Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

We investigated the influence of peptide size on the apparent loss of sequence during collision-induced dissociation (CID) of b ions using a group of peptides containing from between 4 and 10 residues. Although scrambling of sequence for b 3 ϩ generated from tetrapeptides is minimal, significant formation of nondirect sequence ions (i.e., ions for which scrambling has apparently occurred) was observed for all larger b ions included in the study. (J Am Soc Mass Spectrom 2009, 20, 2174 -2181 [3,4]. Implementation of more detailed peptide fragmentation mechanisms into these sequencing algorithms should lead to improved bioinformatics-based MS/MS sequencing.Fragmentation of protonated peptides under lowenergy collision conditions typically involves charge (proton) mediated reactions, in which b, y, and a ions are generated by cleavage of amide bonds [5,6]. Extensive research has been focused on the energetics and kinetics of proton mobilization, which has led to the mobile proton model [7,8] and its associated amide bond cleavage pathways [9 -14]. The more recently introduced pathways in competition (PIC) fragmentation model [14] builds on the mobile proton model by including consideration of the structures and reactivity of the primary fragments.The N-terminal b n -type fragment ions are thought to have a 5-membered oxazolone ring [9,15] structure that maintains much of the sequence of the precursor peptide ion. However, recent experiments [16,17] suggest that cyclization of the linear, oxazolone-terminated b ions can occur to generate a macrocyclic b ion isomer, which can then open at several different amide bonds to re-form linear, oxazolone-terminated ions with concomitant scrambling of the original primary sequence (Scheme 1). We refer to this type of process as b-type scrambling of peptide fragment ions [16]. In the present study, we investigated the tendency for sequence scrambling to occur during collision-induced dissociation (CID) of b n ions of varying sequence and size (3 to 9 amino acid residues). In our experiments, two observations/criteria were used to identify cases in which scrambling of sequence is pronounced: (1) apparent elimination of internal residues from b n ϩ and (2) the similarity of fragmentation patterns for peptides with a given size and their permuted isomers. Experimental Peptide Synthesis and PreparationAll peptides were prepared by conventional solid-phase synthesis methods [18] using 9-fluorenylmethoxycarbonyl (Fmoc) amino acid loaded Wang resin and a custombuilt, multiple reaction vessel peptide synthesis apparatus: sequences were confirmed using multiple-stage CID of Na ϩ and Ag ϩ cationized versions [19]. Solutions of each peptide were prepared by dissolving the appropriate amount of solid material in a 1:1 (vol:vol) mixture of high-performance liquid chromatography grade MeOH (Aldrich Chemical, St. Louis, MO, USA) and deionized H 2 O, to produce final concentrations of 10 Ϫ5 to 10 Ϫ4 M. Mass SpectrometryAll electrospray ionization (ESI) mass spectra were collected using a Finn...

Section: Discussionsupporting

confidence: 91%

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confidence: 99%

Influence of size on apparent scrambling of sequence during CID of b-type ions

Molesworth

Osburn

Stipdonk

2009

“…In a recent paper, Harrison and coworkers [20] proposed that oxazolone terminated linear b ions can cyclize by nucleophilic attack of the N-terminal amino group on the charged oxazolone ring. This reaction leads to a macro-cyclic b isomer [21], the existence of which has recently been demonstrated by Gaskell and coworkers [22] using ion mobility spectroscopy. Cyclic b ion structures can in principle open up at any amide bond (b-type scrambling) and this reaction can lead to linear isomers with scrambled primary structures.…”

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confidence: 80%

“…Quantum chemical calculations indicated that the cyclic form is energetically more favored than the linear structures. Ion mobility experiments [22,31] confirmed that a ions have multiple structures. A recent CID and theoretical study [32] from our laboratories indicated that the CID spectra of a 5 fragments of YAGFL-NH 2 can reasonably be understood by assuming an interplay of b-type scrambling [20] of the corresponding b parent population and a ¡ a* type rearrangements [30].…”

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confidence: 85%

Structure and reactivity of a_n and a*_n peptide fragments investigated using isotope labeling, tandem mass spectrometry, and density functional theory calculations

Bythell

Molesworth

Osburn

et al. 2008

Extensive 15 N labeling and multiple-stage tandem mass spectrometry were used to investigate the fragmentation pathways of the model peptide FGGFL during low-energy collisioninduced-dissociation (CID) in an ion-trap mass spectrometer. Of particular interest was formation of a 4 from b 4 and a 4 * (a 4 -NH 3 ) from a 4 ions correspondingly, and apparent rearrangement and scrambling of peptide sequence during CID. It is suggested that the original FGGF oxa b 4 structure undergoes b-type scrambling to form GGFF oxa . These two isomers fragment further by elimination of CO and 14 NH 3 or 15 NH 3 to form the corresponding a 4 and a 4 * isomers, respectively. For ( 15 N-F)GGFL and FGG( 15 N-F)L the a 4 * ion population appears as two distinct peaks separated by 1 mass unit. These two peaks could be separated and fragmented individually in subsequent CID stages to provide a useful tool for exploration of potential mechanisms along the a 4 ¡ a 4 * pathway reported previously in the literature . In most MS/MS experiments, protonated peptides are excited collisionally to induce dissociation (collision-induced-dissociation, CID) and the fragment ion spectrum is used to elucidate peptide sequences. The CID spectra of peptides in proteomics studies are commonly assigned by bioinformatics tools that implement sequencing algorithms and peptide fragmentation models. Regrettably, the existing sequencing programs are based on rather limited fragmentation models that poorly approximate the rich dissociation chemistry of protonated peptides [3]. These limitations often lead to erroneous assignment of peptides and proteins, and the resulting uncertainty in the evaluation of the raw MS/MS data is one of the major limiting factors in large-scale protein identification studies [4,5]. The incorporation of more detailed peptide fragmentation mechanisms and spectral characteristics into these sequencing algorithms would undoubtedly place MS/MS based sequencing on a much more robust basis.In general, fragmentation of protonated peptides under low-energy collision conditions involves protondriven reactions in which amide bonds are cleaved along the peptide backbone and b, y, and a ions [6,7] are formed. The energetics and kinetics of the necessary proton mobilization (mobile proton model [8,9]) and amide bond cleavage pathways [3, 10 -14] have received significant research interest. On the other hand, much less attention has been devoted to the structure and reactivity of the primary fragments formed by backbone cleavages. According to the recent pathways in competition (PIC) fragmentation model [3], the thermodynamic properties and the reactivity of these fragAddress reprint requests to Dr. M. Van Stipdonk,

“…After many years of characterization by MS/MS, the study of isomeric gas-phase ion structures has been recently the domain of ion mobility-mass spectrometry (IM-MS) and action IRMPD spectroscopy. 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].…”

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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

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.