as scrambled fragments of the b/a type ions resulting in the loss of an amino acid residue from the interior of the peptide chain [21,26]. The first step of such loss of an internal amino acid by sequence scrambling fragmentation pathway was proposed [21,[27][28][29][30][31][32] to involve a nucleophilic attack by N-terminal α-NH 2 group on the acylium carbocation of the b-ion (a primary fragment) forming a protonated cyclicpeptide intermediate (CPI). This CPI subsequently undergoes ring opening at different amide bonds producing sequence scrambled linear b-ions.…”

“…This CPI subsequently undergoes ring opening at different amide bonds producing sequence scrambled linear b-ions. These scrambled linear b-ions undergo further dissociation by conventional fragmentation pathway to produce corresponding daughter ions that are not related to the original sequence of the peptide [21,[27][28][29][30][31][32]. Thus such ions, called the nondirect sequence ions, would be related to all the possible scrambled sequences of the peptide, and therefore would loss the memory of the original sequence.…”