IR action spectroscopy shows competitive oxazolone and diketopiperazine formation in peptides depends on peptide length and identity of terminal residue in the departing fragment.

The interplay between the entropically and enthalpically favored products of peptide fragmentation is probed using a combined experimental and theoretical approach. These b2 ion products can take either an oxazolone or diketopiperazine structure. Cleavage after the second amide bond is often a favorable process because the products are small ring structures that are particularly stable. These structures are structurally characterized by action IRMPD spectroscopy and semi-quantified using gas-phase hydrogen-deuterium exchange. The formation of the oxazolone and diketopiperazine has been thought to be largely governed by the identity of the first two residues at the N-terminus of the peptide. We show here that the length of the precursor peptide and identity of the third residue play a significant role in the formation of the diketopiperazine structure in peptides containing an N-terminal asparagine residue. This is additionally the first instance showing an N-terminal residue with an amide side chain can promote formation of the diketopiperazine b2 ion structure.

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

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.

Surface-induced dissociation of singly and multiply protonated polypropylenamine dendrimers.

The ease of fragmentation of various charge states of protonated polypropylenamine (POPAM) dendrimers is investigated by surface-induced dissociation. Investigated are the protonated diaminobutane propylenamines [DAB(PA)n] DAB(PA)8 (1+ and 2+), DAB(PA)16 (2+ and 3+), and DAB(PA)32 (3+ and 4+). These ions have been proposed to fragment by charge-directed intramolecular nucleophilic substitution (SNi) reactions. Differences in relative fragment ion abundances between charge states can be related to the occupation of different protonation sites. These positions can be rationalized based on estimates of Coulomb energies and gas-phase basicities of the protonation/fragmentation sites. The laboratory collision energies at which the fragment ion current is approximately 50% of the total ion current were found to increase with the size, but to be independent of charge state of the protonated POPAM dendrimers. It is suggested that intramolecular Coulomb repulsion within the multiply protonated POPAM dendrimers selected for activation does not readily result in easier fragmentation, which is in accordance with the proposed fragmentation mechanism.

Investigation of the trans effect in the fragmentation of dinuclear platinum complexes by electrospray ionization surface-induced dissociation tandem mass spectrometry.

Cis and trans isomers of two dinuclear platinum complexes, [cis-¿Pt(NH3)2Cl¿2 mu-(NH2(CH2)nNH2)](NO3)2 (1,1/c,c) and [trans-¿Pt(NH3)2Cl¿2 mu-(NH2(CH2)nNH2)](NO3)2 (1,1/t,t), where the diamine was 1,4-butanediamine (n = 4) or 1,6-hexanediamine (n = 6), were studied using electrospray ionization surface-induced dissociation (ESI/SID) tandem mass spectrometry (MS/MS). The same fragment ions were observed for both the cis and trans isomers of each complex (n = 4 or 6), but the relative intensities were dependent on the isomer studied. The ESI/SID data and energy-resolved mass spectra show that the position of the chloride plays a significant role in the fragmentation of these ions. Two major fragmentation pathways were detected for the complexes. The cleavage of the Pt-N bond trans to chloride was the most favorable pathway for both isomers of the complexes following the ion-surface collision. The differences in the ESI/SID spectra between the cis and trans isomers can be explained by the trans effect, namely that the Pt-N bond trans to chloride is the most labile bond.

Tandem fourier transform mass spectrometry studies of surface-induced dissociation of benzene monomer and dimer ions on a self-assembled fluorinated alkanethiolate monolayer surface.

