Spectroscopic and theoretical evidence for oxazolone ring formation in collision-induced dissociation of peptides

Infrared spectroscopy of the b4+ fragment of Leu-enkephalin demonstrates that the oxazolone ring is formed during collision-induced dissociation of protonated peptides, whereas the linear acylium structure is not observed. Three distinct oxazolone structures are identified, based on the highly conformer-diagnostic C=O stretching mode of the oxazolone ring, clearly showing that proton transfer from the oxazolone ring to the N-terminus takes place. Note that the presence of a cyclic peptide b4+ isomer cannot be excluded.     

Scrambling of sequence information in collision-induced dissociation of peptides

Collision-induced dissociation (CID) of protonated YAGFL-NH2 leads to nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicate that primary fragmentation of the intact peptide leads to the linear YAGFLoxa b5 ion with a C-terminal oxazolone ring that is attacked by the N-terminal amino group to induce formation of a cyclic peptide b5 isomer. The latter can undergo various proton transfer reactions and opens up to form something other than the YAGFLoxa linear b5 isomer, leading to scrambling of sequence information in the CID of protonated YAGFL-NH2.     

Towards understanding the tandem mass spectra of protonated oligopeptides. 1: Mechanism of amide bond cleavage

The mechanism of the cleavage of protonated amide bonds of oligopeptides is discussed in detail exploring the major energetic, kinetic, and entropy factors that determine the accessibility of the b(x)-y(z) (Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2002, 16, 375) and "diketopiperazine" (Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993, 65, 1594) pathways. General considerations indicate that under low-energy collision conditions the majority of the sequence ions of protonated oligopeptides are formed on the b(x)-y(z) pathways which are energetically, kinetically, and entropically accessible. This is due to the facts that (1).the corresponding reactive configurations (amide N protonated species) can easily be formed during ion excitation, (2). most of the protonated nitrogens are stabilized by nearby amide oxygens making the spatial arrangement of the two amide bonds (the protonated and its N-terminal neighbor) involved in oxazolone formation entropically favored. On the other hand, formation of y ions on the diketopiperazine pathways is either kinetically or energetically or entropically controlled. The energetic control is due to the significant ring strain of small cyclic peptides that are co-formed with y ions (truncated protonated peptides) similar in size to the original peptide. The entropy control precludes formation of y ions much smaller than the original peptide since the attacking N-terminal amino group can rarely get close to the protonated amide bond buried by amide oxygens. Modeling the b(x)-y(z) pathways of protonated pentaalanine leads for the first time to semi-quantitative understanding of the tandem mass spectra of a protonated oligopeptide. Both the amide nitrogen protonated structures (reactive configurations for the amide bond cleavage) and the corresponding b(x)-y(z) transition structures are energetically more favored if protonation occurs closer to the C-terminus, e.g., considering these points the Ala(4)-Ala(5) amide bond is more favored than Ala(3)-Ala(4), and Ala(3)-Ala(4) is more favored than Ala(2)-Ala(3). This fact explains the increasing ion abundances observed for the b(2)/y(3), b(3)/y(2), and b(4)/y(1) ion pairs in the metastable ion and low-energy collision induced mass spectra (Yalcin, T.; Csizmadia, I. G.; Peterson, M. B.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1996, 7, 233) of protonated pentaalanine. A linear free-energy relationship is used to approximate the ratio of the b(x) and y(z) ions on the particular b(x)-y(z) pathways. Applying the necessary proton affinities such considerations satisfactorily explain for example dominance of the b(4) ion over y(1) and the similar b(3) and y(2) ion intensities observed for the metastable ion and low-energy collision induced mass spectra.     

Dissociation of the peptide bond in protonated peptides

The dissociation of the amide (peptide) bond in protonated peptides, [M + H](+), is discussed in terms of the structures and energetics of the resulting N-terminal b(n) and C-terminal y(n) sequence ions. The combined data provide strong evidence that dissociation proceeds with no reverse barriers through interconverting proton-bound complexes between the segments emerging upon cleavage of the protonated peptide bond. These complexes contain the C-terminal part as a smaller linear peptide (amino acid if one residue) and the N-terminal part either as an oxazolone or a cyclic peptide (cyclic amide if one residue). Owing to the higher thermodynamic stability but substantially lower gas-phase basicity of cyclic peptides vs isomeric oxazolones, the N-terminus is cleaved as a protonated oxazolone when ionic (b(n) series) but as a cyclic peptide when neutral (accompanying the C-terminal y(n) series). It is demonstrated that free energy correlations can be used to derive thermochemical data about sequence ions. In this context, the dependence of the logarithm of the abundance ratio log[y(1)/b(2)], from protonated GGX (G, glycine; X, varying amino acid) on the gas-phase basicity of X is used to obtain a first experimental estimate of the gas-phase basicity of the simplest b-type oxazolone, viz. 2-aminomethyl-5-oxazolone (b(2) ion with two glycyl residues).     

