Fragmentation of a novel marine peptide, plicatamide, involves an unusual gas-phase intramolecular rearrangement

During our characterization of plicatamide 1, a modified octapeptide: Phe-Phe-His-Leu-His-Phe-His-dc deltaDOPA (where dc deltaDOPA = decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine) from the blood cells of the ascidian Styela plicata, we noted a series of fragment ions from the [M + H]+ ion which could not be assigned. There was no evidence in the 1H NMR spectrum to support an alternative molecular structure and the series of fragment ions were not present in the tandem mass spectrometry analysis of the [M + Na]+ ion. In addition, there was no evidence that the sample was a mixture of isobaric compounds. We propose that an unusual C-terminal to N-terminal rearrangement is responsible for the series of fragment ions from the [M + H]+ ion. This rearrangement was not observed in peptide analogs of plicatamide which did not contain the dc deltaDOPA at the C-terminus suggesting that this moiety is critical for the rearrangement. The proposed reaction is analogous to that recently reported by Vachet et al. involving a fragment ion formed from leucine enkephalin.     

Studies on azaspiracid biotoxins. II. Mass spectral behavior and structural elucidation of azaspiracid analogs

In this report, the mass spectral analysis of azaspiracid biotoxins is described. Specifically, the collision-induced dissociation (CID) behavior and differences between CID spectra obtained on a triple-quadrupole, a quadrupole time-of-flight, and an ion-trap mass spectrometer are addressed here. The CID spectra obtained on the triple-quadrupole mass spectrometer allowed the classification of the major product ions of the five investigated compounds (AZA 1-5) into five distinct fragment ion groups, according to the backbone cleavage positions. Although the identification of unknown azaspiracids was difficult based on CID alone, the spectra provided sufficient structural information to allow confirmation of known azaspiracids in marine samples. Furthermore, we were able to detect two new azaspiracid analogs (AZA 1b and 6) in our samples and provide a preliminary structural analysis. The proposed dissociation pathways under tandem mass spectrometry (MS/MS) conditions were confirmed by a comparison with accurate mass data from electrospray quadrupole time-of-flight MS/MS experiments. Regular sequential MS(n) analysis on an ion-trap mass spectrometer was more restricted in comparison to the triple-quadrupole mass spectrometer, because the azaspiracids underwent multiple [M + H - nH(2)O](+) (n = 1-6) losses from the precursor ion under CID. Thus, the structural information obtained from MS(n) experiments was somewhat limited. To overcome this limitation, we developed a wide-range excitation technique using a 180-u window that provided results comparable to the triple-quadrupole instrument. To demonstrate the potential of the method, we applied it to the analysis of degraded azaspiracids from mussel tissue extracts.     

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning

An API 3000 triple-quadrupole instrument and a QSTAR Pulsar quadrupole time-of-flight (TOF) mass spectrometer were compared for the determination of phosphopeptides by precursor ion scanning in both the positive and negative nanoelectrospray ionization modes. The limits of detection for synthetic phosphopeptides were similar (500 amol microl(-1)) for both types of instruments when monitoring precursors of -79 Da (PO(3)(-)). However, the quadrupole TOF system was approximately fivefold more sensitive (1 fmol microl(-1)) than the triple-quadrupole instrument (5 fmol microl(-1)) when monitoring precursors of 216 Da (immonium ion of phosphotyrosine). The recently introduced Q(2)-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF part, improved the sensitivity of precursor ion scans on a quadrupole TOF instrument. The selectivity of precursor ion scans is much better on quadrupole TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. As a result, the characteristic immonium ion of phosphotyrosine at m/z 216.043 can be utilized for the selective detection of tyrosine phosphorylated peptides. Our data suggest that, in addition to their superior performance for peptide sequencing, quadrupole TOF instruments also offer a very viable alternative to triple quadrupoles for precursor ion scanning, thus combining high sensitivity and selectivity for both MS and MS/MS experiments in one instrument.     

Formation of [b(n−1)+OH+H]+ ion structural analogs by solution-phase chemistry

Derivatization of a variety of peptides by a method known to enhance anhydride formation is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation. The [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b(n-1) + OH + H]+ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b(n-2) + OH + H]+ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b(n-1) + OH + H]+ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS(n) experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b(n-1) + OH + H]+ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b(n-1) + OH + H]+ ions is proposed.     

