Fragmentation of protonated dipeptides containing arginine. Effect of activation method

The fragmentation reactions of the protonated dipeptides Gly-Arg and Arg-Gly have been studied using collision-induced dissociation (CID) in a quadrupole ion trap, by in-source CID in a single-quadrupole mass spectrometer and by CID in the quadrupole cell of a QqTOF mass spectrometer. In agreement with earlier quadrupole ion trap studies (Farrugia, J. M.; O'Hair, R. A. J., Int. J. Mass Spectrom., 2003, 222, 229), the CID mass spectra obtained with the ion trap for the MH(+) ions and major fragment ions are very similar for the two isomers indicating rearrangement to a common structure before fragmentation. In contrast, in-source CID of the MH(+) ions and QqTOF CID of the MH(+), [MH - NH(3)](+) and [MH <23 HN = C(NH(2))(2)](+) ions provide distinctly different spectra for the isomeric dipeptides, indicating that rearrangement to a common structure has not occurred to a significant extent under these conditions even near the threshold for fragmentation in the QqTOF instrument. Clearly, under normal operating conditions significantly different fragmentation behavior is observed in the ion trap and beam-type experiments. This different behavior probably can be attributed to the shorter observation times and concomitant higher excitation energies in the in-source and QqTOF experiments compared to the long observation times and lower excitation energies relevant to the ion trap experiments. Based largely on elemental compositions derived from accurate mass measurements in QqTOF studies fragmentation schemes are proposed for the MH(+), [MH - NH(3)](+), and [MH - (HN = C(NH(2))(2))](+) ions.     

Cyclization of peptide b9 ions

The product ion mass spectra obtained by CID of the b₉ ions derived by loss of neutral alanine from the MH⁺ ion of the peptides Tyr(Ala)₉, (Ala)₄Tyr(Ala)₅, and (Ala)₈TyrAla are essentially identical, indicative of full cyclization reaction to a common intermediate before fragmentation. This leads to abundant nondirect sequence ions in the product ion mass spectra of the b₉ ions. The product ion mass spectra of the b₈ ions from the first two peptides also are essentially identical. The fragmentation of the MH⁺ ions also leads to low intensity nondirect sequence ions in the product ion mass spectra. N-terminal acetylation blocks the cyclization and eliminates nondirect sequence fragment ions in the product ion mass spectra.     

A Systematic Study of Acidic Peptides for b-Type Sequence Scrambling

A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of b _n (where n = 7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH⁺ derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b ₇ and b ₈ ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides. III. The linoleate and arachidonate series

A series of positionally pure triglycerides (TAGs) of the form LXL, YLY, AXA, and YAY was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. L and A represent the linoleate and arachidate moieties, respectively, and X and Y represent large arrays of fatty acid moieties of various chain lengths, degree of unsaturations, double-bond positions, and cis/trans configurations. The abundances of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of these parameters. The major CID products, the diglyceride (DAG) product ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. The following trends are observed in the data. TAGs with higher degrees of unsaturation tend to show greater relative abundances of MH(+) in the CID spectra of their ammoniated precursor ions. The position of the fatty acid constituents along the glycerol backbone also seems to influence the abundances of the MH(+) ion in the CID spectra of the ammoniated precursor ions. A fatty acid constituent with double bonds along the fatty acid chain positioned close to the carbonyl promotes the formation of the DAG product ion that corresponds to its loss upon CID of the ammoniated precursor ion. Linoleic acid substituents also seem to promote the formation of DAG product ions, but to a lesser extent. Data for the YAY TAGs are used to predict the abundances of the product ions in the CID spectra of ammoniated YAX TAGs. These data are discussed in context of a broader project to develop and validate software algorithims to support a platform for comprehensive analysis of complex mixtures of TAGs.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. II. The PXP/YPY series

A series of positionally pure triglycerides (TAGs) of the form PXP and YPY, where P is the palmitate moiety and X and Y are large arrays of different fatty acid moieties, is synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride (DAG) fragment ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. Different trends for each of these series are observed. Trends in the intensities of these fragment ions are also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional intensities of MH(+) increase with increasing degree of unsaturation in the TAGs. MH(+) is absent in the CID spectra of triglycerides containing three saturated fatty acid moieties, suggesting that the presence of double bonds fosters the formation of MH(+). Double bonds positioned close to the carbonyl carbon along the fatty acid chain promote the formation of the DAG fragment ion corresponding to the loss of the fatty acid. The collection of PXP/YPY data produced in this work is used to test the mechanisms of the formation and decomposition of ammoniated TAGs that were previously presented. The YPY data are used to predict the intensities of the fragment ions in the CID spectra of YPX-type TAGs. The limitations of the mathematical approach used in these predictions are discussed in context of a broader plan to develop a software platform for comprehensive analysis of complex TAG mixtures.     

