Dissociation of the peptide bond in protonated peptides

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

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.     

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

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

Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide.     

Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal ??-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.     

Multistep tandem mass spectrometry for Sequencing Cyclic Peptides in an Ion-Trap Mass Spectrometer

Collisionally activated decomposition (CAD) of a protonated cyclic peptide produces a superposition spectrum consisting of fragments produced following random ring opening of the cyclic peptide to give a set of acylium ions (or isomeric equivalents) of the same m/z. Assignment of the correct sequence is often difficult owing to lack of selectivity in the ring opening. A method is presented that utilizes multiple stages of CAD experiments in an electrospray ion-trap mass spectrometer to sequence cyclic peptides. A primary acylium ion is selected from the primary product-ion spectrum and subjected to several stages of CAD. Amino-acid residues are sequentially removed, one at each stage of the CAD, from the C-terminus, until a b2 ion is reached. Results are presented for seven cyclic peptides, ranging in sizes from four to eight amino-acid residues. This method of sequencing cyclic peptides eliminates ambiguities encountered with other MS/MS approaches. The power of the strategy lies in the capability to execute several stages of CAD upon a precursor ion and its decomposition products, allowing the cyclic peptide to be sequenced in an unambiguous, stepwise manner.     

Structure investigation of Maltacine C1a, C1b, C2a and C2b-cyclic peptide lactones of the Maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides, which gave uninterrupted sequences of Bn and Y'n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesised. The structure of the four peptides were found to be: C1a and C2a: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-Orn-103-Y-I-OH) and C1b/C2b: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-K-103-Y-I-OH). Adip = aminodihydroxy pentanoic acid.     

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

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

Mass 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.     

Structure investigation of Maltacine D1a, D1b and D1c-cyclic peptide lactones of the Maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines has recently been described. The structure elucidation of three of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of four unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2 + ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides is tentatively suggested to be: D1a: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-HGly-Y-I-OH, D1b: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-S-Y-I-OH and D1c: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-K-S-Y-I-OH. Adip = aminodihydroxy pentanoic acid and HGly = hydroxyglycine.     

Sequencing of peptides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the era of complete genome sequences, biochemical and medical research will focus more on the dynamic proteome of a cell. Regulation of proteins by post-translational modifications, which are not determined by the gene sequence, are already intensively studied. One example is phosphorylation of serines and threonines, probably the single most common cellular regulatory mechanism. In this paper we describe the sequencing of mono- and bisphosphorylated peptides, including identification of the phosphorylation sites, by post-source decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to dephosphorylation of the parent ions, we studied the influence of the phosphate group on the fragmentation of peptides. Generally, peptides phosphorylated on serine and threonine residues displayed no difference in their fragmentation patterns. The intensities of the resulting fragment ion signals depend only on the peptide sequence and not on either the phosphorylated amino acid or its position in the peptide chain. Phosphorylation increased the bond cleavage C-terminal to the phosphorylation site more than 10-fold, resulting in abundant signals, which typically dominated the PSD spectra. The produced C-terminally phosphorylated b-type fragment ions showed characteristic dephosphorylated fragment ions b(n) -H(3)PO(4) (-98 Da) and b(n) -HPO(3) (-80 Da) of higher abundances than the phosphorylated fragment ion. As a second layer to identify the phosphorylation site, all internally phosphorylated fragment ions were accompanied by minor, but always detectable, signals of the dephosphorylated fragment ions. Interpretation of PSD spectra of phosphopeptides was not more complicated than for unphosphorylated peptides, despite the increased number of obtained fragment ion signals.     

Structure investigation of maltacine B1a, B1b, B2a and B2b: cyclic peptide lactones of the maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MSn of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatization with phenyl isothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure were revealed by a chemoselective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides were found to be as follows: B1a/B2a, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-Orn-103-Y-I-OH); and B1b/B2b, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-K-103-Y-I-OH).     

Chemical ligation of S-scylated cysteine peptides to form native peptides via 5-, 11-, and 14-membered cyclic transition states

Cysteine-containing dipeptides 3a-l, (3b+3b') (compound numbers in parentheses are used to indicate racemic mixtures; thus (3b+3b') is the racemate of 3b and 3b'), and tripeptide 13 were synthesized in 68-96% yields by acylation of cysteine with N-(Pg-α-aminoacyl)- and N-(Pg-α-dipeptidoyl)benzotriazoles (where Pg stands for protecting group in the nomenclature for peptides throughout the paper) in the presence of Et(3)N. Cysteine-containing peptides 3a-l and 13 were S-acylated to give S-(Pg-α-aminoacyl)dipeptides 5a-l and S-(Pg-α-aminoacyl)tripeptide 14 without racemization in 47-90% yields using N-(Pg-α-aminoacyl)benzotriazoles 2 in CH(3)CN-H(2)O (7:3) in the presence of KHCO(3). (In our peptide nomenclature, the prefixes di-, tri-, etc. refer to the number of amino acid residues in the main peptide chain; amino acid residues attached to sulfur are designated as S-acyl peptides. Thus we avoid use of the prefix "iso".) Selective S-acylations of serine peptide 3k and threonine peptide 3l containing free OH groups were thus achieved in 58% and 72% yield, respectively. S-(Pg-α-aminoacyl)cysteines 4a,b underwent native chemical ligations to form native dipeptides 3f,i via 5-membered cyclic transition states. Microwave irradiation of S-(Pg-α-aminoacyl)tripeptide 15 and S-(Pg-α-aminoacyl)tetrapeptide 17 in the presence of NaH(2)PO(4)/Na(2)HPO(4) buffer solution at pH 7.8 achieved chemical ligations, involving intramolecular migrations of acyl groups, via 11- and 14-membered cyclic transition states from the S-atom of a cysteine residue to a peptide terminal amino group to form native peptides 19 and 20 in isolated yields of 26% and 23%, respectively.     

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.     

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.     

Mobile and localized protons: a framework for understanding peptide dissociation

Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.     

Mass spectrometric investigation of Maltacines E1a and E1b--two members of the Maltacine family of peptide antibiotics

We have recently described the discovery of the Maltacines--a new family of cyclic peptide antibiotics from Bacillus subtilis. In this paper the mass spectrometric characterisation of two of the members is reported. A chemoselective ring opening with base to give the linear peptides was necessary before mass spectrometric characterisation could be performed. MS(n) of the singly and doubly charged protonated molecules gave uninterrupted series of Bn and Y'n ions that allowed determination of the amino acid sequence. By using a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange, the identities of three unknown residues were determined. The nature and position of the cyclic structure were disclosed by a chemoselective ring opening with Na 18OH and it was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. Peptides with different combinations of P/Q and P/K at the N-terminus were synthesised to verify the sequence of the N-terminal B2 ion. The structure of the two peptides is proposed to be: E1a: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-Orn-S-Y-I-OH) and E1b: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-K-S-Y-I-OH). Adip = (2-amino-4,5-dihydroxypentanoic acid).     

Structural influences on preferential oxazolone versus diketopiperazine b(2+) ion formation for histidine analogue-containing peptides

Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b(2)(+) ion structures have been of recent interest. While previously studied b(2)(+) ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b(2)(+) ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analogue-containing Xxx-Ala b(2)(+) ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen-deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b(2)(+) ion structures formed. The b(2)(+) ions studied include His-Ala (HA), methylated histidine analogues, including π-methyl-HA and τ-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid-Ala (DabA) and Lys-Ala (KA). The location and accessibility of the histidine π-nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the τ-nitrogen did not change the b(2)(+) ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans-cis isomerization of the first amide bond that is necessary for diketopiperazine formation.