Oxazolone versus macrocycle structures for leu-enkephalin b<Subscript>2</Subscript>–b<Subscript>4</Subscript>: Insights from infrared multiple-photon dissociation spectroscopy and gas-phase hydrogen/deuterium exchange

The collision-induced dissociation (CID) products b₂-b₄ from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b₂ exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b₃ and b₄, the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone structures for b₃ and b₄, using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc. 2009, 131(51), 18272–18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance of the macrocycle structure increases as a function of b fragment size, going from 0% for b₂ to ∼6% for b₃, and culminating in 31% for b₄. Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity in forming macrocycle structures. This indicates that the chemistry of “head-to-tail” cyclization depends on subtle differences in the sequence as well as the size of the b fragment.     

Proton affinities of the N- and C-terminal segments arising upon the dissociation of the amide bond in protonated peptides

Dissociation of the amide bonds in a protonated peptide leads to N-terminal sequence fragments with cyclic structures and C-terminal sequence fragments with linear structures. The ionic fragments containing the N-terminus (b _n ) have been shown to be protonated oxazolones, whereas those containing the C-terminus (y _n ) are protonated linear peptides. The coproduced neutral fragments are cyclic peptides from the N-terminus and linear peptides from the C-terminus. A likely determinant of these structural choices is the proton affinity (PA) of the described peptide segments. This study determines the PA values of such segments (Pep), i.e., cyclic and linear dipeptides and a relevant oxazolone, based on the dissociations of proton-bound dimers [Pep + B _i ]H⁺ in which B _i is a reference base of known PA value (Cooks kinetic method). The dissociations are assessed at different internal energies to thereby obtain both proton affinities as well as entropies of protonation. For species with comparable amino acid composition, the proton affinity (and gas phase basicity) follows the order cyclic peptide ≪ oxazolone ≈ linear peptide. This ranking is consistent with dissociation of the protonated peptide via interconverting proton-bound complexes involving N-terminal oxazolone (O) or cyclopeptide (C) segments and C-terminal linear peptide segments (L), viz. O ⋯ H⁺ ⋯ L ⇄ C ⋯ H⁺ ⋯ L. N-terminal sequence ions (b _n ) are formed with oxazolone structures which can efficiently compete for the proton with the linear segments. On the other hand, N-terminal neutral fragments detach as cyclic peptides, with H⁺ now being retained by the more basic linear segment from the C-terminus to yield y _n .     

Mass spectrometric and quantum mechanical analysis of gas-phase formation, structure, and decomposition of various b2 ions and their specifically deuterated analogs

B ions represent an important type of fragment ions derived from protonated peptides by cleavage of an amide bond with N-terminal charge retention. Such species have also been discussed as key intermediates during cyclic peptide fragmentation. Detailed structural information on such ion types can facilitate the interpretation of multiple step fragmentations such as the formation of inner chain fragments from linear peptides or the fragmentation of cyclic peptides. The structure of different b₂ ion isomers was investigated with collision-induced dissociations (CID) in combination with hydrogen/deuterium (H/D) exchange of the acidic protons. Special care was taken to investigate fragment ions derived from pure gas-phase processes. Structures deduced from the results of the CID analysis were compared with structures predicted on the basis of quantum chemical density functional theory (DFT) calculations to be most stable. The results pointed to different types of structures for b₂ ion isomers of complementary amino acid sequences. Either the protonated oxazolone structure or the N-terminally protonated immonium ion structure were proposed on the basis of the CID results and the DFT calculations. In addition, the analysis of different selectively N-alkylated peptide analogs revealed mechanistic details of the processes generating b ions.     

Defying entropy: forming large head-to-tail macrocycles in the gas phase

Spectral fingerprints: Collision-induced dissociation (CID) of protonated peptides in the gas phase results in linear fragment ions with a five-membered oxazolone ring on their C-terminal side. Infrared spectroscopy confirms that smaller fragments adopt oxazolone structures. Conversely, in mid-sized and larger fragments an isomerization to "head-to-tail" macrocycles is observed (see picture).     

Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains

The [M - H]- ions of a variety of di- to pentapeptides containing H or alkyl side chains have been prepared by electrospray ionization and low-energy collision-induced dissociation (CID) of the deprotonated species carried out in the interface region between the atmospheric pressure source and the quadrupole mass analyzer. Using the nomenclature applied to the fragmentation of protonated peptides, deprotonated dipeptides fragment to give a2 ions (CO2 loss) and y1 ions, where the y1 ion has two fewer hydrogens than the y"1 ions formed from protonated peptides. Deprotonated tri- and tetrapeptides fragment to give primarily y1, c1, and "b2 ions, where the "b2 ion has two fewer hydrogens than the b2 ion observed for protonated peptides. More minor yields of y2, c2, and a2 ions also are observed. The a ion formed by loss of CO2 from the [M - H]- ion shows loss of the N-terminal residue for tripeptides and sequential loss of two amino acid residues from the N-terminus for tetrapeptides. The formation of c(n) ions and the sequential loss of N-terminus residues from the [M - H - CO2]- ion serves to sequence the peptide from the N-terminus, whereas the formation of y(n) ions serves to sequence the peptide from the C-terminus. It is concluded that low-energy CID of deprotonated peptides provides as much (or more) sequence information as does CID of protonated peptides, at least for those peptides containing H or alkyl side chains. Mechanistic aspects of the fragmentation reactions observed are discussed.     

What is the Structure of b2 Ions Generated from Doubly Protonated Tryptic Peptides?

A recent statistical study (Savitski, M. M.; Falth, M.; Eva Fung, Y. M.; Adams, C. M.; Zubarev, R. A. J. Am. Soc. for Mass Spectrom. doi: 10.1016/j.jasms.2008.08.003) of a large spectral database indicated that the product ion spectra of doubly protonated tryptic peptides fall into two distinct classes. The main factor distinguishing the two classes is the relative abundance of the y _N-2 fragment: for Class I spectra y _N-2 is the most abundant y fragment while for Class II other y ions dominate the corresponding spectra. To explain the dominance of y _N-2 for Class I spectra formation of a nontraditional b ₂ ion with a diketopiperazine (6-membered cyclic peptide) rather than an oxazolone structure was proposed. Here we present evidence from tandem mass spectrometry, hydrogen/deuterium exchange, and density functional calculations that do not support this proposal. Namely, that CID of doubly protonated YIGSR, YGGFLR, and YIYGSFK produce Class I product ion spectra, yet the b ₂ fragment is shown to have the traditional oxazolone structure.     

Derivatization of protonated peptides via gas phase ion—molecule reactions with acetone

The protonated [M + H]+ ions of glycine, simple glycine containing peptides, and other simple di- and tripeptides react with acetone in the gas phase to yield [M + H + (CH3)2CO]+ adduct ion, some of which fragment via water loss to give [M + H + (CH3)2CO - H2O]+ Schiff's base adducts. Formation of the [M + H + (CH3)2CO]+ adduct ions is dependent on the difference in proton affinities between the peptide M and acetone, while formation of the [M + H + (CH3)2CO - H2O]+ Schiff's base adducts is dependent on the ability of the peptide to act as an intramolecular proton "shuttle." The structure and mechanisms for the formation of these Schiff's base adducts have been examined via the use of collision-induced dissociation tandem mass spectrometry (CID MS/MS), isotopic labeling [using (CD3)2CO] and by comparison with the reactions of Schiff's base adducts formed in solution. CID MS/MS of these adducts yield primarily N-terminally directed a- and b-type "sequence" ions. Potential structures of the b1 ion, not usually observed in the product ion spectra of protonated peptide ions, were examined using ab initio calculations. A cyclic 5 membered pyrrolinone, formed by a neighboring group participation reaction from an enamine precursor, was predicted to be the primary product.     

Conformation-Specific Spectroscopy of Peptide Fragment Ions in a Low-Temperature Ion Trap

We have applied conformer-selective infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy at low temperatures in an ion trap mass spectrometer for the spectroscopic characterization of peptide fragment ions. We investigate b- and a-type ions formed by collision-induced dissociation from protonated leucine-enkephalin. The vibrational analysis and assignment are supported by nitrogen-15 isotopic substitution of individual amino acid residues and assisted by density functional theory calculations. Under such conditions, b-type ions of different size are found to appear exclusively as linear oxazolone structures with protonation on the N-terminus, while a rearrangement reaction is confirmed for the a (4) ion in which the side chain of the C-terminal phenylalanine residue is transferred to the N-terminal side of the molecule. The vibrational spectra that we present here provide a particularly stringent test for theoretical approaches.     