Structural assignment of isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids by liquid chromatography electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

Positionally isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids give rise to similar electrospray ionization (ESI) and atmosphere pressure chemical ionization (APCI) mass spectra, which show very abundant MH(+) ions and negligible fragmentation. The MH(+) ions of these isomeric acids exhibit notably different behavior under collision-induced dissociation (CID) conditions. The acids with a sulfonic group at position 8' in the quinoline moiety, adjacent to the N-atom, exhibit highly abundant [MH - H(2)SO(3)](+) ions (m/z 272 for the mono- and m/z 352 for the disulfonic acids), which are of lower abundance in the CID spectra of isomers with the SO(3)H group at other positions, remote from the nitrogen atom. The latter isomers undergo efficient eliminations of SO(3) and HSO(3). The isomeric diacids with one SO(3)H group at position 4 of the indene-1,3(2H)-dione moiety, adjacent to one of the carbonyl groups, undergo highly efficient elimination of H(2)O. Mechanistic pathways, involving interactions between adjacent groups, are proposed for the above regiospecific fragmentations. Pronounced different behavior has been also observed in negative ion tandem mass spectrometric measurements of the sulfonic acids. The distinctive behavior of the isomeric acids was strongly pronounced when the measurements were performed with an ion trap mass spectrometer (LCQ), and much less so with a triple-stage quadrupole instrument (TSQ).     

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.     

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.     

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.     

Tandem mass spectrometry investigation of ADP-ribosylated kemptide

Bacterial adenosine diphosphate-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide (LRRASLG) as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron-capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Because of the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a 2-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.     

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.     

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

The fragmentation reactions of the singly-protonated oligoalanines trialanine to hexaalanine have been studied using energy-resolved mass spectrometry in MS(2) and MS(3) experiments. The primary fragmentation reactions are rationalized in terms of the b(x)-y(z) pathway of amide bond cleavage which results in formation of a proton-bound complex of an oxazolone and a peptide/amino acid; on decomposition of this complex the species of higher proton affinity preferentially retains the proton. For protonated pentaalanine and protonated hexaalanine the major primary fragmentation reaction involves cleavage of the C-terminal amide bond to form the appropriate b ion. The lower mass b ions originate largely, if not completely, by further fragmentation of the initially formed b ion. MS(3) energy-resolved experiments clearly show the fragmentation sequence b(n) --> b(n-1) --> b(n-2). A more minor pathway for the alanines involves the sequence b(n) --> a(n) --> b(n-1) --> b(n-2). The a(5) ion formed from hexaalanine loses, in part, NH(3) to begin the sequence of fragmentation reactions a(5) --> a(5)* --> a(4)* --> a(3)* where a(n)* = a(n) - NH(3). The a(3)* ion also is formed from the b(3) ion by the sequence b(3) --> a(3) --> a(3)* with the final step being sufficiently facile that the a(3) ion is not observed with significant intensity in CID mass spectra. A cyclic structure is proposed for the a(3)* ion.     

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H₂N=CH-R]⁺, where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.     

Effect of the his residue on the cyclization of b ions

The MSⁿ spectra of the [M + H]⁺ and b ₅ peaks derived from the peptides HAAAAA, AHAAAA, AAHAAA, AAAHAA, and AAAAHA have been measured, as have the spectra of the b ₄ ions derived from the first four peptides. The MS² spectra of the [M + H]⁺ ions show a substantial series of b_n ions with enhanced cleavage at the amide bond C-terminal to His and substantial cleavage at the amide bond N-terminal to His (when there are at least two residues N-terminal to the His residue). There is compelling experimental and theoretical evidence for formation of nondirect sequence ions via cyclization/reopening chemistry in the CID spectra of the b tons when the His residue is near the C-terminus. The experimental evidence is less clear for ions when the His residue is near the N-terminus, although this may be due to the use of multiple alanine residues in the peptide making identifying scrambled peaks more difficult. The product ion mass spectra of the b ₄ and b ₅ ions from these isomeric peptides with cyclically permuted amino acid sequences are similar, but also show clear differences. This indicates less active cyclization/reopening followed by fragmentation of common structures for b _n ions containing His than for sequences of solely aliphatic residues. Despite more energetically favorable cyclization barriers for the b ₅ structures, the b ₄ ions experimental data show more clear evidence of cyclization and sequence scrambling before fragmentation. For both b ₄ and b ₅ the energetically most favored structure is a macrocyclic isomer protonated at the His side chain.     