A new instrument configuration based on a Finnigan FTMS-2000 platform has been applied to the study of surface-induced dissociation (SID) in this research. Benzene monomer ions C(6)H(6)(+) and dimer ions (C(6)H(6))(2)(+) were impacted on a fluorinated self-assembled monolayer surface at collision energies ranging from 1 to 70 eV. Benzene cations were chosen for this study because the fragmentation characteristics of the molecular cation are well known and its SID has been thoroughly investigated. SID spectra obtained by FTMS-SID are very similar to those reported in the literature for the same surface but exhibit much higher mass resolution. A comparison study of collision-induced dissociation (CID) and SID of benzene molecular cations was performed utilizing the same ICR cell and ion detection protocol. It is demonstrated that SID provides both much higher energy deposition and a narrower internal energy distribution than CID. The present instrument geometry and experimental protocol demonstrate much higher efficiencies than previous SID studies by FTMS and much higher mass resolution than previous SID studies using other types of mass analyzers.

Effect of alkyl substitution at the amide nitrogen on amide bond cleavage: electrospray ionization/surface-induced dissociation fragmentation of substance P and two alkylated analogs.

Doubly protonated substance P and two analogs alkylated at the ninth position was studied to determine the effect of N-alkylation of the amide nitrogen on the electrospray ionization/surface-induced dissociation (ESI/SID) fragmentation pattern. Thermal decomposition experiments and ab initio calculations were also used in conjunction with the ESI/SID experiments. The increase in relative abundances of the product ions resulting from the cleavage of the amide bond at the alkylation site (relative to the corresponding cleavage for substance P) can be explained by the increased basicity of the amide nitrogen in the context of the 'mobile proton' model. The relative abundances of singly charged b ions suggest a rearrangement of the amide hydrogen located N-terminal to the bond cleaved.

Surface-induced dissociation: an effective tool to probe structure, energetics and fragmentation mechanisms of protonated peptides.

The utility of surface-induced dissociation (SID) to probe the structure, energetics and fragmentation mechanisms of protonated peptides is discussed and demonstrated. High internal energy deposition provided by low-energy (eV range) ion-surface collisions yields extensive fragmentation of protonated peptides, allowing relatively uncomplicated and rapid sequence analysis of oligopeptides. SID of multiply protonated peptides is illustrated for peptides with molecular mass of up to approximately 5000 u. It is also illustrated that SID combined with electrospray ionization (ESI) provides a distinctive experimental technique to determine the energetics and mechanisms of peptide fragmentation. The relative position of ESI/SID fragmentation efficiency curves (plots of percentage fragmentation vs. laboratory collision energy) for peptides can be utilized to estimate relative energetics of peptide fragmentation and even to predict proton localization sites. The observed trends support the essential role of the mobile proton model in understanding peptide fragmentation by low-energy tandem mass spectrometry.

Average activation energies of low-energy fragmentation processes of protonated peptides determined by a new approach.

An attempt was made to estimate the average activation energies of low-energy fragmentation processes of protonated oligopeptides by combining RRKM theory and the results of electrospray ionization/surface induced dissociation (ESI/SID). The average internal energy was assumed to be deposited by three processes: thermal energy gained in the heated capillary of the electrospray source, energy gain in the capillary-skimmer region of the electrospray source, and energy deposition by collision with the surface. The latter fraction was calculated based on the position of the ESI/SID fragmentation of efficiency curves and the ratio of kinetic to internal energy conversion in SID. Using the average internal energy estimated from the experimental results, the average activation energies were evaluated by applying RRKM theory. The application of this approach for protonated leucine enkephalin resulted in an average activation energy of 36 +/- 5 kcal/mol for the lowest energy decompositions. The approach has also been applied to several other peptides in the mass range of 200-1200 Da, yielding average activation energies in the range of 35-47 kcal/mol.

Thermal decomposition kinetics of protonated peptides and peptide dimers, and comparison with surface-induced dissociation.

