Cyclization reaction of peptide fragment ions during multistage collisionally activated decomposition: An inducement to lose internal amino-acid residues

Abstract

During characterization of some peptides (linear precursors of the cyclic peptides showing potential to be anticancer drugs) in an ion trap, it was noted that many internal amino acid residues could be lost from singly charged b ions. The phenomenon was not obvious at the first stage of collisionally activated decomposition (CAD), but was apparent at multiple stages of CAD. The unique fragmentation consisting of multiple steps is induced by a cyclization reaction of b ions, the mechanism of which has been probed by experiments of N-acetylation, MS n , rearranged-ion design, and activation-time adjustment. The fragmentation of synthetic cyclic peptides demonstrates that a cyclic peptide intermediate (CPI) formed by b ion cyclization exhibits the same fragmentation pattern as a protonated cyclic peptide. Although no rules for the cyclization reaction were discerned in the experiments of peptide modification, the fragmentations of a number of b ions indicate that the "Pro and Asn/Gln effects" can influence ring openings of CPIs. In addition, large-scale losses of internal residues from different positions of a-type ions have been observed when pure helium was used as collision gas. The fragmentation is initiated by a cyclization reaction forming an a-type ion CPI. This CPI with a fixed-charge structure cannot be influenced by the "Pro effect", causing a selective ring opening at the amide bond Pro-Xxx rather than Xxx-Pro. With the knowledge of the unique fragmentations leading to internal residue losses, the misidentification of fragments and sequences of peptides may be avoided. [5,6] sequencings. For the top-down sequencing, the protein sample without enzymatic digestion is transferred into the gas phase intact, which has the potential for 100% sequence coverage of the protein and improved detection of post-translational modifications [4]. The bottom-up sequencing requires MS/MS analysis of the proteolytic fragments and database searching using algorithms such as Mascot and Sequest [5,6]. In addition, structural elucidations of natural or synthetic peptides, including linear peptides [7,8], cyclic peptides [9,10], and peptide analogues [11] also rely on MS/MS analysis. Undoubtedly, all of the applications described above are based on understanding the dissociation mechanisms of peptides, which have been well summarized in recent reviews [12][13][14][15][16]. However, conventional knowledge of fragmentation pathways and rules cannot reasonably explain many anomalous fragmentations. For example, some groups have reported that certain peptides show enhanced cleavage at special amino acid residues to produce incomplete sequence information [15]. Hence, the study on fragmention intensity relationship was of further concern and specially reviewed by Paizs and Suhai [16]. Also, another class of anomalous fragmentations introduced in this paper should receive attention.For peptide sequencing, the ideal fragmentation pattern of peptides is that the amino acid residues are sequentially lost from the C/N-terminus. However,...

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

“…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. Selective deletion of internal lysine by sequence scrambling following the head-to-tail macrocyclization cannot indeed be completely ruled out in the present case, though no evidence of the deletion of internal alanine as anticipated from the proposed random scrambling [21,[27][28][29][30][31][32] of the peptide sequence was observed in our results. In order to assess whether head-to-tail macrocyclization of the b n -ion (as discussed above) could be a major pathway for the internal lysine deletion, we selectively acetylated the N-terminal α-amine of the peptides since N-acetylation blocks the macrocyclization [28,47].…”

“…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.…”