Cyclization reaction of peptide fragment ions during multistage collisionally activated decomposition: An inducement to lose internal amino-acid residues

During characterization of some peptides (linear precursors of the cyclic peptides showing potential to be anticancer drugs) in an ion trap, it was noted that many internal amino acid residues could be lost from singly charged b ions. The phenomenon was not obvious at the first stage of collisionally activated decomposition (CAD), but was apparent at multiple stages of CAD. The unique fragmentation consisting of multiple steps is induced by a cyclization reaction of b ions, the mechanism of which has been probed by experiments of N-acetylation, MS(n), rearranged-ion design, and activation-time adjustment. The fragmentation of synthetic cyclic peptides demonstrates that a cyclic peptide intermediate (CPI) formed by b ion cyclization exhibits the same fragmentation pattern as a protonated cyclic peptide. Although no rules for the cyclization reaction were discerned in the experiments of peptide modification, the fragmentations of a number of b ions indicate that the "Pro and Asn/Gln effects" can influence ring openings of CPIs. In addition, large-scale losses of internal residues from different positions of a-type ions have been observed when pure helium was used as collision gas. The fragmentation is initiated by a cyclization reaction forming an a-type ion CPI. This CPI with a fixed-charge structure cannot be influenced by the "Pro effect", causing a selective ring opening at the amide bond Pro-Xxx rather than Xxx-Pro. With the knowledge of the unique fragmentations leading to internal residue losses, the misidentification of fragments and sequences of peptides may be avoided.     

Negative ion electrospray tandem mass spectrometric structural characterization of leukotriene B4 (LTB4) and LTB4-derived metabolites

The low energy collision induced dissociation (CID) of the carboxylate anions generated by electrospray ionization of leukotriene B₄ (LTB₄) and 16 of its metabolites was studied in a tandem quadrupole mass spectrometer. LTB₄ is a biologically active lipid mediator whose activity is terminated by metabolism into a wide variety of structural variants. The collision-induced dissociation spectra of the carboxylate anions revealed structurally informative ions whose formation was determined by the position of hydroxyl substituents and double bonds present in the LTB₄ metabolite. Major ions resulted from charge remote α-hydroxy fragmentation or charge directed α-hydroxy fragmentation. The conjugated triene moiety present in some metabolites was proposed to undergo cyclization to a 1,3-cyclohexadiene structure prior to charge remote or charge driven a-hydroxy fragmentation. The mechanisms responsible for all major ions observed in the CID spectra were studied using stable isotope labeled analogs of the LTB₄ metabolites. In general, the collision-induced decomposition of carboxylate anions produced unique spectra for all LTB₄ derived metabolites. The observed decomposition product ions from the carboxylate anion could be useful in developing assays for these molecules in biological fluids.     

Collision-induced dissociation of ring-opened cyclic depsipeptides with a guanidino group by electrospray ionization/ion trap mass spectrometry

The characteristics shown in the electrospray ionization/ion trap mass spectra of ring-opened LI-F antibiotics (cyclic depsihexapeptides with a 15-guanidino-3-hydroxypentadecanoic group as a side-chain) were examined. Collision-induced dissociation (CID) MS of protonated molecules of the depsipeptides produced many fragment ions. Most of these fragment ions contained information for determining the amino acid sequences of antifungal antibiotics. The fragment ions were classified into six groups (b(n'), B(n'), B'(n'), beta(n'), y(n) and Y(n)). According to MS(3) spectra, the B(n'), B'(n) and beta(n) ions can be considered to be derived with a cleavage at each CO--NH in the peptide bonds of [MH--NH(3)](+),[MH--NH(3)--OH](+) and [MH--NH(3)--2H(2)O](+), respectively, in ion trap MS. Losses of NH(3) and H(2)O from the amino acid residues of the depsipeptides in ion trap MS are likely to be smaller than those from the side-chain. The measurements with electrospray ionization (ESI)/ion trap MS of depsipeptides with a side chain containing polar groups may provide useful information for structural determination.     

