Collision-induced Dissociation Spectra Research Articles

Investigation of the mechanism of electron capture and electron transfer dissociation of peptides with a covalently attached free radical hydrogen atom scavenger

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280kJ/mol) and low electron affinity (ca. 0.45eV), and was derivatized to the model peptide, FQXTEMPOEEQQQTEDELQDK. The XTEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882kJ/mol) of TEMPO is similar to backbone amide carbonyls (889kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision-induced dissociation (CID) of the TEMPO-tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO-labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of NCα backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO-tagged peptide dication produced a series of b+H (bH) and y+H (yH) ions along with some c ions having suppressed intensities, consistent with stable OH bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

Identification of position isomers by energy-resolved mass spectrometry.

This study reports an energy-resolved mass spectrometric (ERMS) strategy for the characterization of position isomers derived from the reaction of hydroxyl radicals ((●)OH) with diphenhydramine (DPH) that are usually hard to differentiate by other methods. The isomer analogues formed by (●)OH attack on the side chain of DPH are identified with the help of a specific fragment ion peak (m/z 88) in the collision-induced dissociation (CID) spectrum of the protonated molecule. In the negative ion mode, the breakdown curves of the deprotonated molecules show an order of stability (supported by density functional theory (DFT) calculations) ortho > meta > para of the positional isomers formed by the hydroxylation of the aromatic ring. The gas phase stability of the deprotonated molecules [M - H](-) towards the benzylic cleavage depends mainly on the formation of intramolecular hydrogen bonds and of the mesomeric effect of the phenol hydroxyl. The [M - H](-) molecules of ortho and meta isomers result a peak at m/z 183 with notably different intensities because of the presence/absence of an intramolecular hydrogen bonding between the OH group and C9 protons. The ERMS approach discussed in this report might be an effective replacement for the conventional methods that requires very costly and time-consuming separation/purification methods along with the use of multi-spectroscopic methods.

Formation of c- and z-ions due to preferential cleavage at the N[sbnd]C bond of Xxx-Asp/Asn residues in negative-ion CID of peptides

In collision-induced dissociation (CID) experiments of peptides, the formation of c- and z-ions originating from NCα bond cleavage of the peptide backbone is unusual, whereas b- and y-ions are often observed in positive-ion CID spectra. Here we report that characteristic product c- and z-ions can be observed in negative-ion CID spectra of peptides with Asp and Asn residues. The negative c- and z-ions are mainly generated at the NCα bond of Xxx-Asp and Xxx-Asn residues that are classified as the flexible amino acids in protein chemistry. In the absence of any acidic amino acid residues, negative-ion CID spectra show dominant c-ion peaks originating from cleavage at the NCα bond lying in between the 3rd and 4th residues from the C-terminus of analyte peptides. It is proposed that the NCα bond cleavage leading to the formation of c- and/or z-ions is caused by the negative charge located on the amide nitrogen, which generated with an intra-molecular hydrogen abstraction from the backbone amide regions due to negative-charged acidic functional groups.

Tandem mass spectrometry analysis of linoleic and arachidonic acid hydroperoxides via promotion of alkali metal adduct formation.

Recently, we demonstrated that tandem mass spectrometry (MS/MS) analysis in the presence of sodium ions was useful for identification of the position of the hydroperoxy group in phosphatidylcholine hydroperoxide (PCOOH). Likewise, MS/MS may enable identification of the hydroperoxy group position in various lipid hydroperoxides (LOOHs). To this end, we prepared major LOOHs, namely hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE), and analyzed them by quadrupole-time-of-flight MS/MS in both the absence and presence of alkali metals. Photo-oxidation (singlet oxygen-induced oxidation) of linoleic acid (LA) was used to prepare 9-10E,12Z-HPODE, 9-10E,12E-HPODE, 10-8E,12Z-HPODE, 12-9Z,13E-HPODE, 13-9Z,11E-HPODE, and 13-9E,11E-HPODE. Each isomer was analyzed under various MS/MS conditions (e.g., absence and presence of sodium). We found that in the presence of alkali metals, especially sodium, collision-induced dissociation (CID) of all HPODE isomers yielded structure-diagnostic fragment ions that were highly useful in identifying the position of the hydroperoxy group. For instance, CID spectra of sodiated 13-9Z,11E-HPODE revealed a neutral loss of 88 Da arising from fragmentation of the hydroperoxy group. Similar results were observed for HPETE isomers. Following oxidation of LA (or arachidonic acid) by lipoxygenase, the hydroperoxy group position of the resultant HPODE (or HPETE) was easily identified using this method, without any chromatographic separation processes. As information on the position of the hydroperoxy group provides insight into the processes that initiate lipid peroxidation (e.g., enzymatic oxidation, auto-oxidation and singlet oxygen-induced oxidation), the proposed method may be useful in elucidating the involvement and mechanism of lipid peroxidation in food deterioration and pathophysiological processes.

