Evidence of Diketopiperazine and Oxazolone Structures for HA b 2 + Ion

Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b 2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm -1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO 2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm -1 , corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH 2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

mentioning

confidence: 68%

“…Most of the attention concerning peptide fragments has been devoted to b n ions [12,[17][18][19][20][21][22][23][24][25]. Nonetheless, a n CID fragments [26][27][28] as well as c n fragment ions [29] generated via electron capture dissociation have also been investigated by IR spectroscopy.…”

mentioning

confidence: 99%

Diagnosing the Protonation Site of b₂ Peptide Fragment Ions using IRMPD in the X–H (X = O, N, and C) Stretching Region

Sinha

Erlekam

Bythell

et al. 2011

“…IRMPD action spectra were acquired at the CLIO Free Electron Laser Facility (Orsay, France) as described previously [26]. Briefly, peptides were fragmented in a modified Bruker Esquire 3000ϩ ion trap mass spectrometer by using CID.…”

Section: Methodsmentioning

confidence: 99%

“…Gaskell and coworkers used IM-MS to identify both oxazolone and macrocyclic structures for the b 5 ion from YAGFL-NH 2 , while Clemmer and coworkers used a similar approach to identify linear and macrocyclic structures for the a 4 and b 4 fragment ions from leucine enkephalin [21,22]. Variable wavelength action IRMPD spectroscopy has been recently employed by several research groups for the study of gas-phase ions [23][24][25][26] and has also been the subject of two recent, extensive reviews [27,28]. Action IRMPD spectroscopy is ideal for structural characterization of ions, as the IR spectra generated depict the vibrational modes of selected ions, which can be directly correlated to structural features.…”

mentioning

confidence: 99%

Separation and identification of structural isomers by quadrupole collision-induced dissociation-hydrogen/deuterium exchange-infrared multiphoton dissociation (QCID-HDX-IRMPD)

Gucinski

Somogyi

Chamot‐Rooke

et al. 2010

Self Cite

A new approach that uses a hybrid Q-FTICR instrument and combines quadrupole collisioninduced dissociation, hydrogen-deuterium exchange, and infrared multiphoton dissociation (QCID-HDX-IRMPD) has been shown to effectively separate and differentiate isomeric fragment ion structures present at the same m/z. This method was used to study protonated YAGFL-OH (free acid), YAGFL-NH 2 (amide), cyclic YAGFL, and YAGFL-OCH 3 (methyl ester). QCID-HDX of m/z 552.28 (C 29 T andem mass spectrometry is widely used for the identification of peptides and proteins [1,2]. Generally, protonated peptides at a given m/z are selected and fragmented via collision-induced dissociation (CID), generating 'b' and 'y' fragment ions, which allows the peptide sequence to be determined.In the course of a common proteomics experiment, several hundred to several thousand spectra are generated for a single sample, making manual interpretation of the peptide fragmentation spectra impractical. To speed up the assignment of MS/MS spectra, protein identification algorithms are often used [3,4]. These algorithms generate theoretical peptide spectra or m/z lists based loosely on prevalent fragmentation models, including the mobile proton model [5] and the pathways-in-competition model [6], and compare them with experimental spectra to determine peptide sequences. While these models can help to predict many trends in peptide fragmentation, actual dissociation chemistry is much more complex, and the complete dissociation chemistry in an MS/MS spectrum cannot always be accurately predicted by using existing theoretical models. As a result, false and missed identifications frequently occur, with only a small percentage of the spectra being correctly assigned [7][8][9][10]. Incorporation of additional chemical information and fragmentation models into sequencing algorithms could depict fragmentation processes more completely, potentially allowing more accurate kinetic modeling of spectra to be possible, and the success of identification algorithms would likely improve.One potential cause for missed or false identifications of peptide fragmentation spectra is the presence of multiple isomeric ion structures at a single m/z. If multiple isomers are present at one m/z, the MS/MS spectrum of that m/z could contain fragments from each of the structures present. For example, the b n ions from peptides of length n and the corresponding [MH Ϫ H 2 O] ϩ ions are isomeric. While water loss at the peptide C-terminus results in b 5 ion formation for pentapeptides, water can also be lost involving oxygen from an Address reprint requests to Dr.

“…Paizs and Suhai [1] have pointed out that such a cyclization involves a trans-cis isomerization of the first amide bond that is not being broken and that such an isomerization has a significant energy barrier. However, there are a number of cases [26][27][28][29][30] where diketopiperazine formation has been observed. These involve systems where a side-chain group, such as in His and Arg, acts as a catalyst for the isomerization.…”

Section: Introductionmentioning

confidence: 99%

Formation of a₁ Ions Directly from Oxazolone b₂ Ions: an Energy-Resolved and Computational Study

Bythell

Harrison

2015

Abstract. It is well-known that oxazolone b 2 ions fragment extensively by elimination of CO to form a 2 ions, which often fragment further to form a 1 ions. Less well-known is that some oxazolone b 2 ions may fragment directly to form a 1 ions. The present study uses energy-resolved collision-induced dissociation experiments to explore the occurrence of the direct b 2 →a 1 fragmentation reaction. The experimental results show that the direct b 2 →a 1 reaction is generally observed when Gly is the C-terminal residue of the oxazolone. When the C-terminal residue is more complex, it is able to provide increased stability of the a 2 product in the b 2 →a 2 fragmentation pathway. Our computational studies of the relative critical reaction energies for the b 2 →a 2 reaction compared with those for the b 2 →a 1 reaction provide support that the critical reaction energies are similar for the two pathways when the C-terminal residue of the oxazolone is Gly. By contrast, when the nitrogen of the oxazolone ring in the b 2 ion does not bear a hydrogen, as in the Ala-Sar and Tyr-Sar (Sar=N-methylglycine) oxazolone b 2 ions, a 1 ions are not formed but rather neutral imine elimination from the N-terminus of the b 2 ion becomes a dominant fragmentation reaction. The M06-2X/6-31+G(d,p) density functional theory calculations are in general agreement with the experimental data for both types of reaction. In contrast, the B3LYP/6-31+G(d,p) model systematically underestimates the barriers of these S N 2-like b 2 →a 1 reaction. The difference between the two methods of barrier calculation are highly significant (PG0.001) for the b 2 →a 1 reaction, but only marginally significant (P=0.05) for the b 2 →a 2 reaction. The computations provide further evidence of the limitations of the B3LYP functional when describing S N 2-like reactions.