(+)-Silybin, a pharmacologically active constituent of Silybum marianum: fragmentation studies by atmospheric pressure chemical ionization quadrupole time-of-flight tandem mass spectrometry

The fragmentation behavior of (+)-silybin (1) and (+)-deuterosilybin (2), as well as of their flavanone-3-ol-type building blocks, such as 3,5,7-trihydroxy-2-phenyl-4-chromanone (3) and 2-(1,4-benzodioxolanyl)-3,5,7-trihydroxy-4-chromanone (4), were investigated by atmospheric pressure chemical ionization quadropole time-of-flight tandem mass spectrometry in the positive ion mode (APCI(+)-QqTOF MS/MS). The product ion spectra of the protonated molecules of 1 revealed a rather complicated fragmentation pattern with product ions originating from consecutive and competitive loss of small molecules such as H2O, CO, CH2O, CH3OH and 2-methoxyphenol, along with the A+- and B+-type ions arising from the cleavage of the C-ring of the flavanone-3-ol moiety. The elucidation of the fragmentation behavior of 1 was facilitated by acquiring information on the fragmentation characteristics of the flavanone-3-ol moieties and 2. The capability of the accurate mass measurement on the quadrupole time-of-flight mass spectrometer allowed us to determine the elemental composition of each major product ion. Second-generation product ion spectra obtained by combination of in-source collision induced dissociation (CID) with selective CID (pseudo-MS(3)) was also helpful in elaborating the fragmentation pathways and mechanism. Based on the experimental results, a fragmentation mechanism as well as fragmentation pathways for 1 and its flavanone-3-ol building blocks (3, 4) are proposed and discussed.     

Differentiation of three pairs of positional isomers of hybrid peptides with repeats of phenylalanine-beta3-h-valine/beta3-valine-phenylalanine by electrospray ionization tandem mass spectrometry

Electrospray ionization ion trap mass spectrometry has been used to distinguish three pairs of positional isomers of a new series of N-blocked hybrid peptides derived from repeats of phenylalanine(D)-beta3-h-valine/beta3-h-valine-phenylalanine(D) (FbetaV/betaVF) non-natural amino acids. MSn of protonated isomeric peptides produces characteristic fragmentation involving the peptide backbone, the Boc group and the side chain. FbetaV-peptides can be distinguished from betaVF-peptides by the loss of R-OH from [M+H-Boc+H]+, which is either of relatively low abundance or totally absent for the latter peptides. In contrast, betaVF-peptides show abundant Mannich base characteristic ions by the elimination of ammonia, and imine due to a retro-Mannich cleavage. This fragmentation is absent for FbetaV-peptides. When beta-valine is at the C-terminus, abundant b+(n-1) ions are produced. This is ascribed to the probable formation of a stable diketopiperazine structure, and this has been supported by the loss of H2O and CO in the CID spectra of b+(n-1) ions. The hybrid dipeptide acids have also been distinguished in negative ion mass spectrometry.     

Does the reagent gas influence collisional activation when performing in situ chemical ionization with an ion trap mass spectrometer?

Users of ion trap mass spectrometers frequently develop methods that associate chemical ionization with tandem mass spectrometry detection. With apparatus using internal ionization, the chemical reagent is present in the trap during the collision induced dissociation (CID) step and one may wonder if the reagent influences the fragmentation ratios in MS/MS. We report a comparison of the fragmentation ratios of protonated molecules when using the most common reagents (methane, ammonia, methanol, acetonitrile, isobutane) for performing in situ chemical ionization. Four molecules were chosen in the medical field to serve as models: alprazolam, diazepam, flunitrazepam and acetaminophen. In the non-resonant CID mode, the influence of the reagent mass is clearly seen in spite of its low partial pressure in the ion trap; the reagent acts as a "heavy target": the degree of fragmentation increases with the molecular weight of the reagent. In the resonant CID mode, there is no evident correlation between the fragmentation ratio of MH(+) ions and the nature of the CI reagent; a slight shift of the secular frequency of the precursor ion, which tends to reduce the CID efficiency, could compensate for the "heavy target" effect underscored in the non-resonant mode.