Strategies for generating peptide radical cations via ion/ion reactions.

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266-nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process.

Obtaining complementary polypeptide sequence information from a single precursor ion packet via sequential ion mobility-resolved electron transfer and vibrational activation.

Tandem mass spectrometry (MS/MS) is now well-known as a powerful tool for characterizing the primary structures of peptides and proteins; however, in many cases the use of but a single dissociation method provides only a partial view of the amino acid sequences and post-translational modification patterns of polypeptides. While the application of multiple fragmentation methods can be more informative, this introduces the burden of acquiring multiple MS/MS spectra per analyte, thus reducing the effective duty cycle of such methods. In this work, initial proof-of-concept is provided for a method designed to overcome these barriers. This method relies on the complementary fragmentation information that can be provided by performing collision-induced dissociation (CID) and electron transfer dissociation (ETD) in concert, while also taking advantage of an ion mobility (IM) dimension to temporally resolve the occurrence of CID and ETD when applied to a single accumulated packet of precursor ions. In this way, the significant proportion of the precursor ion population that remains unreacted in ETD experiments is subjected to CID rather than being fruitlessly discarded. In addition, the two distinct fragmentation spectra can be extracted from their corresponding IM domains to render readily interpretable individual fragmentation spectra. This scheme was demonstrated for several polypeptides ranging from 1.3 to 8.6 kDa in molecular weight. In each case, IM-resolved CID and ETD events resulted in b/y and c/z ions, respectively, which each covered both unique and overlapping sequence information. These findings demonstrate that the combination of CID and ETD can be achieved with greater utilization of the available ion population and little or no loss of duty cycle.

Mass Spectral Profile for Rapid Differentiating Beta-Lactams from Their Ring-Opened Impurities.

High performance liquid chromatography tandem mass spectrometry (HPLC MS) has been widely used for β-lactam antibiotics determination. However, its application to identify impurities of these frequently used drugs is not sufficient at present. In this job, characteristic profiles of the collision induced dissociation (CID) spectra of both β-lactams and ring-opened β-lactams were extracted from the MS data of six β-lactam antibiotics and their forty-five impurities, and were confirmed by the MS data reported in the literature. These characteristics have been successfully applied to rapid differentiation of β-lactam and ring-opened β-lactam impurities in cefixime, cefdinir, and cefaclor. However, these characteristic profiles can only be obtained under low activating voltage. They did not display in the high energy activated CID spectra. Diagnostic fragmentations for determining the localization of double bond and substituents on the thiazine ring and the side chain were also observed. In addition, several characteristic fragmentations are hopeful to be used to differentiate the configurations of C-2 on the thiazine ring of ring-opened impurities, which is generally disadvantageous of mass spectrometry. Taken together, forty-five impurities were identified from the capsules of cefixime, cefdinir, and cefaclor.

Examination of small molecule losses in 5-methylpyranopelargonidin MS/MS CID spectra by DFT calculations.