Rate constants for the unimolecular decomposition of peptide monomer and dimer ions by thermal and surface-induced dissociation (SID) are measured and compared. Rate constants for thermal dissociation are measured in a heated wide-bore capillary flow reactor attached in front of the capillary leading into the mass spectrometer. Thermal decomposition of the leucine enkephalin ion (YGGFL)H+ is observed between 600 and 680 K with rate constants of 20-200 s-1, and yields many of the same fragments as SID at 35 eV, although with different relative intensities. The thermal decomposition yields the Arrhenius parameters Ea = 38.3 kcal/mol, log A = 15.7. The decomposition of the monomer and dimer ions are also observed by using SID on C18 and fluorinated hydrocarbon surfaces, with rate constants of 2 x 10(4) to 40 x 10(4) s-1. The SID activated monomer ions are assigned equivalent temperatures of 710-840 K by extrapolation of the thermal activation parameters. The protonated dimer ion (YGGFL)2 H+ decomposes thermally at 500-540 K to yield the monomer ion. The dimer also decomposes by SID at low collision energies 10-20 eV on both surfaces to yield the monomer ion, and at much higher energies of 60-80 eV to yield fragments identical to the decomposition of the monomer. The large energy requirement for fragmentation from the dimer is due to energy deposition into more degrees of freedom plus the additional energy required for dissociation of the dimer to the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of protonation site on bond strengths in simple peptides: Application of Ab initio and modified neglect of differential overlap bond orders and modified neglect of differential overlap energy partitioning.

A comparative study of ab initio 6-31G(*) and semiempirical modified neglect of differential overlap (MNDO) bond orders and MNDO diatomic energy contributions for the description of bond strengths in neutral and protonated glycine, diglycine, triglycine, and dialanine is presented. Good correlations were found between 6-31G(*) and MNDO bond orders and between MNDO bond orders and diatomic energy contributions. Although bond orders and diatomic energy contributions are inherently different quantities, both predict the changes in bond strengths due to protonation to be qualitatively the same. The theoretically predicted differences in bond strengths for different protonated forms clearly indicate that in peptide fragmentation schemes one should consider even those protonated forms whose formation is not preferred energetically.

Fragmentation of protonated peptides: surface-induced dissociation in conjunction with a quantum mechanical approach.

This paper describes the results of a systematic investigation designed to assess the utility of surface-induced dissociation in the structural analysis of small peptides (500-1800u). A number of different peptides, ranging in mass and amino acid sequence, are fragmented by collision with a surface in a tandem mass spectrometer and the spectra are compared with data obtained by gas-phase collisional activation. The surface-induced dissociation spectra provide ample sequence information for the peptides. Side-chain cleavage ions of type w, which are generally detected upon kiloelectronvolt collisions with gaseous targets but not upon electronvolt collisions with gaseous targets, are detected in the ion-surface collision experiments. A theoretical approach based on MNDO bond order calculations is suggested for the description of peptide fragmentation. This model, supplemented by ab initio calculations, serves as a complement to the experimental work described in the paper and explains (i) the easy cleavage of the amide bond, (ii) charge-remote backbone and side-chain cleavages, and (iii) the influence of intramolecular H-bonding.

Surface-induced dissociation of multiply protonated peptides.

We report here surface-induced dissociation spectra of three multiply charged peptides: doubly protonated angiotensin I, doubly protonated renin substrate, and triply protonated melittin. For comparison, the collision-activated dissociation spectra of renin substrate and melittin are also presented. The spectra show that surface-induced dissociation provides structural information on multiply charged peptides at the picomole per microliter sample concentrations compatible with electrospray ionization. For multiply protonated angiotensin I, renin substrate, and melittin, surface collisions (l00-165 eV) favor a limited number of fragmentation pathways, which are the same as those favored in collision-activated dissociation experiments.

Surface-induced dissociation in tandem quadrupole mass spectrometers: A comparison of three designs.

Three different devices that-can be used for surface-induced dissociation (SID) m tandem quadrupole instruments are compared here. The designs were compared by examining the fragmentation of several compounds including benzene, W(CO)6, and (CH3)4N(+). These studies show that SID can be readily implemented on a variety of tandem quadrupoIe instruments and that the spectra obtained with the in-line and 90° instruments are similar. Evidence is presented that confirms that high average internal energies and narrow distributions of internal energy are available by this technique. Efficiencies for fragmentation of odd-electron ions are on the order of those previously reported by others. The overall SID efficiency for even-electron ions is higher than that for odd-electron ions of similar structure.