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of non-steroidal antiinflammatory drugs in surface water by liquid chromatography/tandem mass spectrometry

A triple-quadrupole instrument and a hybrid quadrupole/time-of-flight (TOF) mass spectrometer were compared for the determination of pharmaceutical compounds in water samples. The drugs investigated were the analgesics Ibuprofen, Fenoprofen, Ketoprofen, Naproxen, and Diclofenac. The recently introduced Q2-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF analyzer, improved the sensitivity of product ion scans on the quadrupole/TOF instrument. The selectivity is much better on quadrupole/TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection. This minimizes interferences from environmental matrices and allows acquisition of full spectra for selected analytes with better signal-to-noise characteristics than comparable spectra obtained with a scanned quadrupole. The qualitative information obtained (mass accuracy, resolution and full-scan spectra) by hybrid quadrupole/TOF mass spectrometry allows a more certain identification of analytes in environmental matrices at trace levels. Sample enrichment of water samples was achieved by a solid-phase extraction procedure. Average recoveries for loading 1 L of samples varied from 88 to 110%, and the quantification limits were less than 1.2 ng/L for the triple-quadrupole instrument (in MRM mode) and less than 3 ng/L for the quadrupole/TOF instrument.     

Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.     

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Tandem Time-of-Flight Mass Spectrometer with High Precursor Ion Selectivity Employing Spiral Ion Trajectory and Improved Offset Parabolic Reflectron

In this study, we have developed a tandem time-of-flight mass spectrometry (TOF/TOF) technique involving the use of a matrix-assisted laser desorption/ionization ion source that exhibits high precursor ion selectivity. An ion optical system with a 17 m spiral ion trajectory was used in the first time-of-flight mass spectrometer. High precursor ion selectivity was achieved by realizing a 15 m flight path, which is considerably longer than that of the conventional MALDI-TOF/TOF before the precursor ion selection by an ion gate; monoisotopic ions could be selected properly up to m/z 2500. Furthermore, the first time-of-flight mass spectrometer was composed of electrostatic sectors and could eliminate post-source decay (PSD) ions. Precursor ions with 20 keV kinetic energy were selected and injected into a collision cell, leading to the generation of fragment ions by high-energy collision-induced dissociation (HE-CID). The optimized second time-of-flight mass spectrometer included a post-acceleration region and an offset parabolic reflectron to record product ion spectra in the entire mass range. Our system could generate a simple HE-CID product ion spectrum because each fragment pathway could be observed as a single peak by the selection of monoisotopic ions of all precursor ions and HE-CID fragment pathways could be predominantly observed by the PSD ion elimination.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Claisen rearrangement of allyl phenyl ether and its sulfur and selenium analogues on electron impact

The electron impact (EI) mass spectrum of allyl phenyl ether (1) includes an ion at m/z 106 that is formed mainly by the loss of CO from the molecular ion, as supported by high resolution and MS/MS data. The formation of the [M - CO](+) ion from 1 can be explained in terms of the Claisen rearrangement of 1 after ionization in the ion source of the mass spectrometer. Similarly, allyl phenyl sulfide (2) and allyl phenyl selenide (3) showed characteristic ions corresponding to [M - CH(3)](+), [M - XH](+) (X = S or Se) and [M - C(2)H(4)](+.), and the formation of these ions are explained via Claisen rearrangement of 2 and 3 in the ion source of the mass spectrometer resulting in a mixture of rearrangement products. The formation of molecular ions of 2-allyl thiophenol and 2-allyl selenophenol as intermediates, that cannot be isolated as the neutrals from the solution phase Claisen rearrangement of 2 and 3, respectively, is clearly indicated in the gas phase. The mass spectra of the rearrangement products obtained from the solution phase reaction were also consistent with the proposal of formation of these products in the ion source of the mass spectrometer. The formation of characteristic fragment ions attributed to the Claisen rearrangement products are also evident in the collision induced dissociation spectra of the corresponding molecular ions. Copyright 2000 John Wiley & Sons, Ltd.     

Apparent gas-phase acidities of multiply protonated peptide ions: Ubiquitin,insulin B,and renin substrate