Pyranoanthocyanins are formed during food treatment and maturation (e.g. wine, juices), and they can be considered a natural alternative to artificial food colorants. Tandem mass spectrometry (MS/MS) is perhaps the most important technique in analysis of anthocyanin dyes. Knowledge of fragmentation pattern is a key aspect of their successful structural characterization. Polyphenolic compounds are known to lose small molecules during collision-induced dissociation (CID) in MS/MS experiments. However, the specific positions where such losses occur preferentially are unknown. The aim of this communication is to investigate the energetically most preferred places for H2 O and CO losses during the fragmentation of 5-methylpyranopelargonidin molecule by the means of computational chemistry (employing density functional theory) combined with CID MS/MS experiments and infrared multiphoton dissociation spectroscopy. Mechanisms responsible for the fragmentations were investigated, and optimal geometries and transition states were obtained. Cleavage of water as well as carbon monoxide occurs preferentially from the C-ring of flavonoid skeleton. In the most stable structure of 5-methylpyranopelargonidin, B-ring was found to be tilted with respect to the rest of the molecule. Planarization effort of the parent molecule contributes both to its decarbonylation and dehydration.

Addition-elimination versus Tishchenko reaction in the gas phase.

Gas phase reactions of the substituted phenide ions with methyl formate have been studied. It was found that the results of these reactions depend mainly on the basicity of the phenide ion, which is related to the presence of the electron-accepting or electron-donating substituents in the benzene ring. It was shown that the phenide ions substituted with electron-withdrawing groups react with methyl formate in the gas phase in a two-step reaction. The first step that proceeds according to the typical addition-elimination mechanism results in the formation of the anion of the respective benzaldehyde derivative with the negative charge located either in the aldehyde group (acyl anion) or in the benzene ring (phenide anion) in position ortho to an aldehyde moiety. In the second step, the preliminary-formed anion reacts with the second molecule of methyl formate yielding formally product of the second addition-elimination reaction. Theoretical calculations as well as collision induced dissociation spectra of the model compounds suggest that this reaction proceeds according to the Tishchenko reaction mechanism yielding the respective phthalide anion. According to our knowledge, this is the first example of the Tishchenko-type reaction in the gas phase.

Determination of triterpenoids from birch bark by liquid chromatography-tandem mass spectrometry

Pentacyclic triterpenoids from birch bark are of great interest as biologically active substances and raw materials for producing a wide range of drugs. In this work, a method for the highly sensitive determination of betulin, betulinic acid, lupeol, and erythrodiol in plant extracts based on a combination of high-performance liquid chromatography with tandem mass spectrometric detection is proposed. Mass spectra under the conditions of atmospheric pressure chemical ionization and collision-induced dissociation spectra of analytes are obtained. The conditions for mass spectrometric detection in the multiple reaction monitoring mode are optimized. The calculated detection limits are in the range 0.7–1.8 μg/L.

Artificial neural network for charge prediction in metabolite identification by mass spectrometry.

Collision-induced dissociation (CID) is widely used in mass spectrometry to identify biologically important molecules by gaining information about their internal structure. Interpretation of experimental CID spectra always involves some form of in silico spectra of potential candidate molecules. Knowledge of how charge is distributed among fragments is an important part of CID simulations that generate in silico spectra from the chemical structure of the precursor ions entering the collision chamber. In this chapter we describe a method to obtain this knowledge by machine learning.

Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides.

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.

Correction of precursor and product ion relative abundances in order to standardize CID spectra and improve Ecom50 accuracy for non-targeted metabolomics

Quantitative biases in the abundance of precursor and product ions due to mass discrimination in RF-only ion guides results in inaccurate collision induced dissociation (CID) spectra. We evaluated the effects of collision cell RF voltage and collision energy on CID spectra using ten singly protonated compounds (46-854 Da) in an orthogonal acceleration time-of-flight mass spectrometer. The relative ion transfer efficiency, i.e. the relative amount of ions transferred through the ion guide at any particular RF voltage was shown to be dependent on the ion's m/z. We developed an algorithm to correct for the mass discriminating effects of RF voltage on CID spectra. The algorithm was tested for both precursor and product ions at multiple RF voltages and collision energies in order to ensure reliability. Our results suggest that compounds that generate major product ions with m/z values <150 have peak intensities that deviate substantially from their actual abundance. This has implications for small molecule metabolomics research, particularly for studies that rely on CID spectra matching methods for structure identification.

