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...

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...

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,

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...

There is now strong evidence for the existence of macrocyclic isomers of b n ϩ ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b 5 ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N-or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed. (J Am Soc Mass Spectrom 2010, 21, 1322-1328) © 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry C ollision-induced dissociation (CID) and tandem mass spectrometry are now widely used for the identification and characterization of peptides and proteins in proteomics. During low-energy CID, fragmentation of protonated peptides typically involves charge mediated unimolecular reactions, which ultimately result in cleavage of amide bonds and generation of a series of b, y, and a ions [1,2]. Central to the fragmentation of protonated peptides is the mobilization and migration of protons, presumably from the most basic groups on a gas-phase peptide, to the site of an intramolecular nucleophilic attack that leads to cleavage of the amide bond. Consideration of proton mobilization and migration within this context led to the development of the mobile proton model [3, 4] of peptide fragmentation and related amide bond cleavage pathways [5][6][7][8][9][10]. A recently developed "pathways in competition" model [10] builds upon the seminal discussion of proton migration within the mobile proton model and includes consideration of the structures and reactivity of key reactive configurations and primary (post-cleavage) fragments as well as transition states and their energies.Many experimental and theoretical studies of sequence ion formation and structure have produced evidence that N-terminal b n type fragment ions have C-terminal oxazolone rings [5,11] and retain much of the primary sequence of the precursor peptide ion. The existence of the C-terminal oxazolone structure has been confirmed for b 2 ϩ and larger species from protonated peptides by our group and others using IRMPD spectroscopy [12][13][14][15][16]. However, more recent experiments [16 -22] overwhelmingly indicate that a macro-cyclic b ion isomer, or intermediate, can arise through "tail to head" cyclization of linear, oxazolone-terminated b ions. The macrocyclic species then can open at various amide bond positions to regain a linear, oxazolone terminated structure, but with the loss of primary sequence information. This type of pathway is referred to as b-type scrambling of peptide fragment ions [19], and evidence to...