IRMPD Spectroscopy Shows That AGG Forms an Oxazolone b 2 + Ion

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.

Section: Introductionmentioning

confidence: 84%

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

Bythell

Harrison

2015

“…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.…”

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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.

“…Relative energies for all species were calculated by correcting B3LYP/6-311ϩg(d,p) total energies for zeropoint vibrational energy (ZPE) obtained from the unscaled frequencies determined at the same level of theory. Predicted IR spectra were generated at the B3LYP/6-311ϩg(d,p) level of theory and scaled by a factor of 0.98, which is commonly used when comparing DFT and IRMPD data for peptides [29,30,32].…”

Section: Density Functional Theory Calculationsmentioning

confidence: 99%

“…Low-energy CID of protonated peptides promotes rearrangement reactions in which the added proton presumably migrates to the amide bond that is ultimately cleaved [1][2][3][4][5][6][7][8][9][10][11][12], as treated in the "mobileproton" (MP) [13][14][15][16][17][18][19][20][21][22][23][24][25], and "pathways in competition" (PIC) models of peptide dissociation [26]. Experimental studies have established that the C-terminus-containing y n ϩ fragments are truncated peptides [6,27,28], while the N-terminus-containing b n ϩ and a n ϩ species have substituted oxazolone ring and imine structures, respectively [4,5,11,12,29,30].Wavelength-selective infrared multiple photon dissociation (IRMPD) spectroscopy has recently been used to probe and confirm proposed structures of peptides and peptide dissociation products, with studies of the latter focusing primarily on b n ϩ and a n ϩ ions [11,12,29,30]. Our group has designed model peptides and approaches to probe intramolecular migration of protons during peptide dissociation reactions [31,32].…”

mentioning

confidence: 99%

“…Wavelength-selective infrared multiple photon dissociation (IRMPD) spectroscopy has recently been used to probe and confirm proposed structures of peptides and peptide dissociation products, with studies of the latter focusing primarily on b n ϩ and a n ϩ ions [11,12,29,30]. Our group has designed model peptides and approaches to probe intramolecular migration of protons during peptide dissociation reactions [31,32].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Spectroscopic evidence for mobilization of amide position protons during CID of model peptide ions

Molesworth

Leavitt

Groenewold

et al. 2009

Infrared multiple photon dissociation (IRMPD) spectroscopy was used to study formation of b 2 ϩ from nicotinyl-glycine-glycine-methyl ester (NicGGOMe). IRMPD shows that NicGGOMe is protonated at the pyridine ring of the nicotinyl group, and more importantly, that b 2 ϩ from NicGGOMe is not protonated at the oxazolone ring, as would be expected if the species were generated on the conventional b n ϩ /y n ϩ oxazolone pathway, but at the pyridine ring instead. IRMPD data support a hypothesis that formation of b 2 ϩ from NicGGOMe involves mobilization and transfer of an amide position proton during the fragmentation reaction. E ffective application of tandem mass spectrometry, collision induced dissociation (CID), and bioinformatics for protein identification requires a clear understanding of peptide ion fragmentation mechanisms. Low-energy CID of protonated peptides promotes rearrangement reactions in which the added proton presumably migrates to the amide bond that is ultimately cleaved [1][2][3][4][5][6][7][8][9][10][11][12], as treated in the "mobileproton" (MP) [13][14][15][16][17][18][19][20][21][22][23][24][25], and "pathways in competition" (PIC) models of peptide dissociation [26]. Experimental studies have established that the C-terminus-containing y n ϩ fragments are truncated peptides [6,27,28], while the N-terminus-containing b n ϩ and a n ϩ species have substituted oxazolone ring and imine structures, respectively [4,5,11,12,29,30].Wavelength-selective infrared multiple photon dissociation (IRMPD) spectroscopy has recently been used to probe and confirm proposed structures of peptides and peptide dissociation products, with studies of the latter focusing primarily on b n ϩ and a n ϩ ions [11,12,29,30]. Our group has designed model peptides and approaches to probe intramolecular migration of protons during peptide dissociation reactions [31,32]. Versions of these model peptides are the subject of the present study, in which IRMPD was used to determine the structure of protonated nicotinic acid-glycine-glycinemethyl ester (NicGGOMe), and the b 2 ϩ fragment ions from NicGGOMe and benzoic acid-glycine-glycinemethyl ester (BzGGOMe). The pyridine ring of the nicotinic acid residue is used to sequester the "mobile" proton added to the peptide to produce (M ϩ H) ϩ , and inhibit migration to the site of intramolecular nucleophilic attack during fragmentation reactions. IRMPD spectroscopy provides strong evidence for the mobilization and migration of amide-position protons during dissociation reactions of model peptides. Experimental Mass Spectrometry and IRMPD SpectroscopyCID experiments were performed using a ThermoFinnigan (San Jose, CA, USA) LCQ-Deca quadrupole ion trap (QIT) mass spectrometer. IRMPD spectra were collected using the FT-ICR mass spectrometer coupled to the beamline of the free electron-laser user facility (FELIX) infrared free electron laser [33][34][35]. Ions produced by electrospray ionization (ESI) were accumulated in a hexapole ion trap, and isolated and irradiated with FELIX for 2 s at a...