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.

Ion mobility and tandem mass spectrometry of phosphatidylglycerol and bis(monoacylglycerol) phosphate (BMP)

The tandem mass spectrometry, ion mobility, and normal phase HPLC of isomeric phosphatidylglycerol (PG) and bis(monoacylglycerol) phosphate (BMP) have been investigated in this study with the objective of differentiating these unique classes of lipids. Measurement of ion mobility using the traveling wave method for negative molecular and product ions from isomeric PG and BMP yielded identical results, but different ion mobilities were observed for positive product ions arising from collision-induced dissociation (CID). The fastest moving positive product ions from the ion mobility analysis of BMP(18:1/18:1) were monoglyceride-like, and the slowest moving product ions from this BMP corresponded to [M+H−2H2O]+, which were readily observed for BMP but were only at very low abundance in the CID spectra of PG. The major product ions observed from the sodium adduct of PG(18:1/18:1) were consistent with diglyceride-like ion formation, but for BMP(18:1/18:1) only monoglyceride-like product ions were formed. The usefulness of ion mobility separation was tested with the selection of positive product ions derived from the isomeric PG and BMP molecular species in the lipid extract of RAW 264.7 cells. The ion mobility spectra of monoglyceride-like ions derived from BMP species with various esterified fatty acyl groups displayed some separation in ion mobility based on fatty acyl chain length and presence of a double bond in the acyl chain. The mechanism of ion formation of the diglyceride- and monoglyceride-like ions from PG and BMP respectively was examined using deuterium-labeled species including PG(D3116:0/18:1) and PG and BMP labeled by deuterium exchange.

Collision-induced dissociation of fatty acid [M - 2H + Na]- ions: charge-directed fragmentation and assignment of double bond position.

The collision-induced dissociation (CID) of cationic fatty acid-metal ion complexes has been extensively studied and, in general, provides rich structural information. In particular, charge-remote fragmentation processes are commonly observed allowing the assignment of double bond position. In a previous manuscript, we presented two methods to doubly deprotonate polyunsaturated fatty acids to form anionic fatty acid-sodium ion complexes, referred to as [M - 2H + Na] (-) ions. In the current manuscript, the CID behavior of these [M - 2H + Na] (-) ions is investigated for the first time. Significantly, we also present a deuterium-labeling experiment, which excludes the possibility that deprotonation occurs predominately at the α-carbon in the formation of fatty acid [M - H + NaF](-) ions. This supports our original proposal where deprotonation occurs at the bis-allylic positions of polyunsaturated fatty acids. CID spectra of polyunsaturated fatty acid [M - 2H + Na](-) ions display abundant product ions arising from acyl chain cleavages. Through the examination of fatty acid isomers, it is demonstrated that double bond position may be unequivocally determined for methylene-interrupted polyunsaturated fatty acids with three or more carbon-carbon double bonds. In addition, CID of [M - 2H + Na](-) ions was applied to 18:3 isomers of Nannochloropsis oculata and three isomers were tentatively identified: ∆(9,12,15)18:3, ∆(6,9,12)18:3, and ∆(5,8,11)18:3. We propose that structurally-informative product ions are formed via charge-driven fragmentation processes at the site of the resonance-stabilized carbanion as opposed to charge-remote fragmentation processes, which could be inferred if deprotonation occurred predominately at the α-carbon.

