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

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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...

Section: Resultsmentioning

confidence: 98%

Influence of amino acid side chains on apparent selective opening of cyclic b₅ ions

Molesworth

Osburn

Stipdonk

2010

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“…For the latter case, sequestering of the "mobile" proton by the R side group would require mobilization and transfer of an amide position proton. Such a process has been invoked to explain formation of b 2 ϩ from a model glycyl-glycine methyl ester peptide that featured and N-terminal pyridine group introduced to sequester the mobile proton, with evidence supplied by CID with isotope labeling and IRMPD spectroscopy [30]. More recently, Paizs and coworkers invoked the mobilization of an amide position H atom through formation of an iminol tautomer to explain the fragmentation of peptides with R residues [31].…”

Section: Resultsmentioning

confidence: 99%

“…A more indirect effect would be sequestration of the mobile proton by an amino acid side group, such that suppression of proton migration to populate a reactive configuration inhibits formation of a given product ion. We have shown, however, that model peptides continue to produce sequence informative ions even when obvious mobile protons are immobilized [30]. In this case, mobilization of amide position protons has been invoked to explain product ion (including b-type) ion formation [30,31].…”

mentioning

confidence: 95%

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Apparent inhibition by arginine of macrocyclic b ion formation from singly charged protonated peptides

Molesworth

Stipdonk

2010

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...

“…An important overall goal of such studies is to expand the sequence coverage available in proteomic analyses by rationalizing the fragmentation pathways [e.g., observed in collision-induced dissociation (CID)] in a structural context [3][4][5][6][7]. Vibrational spectroscopy is emerging as a powerful means with which to elucidate such structures by revealing peptide conformations and protonation sites through theoretical analysis of the observed band patterns [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. This spectroscopic data is most often available through the integration of tunable infrared laser photodissociation with mass spectrometry and, in most cases, spectra of the mass-selected ions are obtained under ambient temperature conditions using infrared multiple photon dissociation (IRMPD) [28][29][30][31][32][33].…”

Section: Introductionmentioning

confidence: 99%

Characterizing the Intramolecular H-bond and Secondary Structure in Methylated GlyGlyH⁺ with H₂ Predissociation Spectroscopy

Leavitt

Wolk

Kamrath

et al. 2011

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We report vibrational predissociation spectra of the four protonated dipeptides derived from glycine and sarcosine, GlyGlyH•(H 2 ) 2 , and SarSarH + •(D 2 ) 2 , generated in a cryogenic ion trap. Sharp bands were recovered by monitoring photoevaporation of the weakly bound H 2 (D 2 ) molecules in a linear action regime throughout the 700-4200 cm -1 range using a table-top laser system. The spectral patterns were analyzed in the context of the low energy structures obtained from electronic structure calculations. These results indicate that all four species are protonated on the N-terminus, and feature an intramolecular H-bond involving the amino group. The large blue-shift in the H-bonded N-H fundamental upon incorporation of a methyl group at the N-terminus indicates that this modification significantly lowers the strength of the intramolecular H-bond. Methylation at the amide nitrogen, on the other hand, induces a significant rotation (~110 o ) about the peptide backbone.