Collision-induced dissociation of protonated tetrapeptides containing beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residues

The influence of the presence and position of a single beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or 4-aminomethylbenzoic acid residue on the tendency to form b(n)+ -and y(n)+ -type product ions was determined using a group of protonated tetrapeptides with general sequence XAAG, AXAG and AAXG (where X refers to the position of amino acid substitution). The hypothesis tested was that the 'alternative' amino acids would influence product ion signal intensities by inhibiting or suppressing either the nucleophilic attack or key proton transfer steps by forcing the adoption of large cyclic intermediates or blocking cyclization altogether. We found that specific b ions are diminished or eliminated completely when betaA, gammaAbu, Cap or 4AMBz residues are positioned such that they should interfere with the intramolecular nucleophilic attack step. In addition, differences in the relative proton affinities of the alternative amino acids influence the competition between complementary b(n) and y(n) ions. For both the AXAG and the XAAG series of peptides, collision-induced dissociation (CID) generated prominent b ions despite potential inhibition or suppression of intramolecular proton migration by the betaA, gammaAbu, Cap or 4AMBz residues. The prominent appearance of b ions from the AXAG and XAAG peptide is noteworthy, and suggests either that proton migration occurs through larger, 'whole' peptide cyclic intermediates or that fragmentation proceeds through a population of [M+H]+ isomers that are initially protonated at amide O atoms.     

Mass spectral study of alkali-cationized Boc-carbo-beta3-peptides by electrospray tandem mass spectrometry

Electrospray tandem mass spectrometry was used to study the dissociation reactions of [M+Cat]+ (Cat = Na+ and Li+) of Boc-carbo-beta3-peptides. The collision-induced dissociation (CID) spectra of [M+Cat-Boc]+ of these peptides are found to be significantly different from those of [M+H-Boc]+ ions. The spectra are more informative and display both C- and N-terminus metallated ions in addition to characteristic fragment ions of the carbohydrate moiety. Based on the fragmentations observed in the CID spectra of the [M+Cat-Boc]+ ions, it is suggested that the dissociation involves complexes in which the metal ion is coordinated in a multidentate arrangement involving the carbonyl oxygen atoms. The CID spectra of [M+Cat-Boc]+ ions of the peptide acids show an abundant N-terminal rearrangement ion [b(n)+17+Cat]+ which is absent for esters. Further, two pairs of positionally isomeric Boc-carbo-beta3-peptide acids, Boc-NH-Caa(S)-beta-hGly-OH (11) and Boc-NH-beta-hGly-Caa(S)-OH (12), and [Boc-NH-Caa(S)-beta-hGly-Caa(S)-beta-hGly-OH] (13) and [Boc-NH-beta-hGly-Caa(S)-beta-hGly-Caa(S)-OH] (14), were differentiated by the CID of [M+Cat-Boc]+ ions. The CID spectra of compounds 11 and 13 are significantly different from those of 12 and 14, respectively. The abundance of [b(n)+17+Cat]+ ions is higher for peptide acids 12 and 14 with a sugar group at the C-terminus when compared to 11 and 13 which contain a sugar moiety at the N-terminus. The observed differences between the CID spectra of these isomeric peptides are attributed to the difference in the preferential site of metal ion binding and also on the structure of the cyclic intermediate involved in the formation of the rearrangement ion.     

Profiling of cyclic hexadepsipeptides roseotoxins synthesized in vitro and in vivo: a combined tandem mass spectrometry and quantum chemical study

High-performance liquid chromatography and tandem mass spectrometry (HPLC/MS/MS) was used for the detection of cyclic hexadepsipeptides roseotoxins produced by Trichothecium roseum. Roseotoxins were found in both submerged standard cultivation on CzapekDox medium and in vivo cultivation extract obtained from an apple. Roseotoxin chromatographic profiles from these two experiments were compared. Product-ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionisation, ESI) were used for the identification of natural roseotoxins A, B, C and of minor destruxins A and B. The dissociation behavior of roseotoxins is discussed in terms of a fragmentation scheme proposed for describing the dissociation pathways of cyclic peptides. This scheme involves opening of the cyclopeptide ring via formation of oxazolone derivatives and fragmentation of the resulting linear species, which have a free N-terminus and an oxazolone ring at the C-terminus. Some aspects of this fragmentation scheme are underlined by modeling the dissociation channels of roseotoxin A using quantum chemical calculations. The structures of roseotoxin A and destruxin B were verified by nuclear magnetic resonance (NMR) spectroscopy. Structures of three new minor natural roseotoxins [Val(4)]RosA, [MeLxx(4)]RosA and [MeLxx(4)]RosB were deduced by ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT-MS) and ion trap tandem mass spectrometry by examining the pre-separated roseotoxin fraction.     