DART–MS in-source collision induced dissociation and high mass accuracy for new psychoactive substance determinations

The influx of new psychoactive substances is a problem that is challenging the analytical capabilities of enforcement agencies. Cathinone designer drugs are less likely to be included in routine drug screens and typical drug formulations are commonly mixtures with continually shifting components. Ambient ionization mass spectrometry employs relatively mild conditions to desorb and ionize solid samples, imparting much less energy than that associated with conventional mass spectrometry methods. Direct analysis in real time mass spectrometry (DART–MS) is an ambient ionization method that was employed to rapidly screen cathinones, alone and in mixtures, readily enabling differentiation of the active drug(s) from various cutting agents. Accurate mass determinations provided preliminary identification of the various components of drug mixtures. The data generated in forensic mass spectrometry can be used for both elemental composition formulations and isotope abundance calculations for determination of unknown psychoactive substances, and we demonstrate how this data could be applied to the presence of new drugs as the active components shift in response to regulations. Isotope abundance calculations were used to develop a candidate pool of possible molecular formulas associated with cathinones as a specific class of designer drugs. Together, the combination of a time-of-flight (TOF) mass analyzer along with in-source collision-induced dissociation (CID) spectra were used to drastically narrow the pool of candidates to a single molecular formula. The [M+H]+ and product ion peaks provided data for presumptive analysis of various substituted synthetic cathinones in a manner that is complementary to conventional GC–MS analysis of new psychoactive substances.

Fragmentation of negative ions from N-linked carbohydrates: part 6. Glycans containing one N-acetylglucosamine in the core.

Negative ion collision-induced dissociation (CID) spectra of N-glycans contain many diagnostic ions that provide more structural information than positive ion spectra. EndoH or endoS release of glycans from glycoproteins, as used by many investigators, cleaves glycans between the GlcNAc residues of the chitobiose core leaving the glycan without the reducing-terminal GlcNAc residue. However, their negative ion CID spectra do not appear to have been studied in detail. This paper examines the CID and ion mobility properties of these endoH-released glycans to determine if the missing GlcNAc influences the production of diagnostic fragment ions. N-Glycans were released from ribonuclease B, ovalbumin and gp120 with endoH to give high-mannose and hybrid glycans, and from IgG with endoS to produce biantennary complex glycans, all missing the reducing-terminal GlcNAc residue. Negative ion CID and travelling wave ion mobility spectra were recorded with a Waters Synapt G2 mass spectrometer using nanospray sample introduction. The majority of glycans yielded CID spectra exhibiting the same diagnostic fragments, which were equivalently informative, as the fully released structures. However, the ability of ion mobility to separate isomers was generally found to be inferior to its use with the full glycans despite the smaller nature of the compounds. The exception was the partial resolution of a pair of biantennary monogalactosylated glycans from IgG where, as chloride adducts, slight separation of the isomers was observed. The results show that the CID spectra of endoH- and endoS-released glycans are as useful as the corresponding spectra of the intact glycans (as released by PNGase F) in providing structural information on N-glycans.

Negative charge induced dissociation: fragmentation of deprotonated N-benzylidene-2-hydroxylanilines in electrospray ionization mass spectrometry.

Unimolecular reactivities of different N-benzylidene-2-hydroxylaniline anions were investigated in gas phase by electrospray ionization tandem mass spectrometry. All the collision-induced dissociation spectra of N-benzylidene-2-hydroxylaniline anions show similar ions at phenyl anions, neutral loss of benzonitrile and benzoxazole anions, respectively. The possible fragmentation pathway was probed through deuterium labeling and various group substituents experiments. Computational results were applied to shed light on the mechanism of fragmentation patterns. The proton in the CH=N is reactive in the formation of the concerned ions. Its direct transfer to the oxygen results in 2-hydroxyphenyl anion. Proton abstraction between benzoxazole and phenyl anion leads to the formation of benzene and benzoxazole anion.

Fragmentation of peptide radical cations containing a tyrosine or tryptophan residue: structural features that favor formation of [x(n-1) + H]˙⁺ and [z(n-1) + H]˙⁺ ions.

Peptide radical cations A(n)Y(•+) (where n = 3, 4, or 5) and A5W(•+) have been generated by collision-induced dissociation (CID) of [Cu(II)(tpy)(peptide)](•2+) complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H](+) or [z - H](•+) ions and radical-driven fragmentation at the Cα-C bond to give a(+) ions, unusual product ions [x + H](•+) and [z + H](•+) are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H](•+) ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H](•+). Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H](•+) increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG(•+), AAYG(•+), and AWAG(•+).