The gas-phase deprotonation reactions of multiply protonated bovine ubiquitin, insulin chain B, and renin substrate tetradecapeptide ions have been studied in a Fourier transform ion cyclotron resonance mass spectrometer coupled with an external electrospray source. Rate constants were measured for the reactions of these peptide ions with a series of reference compounds of known gas-phase basicities ranging from 195.6 to 232.6 kcal/mol. The apparent gas-phase acidities (GA_app) of the multiply protonated peptide ions [M + nH]ⁿ⁺ were determined with deprotonation reactions. The deduced values of GA_app show a strong dependence on the charge states of the multiply protonated peptide ions. In general, the values decrease as the charge states of the peptide ions increase. For ubiquitin ions, the determined GA_apps values decrease from> 232.6 to 205.0 kcal/mol for n = 4-13; for insulin B ions, the GA_apps decrease from> 232.6 to 198.2 kcal/mol for n = 2-5; for renin substrate ions, the GA_apps decrease from 221.6 to < 195.6 kcal/mol for n = 2-4. Interestingly, at a given mass-to-charge ratio, the GA_apps of these peptide ions agree within 10 kcal/mol despite large differences in their mass and charge. The ubiquitin and insulin B ions generated under the present conditions reveal multiple isomers at certain charge states,n = 4, 5, 6, 12 for ubiquitin and n = 4, 5 for insulin B, as evidenced by the fact that the isomers display distinctively different deprotonation reaction rates with certain reference compounds.     

Sequence and side-chain specific photofragment (193 nm) ions from protonated substance P by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Photodissociation at 193 nm (6. 43 eV) of the protonated substance-P, [M + H]⁺ ions, in a delayed extraction matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometer, is reported. The photofragment ion spectrum of substance P contains a complete series of a-type fragment ions and abundant side-chain cleavage ions. This article focuses on the utility of MALDI-TOF photodissociation for peptide sequencing.     

Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination

In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are systematically varied. Our findings suggest that, under the experimental conditions we use, the extent of intramolecular hydrogen scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier, and therefore most likely by the gas-phase structure of the peptide ion. Moreover, the observed scrambling seems to be independent of the nature of the peptide fragment ions (i.e., protonated B and Y' ' ions, and sodiated A and Y' ions). Our results strongly suggest that scrambling may be reduced by using alkali metal cationization instead of protonation in the ionization process.     

A collinear tandem time-of-flight mass spectrometer for infrared photodissociation spectroscopy of mass-selected ions

An apparatus based on collinear tandem time-of-flight mass spectrometer has been designed for the measurement of infrared photodissociation spectroscopy of mass-selected ions in the gas phase.The ions from a pulsed laser vaporization supersonic ion source are skimmed and mass separated by a Wiley-McLaren time-of-flight mass spectrometer.The ion of interest is mass selected,decelerated and dissociated by a tunable IR laser.The fragment and parent ions are reaccelerated and mass analyzed by the second time-of-flight mass spectrometer.A simple new assembly integrated with mass gate,deceleration and reacceleration ion optics was designed,which allows us to measure the infrared spectra of mass selected ions with high sensitivity and easy timing synchronization.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

N-terminal derivatization and fragmentation of neutral peptides via ion--molecule reactions with acylium ions: toward gas-phase Edman degradation?

The gas-phase ion-molecule reactions of neutral alanylglycine have been examined with various mass-selected acylium ions RCO(+) (R= CH(3), CD(3), C(6)H(5), C(6)F(5) and (CH(3))( 2)N), as well as the transacylation reagent O-benzoylbenzophenone in a Fourier transform ion cyclotron resonance mass spectrometer. Reactions of the gaseous dipeptide with acylium ions trapped in the ICR cell result in the formation of energized [M + RCO](+) adduct ions that fragment to yield N-terminal b-type and C-terminal y-type product ions, including a modified b(1) ion which is typically not observed in the fragmentation of protonated peptides. Judicious choice of the acylium ion employed allows some control over the product ion types that are observed (i.e., b versus y ions). The product ion distributions from these ion--molecule reactions are similar to those obtained by collision-activated dissociation in a triple quadrupole mass spectrometer of the authentic N-acylated alanylglycine derivatives. These data indicate that derivatization of the peptide in the gas-phase occurs at the N-terminal amine. Ab initio molecular orbital calculations, performed to estimate the thermochemistry of the steps associated with adduct formation as well as product ion formation, indicate that (i) the initially formed adduct is energized and hence likely to rapidly undergo fragmentation, and (ii) the likelihood for the formation of modified b(1) ions in preference to y(1) ions is dependent on the R substituent of the acylium ion. The reaction of the tetrapeptide valine--alanine--alanine--phenylalanine with the benzoyl cation was also found to yield a number of product ions, including a modified b(1) ion. This result suggests that the new experimental approach described here may provide a tool to address one of the major limitations associated with traditional mass spectrometric peptide sequencing approaches, that is, determination of the identity and order of the two N-terminal amino acids. Analogies are made between the reactions observed here and the derivatization and N-terminal cleavage reactions employed in the condensed-phase Edman degradation method.