SO<Stack><Subscript>2</Subscript><Superscript>−·</Superscript></Stack> electron transfer ion/ion reactions with disulfide linked polypeptide ions

Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2-* in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)-S* and Chain A (or B)-SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+* ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+* ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+* ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.     

On the dynamics of fragment isomerization in collision-induced dissociation of peptides

The structures of peptide collision-induced dissociation (CID) product ions are investigated using ion mobility/mass spectrometry techniques combined with theoretical methods. The cross-section results are consistent with a mixture of linear and cyclic structures for both b4 and a4 fragment ions. Direct evidence for cyclic structures is essential in rationalizing the appearance of fragments with scrambled (i.e., permutated) primary structures, as the cycle may not open up where it was initially formed. It is demonstrated here that cyclic and linear a4 structures can interconvert freely as a result of collisional activation, implying that isomerization takes place prior to dissociation.     

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala,Ser, Leu,Val, Phe,and Trp)

The product ion spectra of proline-containing peptides are commonly dominated by y _n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y ₂ ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y ₂ /b ₃ abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y ₂ /b ₃ abundance ratio decreases.     

Effect of metal cationization on the low-energy collision-induced dissociation of loganin, epi-loganin and ketologanin studied by electrospray ionization tandem mass spectrometry

The effect of alkali metal and silver cationization on the collision-induced dissociation (CID) of loganin (1), epi-loganin (2) and ketologanin (3) is discussed. Their protonated molecular ions fragment mainly by glycosidic cleavages. The epimeric pairs (1 and 2) show differences in the abundances of the resulting fragment ions. Lithium cationization induces new dissociation pathways such as the retro-Diels-Alder (RDA) fragmentation followed by rearrangement. Unlike the dissociation of protonated molecular ions, the dissociation of lithiated molecules also provides lithiated sugar fragments. The CID of dilithiated molecules is substantially different from that of the monolithiated precursors. RDA reaction appears to be favoured by the presence of the additional lithium atom in the molecule. In addition, other ring cleavages are also induced. The abundances of the various fragment ions are different in the CID spectra of the epimeric pairs. Extensive D labelling and (6)Li labelling experiments confirmed many of the ion structures proposed. The CID spectra of the sodiated ions are generally weaker, although similar to those of the corresponding lithiated species. Higher alkali metal ion (K(+), Rb(+) and Cs(+)) adducts generated only the corresponding metal ions as products of CID. Similar fragmentations were also observed in the CID of the [M + Ag](+) ions of these compounds, the epimeric pairs showing characteristic differences in their CID behaviour. Copyright 2000 John Wiley & Sons, Ltd.     

The role of proton mobility in determining the energy-resolved vibrational activation/dissociation channels of N-glycopeptide ions

Site-specific glycoproteomic analysis largely hinges on the use of tandem mass spectrometry (MS/MS) to identify glycopeptides. Experiments of this type are usually aimed at drawing connections between individual oligosaccharide structures and their specific sites of attachment to the polypeptide chain. These determinations inherently require ion dissociation methods capable of interrogating both the monosaccharide and amino acid connectivity of the glycopeptide. Collision-induced dissociation (CID) shows potential to satisfy this requirement, as the vibrational activation/dissociation of protonated N-glycopeptides has been observed to access cleavage of either glycosidic bonds of the glycan or amide bonds of the peptide in an energy-resolved manner. Nevertheless, the relative energy requirement for these fragmentation pathways varies considerably among analytes. This research addresses the influence of proton mobility on the vibrational energy necessary to achieve either glycan or peptide cleavage in a collection of protonated N-glycopeptide ions. While greater proton mobility of the precursor ion was found to correlate with lower energy requirements for precursor ion depletion and appearance of glycosidic fragments, the vibrational energy deposition necessary for appearance of peptide backbone fragments showed no relation to the precursor ion proton mobility. These results are consistent with observations suggesting that peptide fragments arise from an intermediate fragment which is generally of lower proton mobility than the precursor ion. Such findings have potential to facilitate the rational selection of CID conditions which are best suited to provide either glycan or peptide cleavage products in MS/MS based N-glycoproteomic analysis.