Cations Derived from Fentanyls Generated by Atmospheric Pressure Photoionization in the Presence of Ammonia: An IMS-MS Study.

A set of fentanyl molecules when subjected to vacuum UV atmospheric pressure photoionization (VUV-APPI) in the presence of dopants (ammonia and anisole) shows two major bands in the ion mobility-mass spectrometry (IMS-MS) spectrum corresponding to (a) the protonated fentanyl, [M+H]+ and (b) a unique [M-74]+ ion. For the parent fentanyl, the [M-74]+ ion is at m/z 262 but, in the absence of ammonia, the product ion is shifted to m/z 245, corresponding to a difference of NH3. Collision-induced dissociations (CID) of the [M-74]+ ions for all the different fentanyls examined here show the same pattern of neutral losses, namely NH3 and HN=CH2, and the dominant product ion is at m/z 84 (shifted to m/z 98 for 3-methylfentanyl and m/z 142 and 231 for carfentanyl). Dissociation of the [M-74-NH3]+ ion derived from the fentanyls yields the same product ions as found in the electron impact (EI) ionization spectra of the fentanyls. The dissociation products of the [M-74-HN=CH2]+ ion are different, include the ion at m/z 84, and correspond to the fragmentation products of protonated norfentanyls. Theoretical modeling supports the opening of new fragmentation channels as a result of the reaction of the initially formed iminium cation with ammonia at atmospheric pressure.

Structures of a(n)* ions derived from protonated pentaglycine and pentaalanine: results from IRMPD spectroscopy and DFT calculations.

Infrared multiple-photon dissociation (IRMPD) spectroscopy and DFT calculations have been used to probe the most stable structures of a3(*) and a4(*) ions derived from both protonated pentaglycine (denoted G5) and pentaalanine (A5). The a3(*) and a4(*) ions derived from protonated A5 feature a CHR=N-CHR'- group at the N-terminus and an oxazolone ring at the C-terminus, as proposed previously [J. Am. Soc. Mass Spectrom. 19, 1788-1798 (2008)]. The isomeric a4(*) ion derived from A5 with a 3,5-dihydro-4H-imidazol-4-one ring structure was calculated to have a slightly better energy than the oxazolone, but the barrier to its formation is higher and there was no evidence of this ion in the IRMPD spectrum. By contrast, the a4(*) and [a4 - H2O](+) (denoted a4(0)) ions from G5 gave strikingly similar IRMPD spectra and both have the 3,5-dihydro-4H-imidazol-4-one ring structure similar to that recently reported for the [GGGG + H - H2O](+) ion [Int. J. Mass Spectrom. 316-318, 268-272 (2012)]. In the absence of a solvent molecule, the pathway to the oxazolone is calculated to be lower than those to thermodynamically more stable products, the a4(0) and the a4(*) with the 3,5-dihydro-4H-imidazol-4-one ring structure. Incorporation of one water molecule is sufficient to reduce the barrier to formation of the a4(0) of G5 to below that for formation of the oxazolone. On the equivalent potential energy surface for protonated A5 the barrier to formation of the a4(0) ion is 12.3 kcal mol(-1) higher than that for oxazolone formation and the a4(0) ion is not observed experimentally.

Metal ion complexes with HisGly: comparison with PhePhe and PheGly.

Gas-phase complexes of five metal ions with the dipeptide HisGly have been characterized by DFT computations and by infrared multiple photon dissociation spectroscopy (IRMPD) using the free electron laser FELIX. Fine agreement is found in all five cases between the predicted IR spectral features of the lowest energy structures and the observed IRMPD spectra in the diagnostic region 1500-1800 cm(-1), and the agreement is largely satisfactory at longer wavelengths from 1000 to 1500 cm(-1). Weak-binding metal ions (K(+), Ba(2+), and Ca(2+)) predominantly adopt the charge-solvated (CS) mode of chelation involving both carbonyl oxygens, an imidazole nitrogen of the histidine side chain, and possibly the amino nitrogen. Complexes with Mg(2+) and Ni(2+) are found to adopt iminol (Im) binding, involving the deprotonated amide nitrogen, with tetradentate chelation. This tetradentate coordination of Ni(II) is the preferred binding mode in the gas phase, against the expectation under condensed-phase conditions that such binding would be sterically unfavorable and overshadowed by other outcomes such as metal ion hydration and formation of dimeric complexes. The HisGly results are compared with corresponding results for the PheAla, PheGly, and PhePhe ligands, and parallel behavior is seen for the dipeptides with N-terminal Phe versus His residues. An exception is the different chelation pattern determined for PhePhe versus HisGly, reflecting the intercalation-type cation binding pocket of the PhePhe ligand. The complexes group into three well-defined spectroscopic patterns: nickel and magnesium, calcium and barium, and potassium. Factors leading to differentiation of these distinct spectroscopic categories are (1) differing propensities for choosing the iminol binding pattern, and (2) single versus double charge on the metal center. Nickel and magnesium ions show similar gas-phase binding behavior, contrasting with their quite different patterns of peptide interaction in condensed phases.

Structure and reactivity of the distonic and aromatic radical cations of tryptophan.

In this work, we regiospecifically generate and compare the gas-phase properties of two isomeric forms of tryptophan radical cations-a distonic indolyl N-radical (H3N(+) - TrpN(•)) and a canonical aromatic π (Trp(•+)) radical cation. The distonic radical cation was generated by nitrosylating the indole nitrogen of tryptophan in solution followed by collision-induced dissociation (CID) of the resulting protonated N-nitroso tryptophan. The π-radical cation was produced via CID of the ternary [Cu(II)(terpy)(Trp)](•2+) complex. CID spectra of the two isomeric species were found to be very different, suggesting no interconversion between the isomers. In gas-phase ion-molecule reactions, the distonic radical cation was unreactive towards n-propylsulfide, whereas the π radical cation reacted by hydrogen atom abstraction. DFT calculations revealed that the distonic indolyl radical cation is about 82 kJ/mol higher in energy than the π radical cation of tryptophan. The low reactivity of the distonic nitrogen radical cation was explained by spin delocalization of the radical over the aromatic ring and the remote, localized charge (at the amino nitrogen). The lack of interconversion between the isomers under both trapping and CID conditions was explained by the high rearrangement barrier of ca.137 kJ/mol. Finally, the two isomers were characterized by infrared multiple-photon dissociation (IRMPD) spectroscopy in the ~1000-1800 cm(-1) region. It was found that some of the main experimental IR features overlap between the two species, making their distinction by IRMPD spectroscopy in this region problematic. In addition, DFT theoretical calculations showed that the IR spectra are strongly conformation-dependent.

Infrared multiple-photon dissociation spectroscopy of tripositive ions: lanthanum-tryptophan complexes.

Collision-induced charge disproportionation limits the stability of triply charged metal ion complexes and has thus far prevented successful acquisition of their gas-phase IR spectra. This has curtailed our understanding of the structures of triply charged metal complexes in the gas phase and in biological environments. Herein we report the first gas-phase IR spectra of triply charged La(III) complexes with a derivative of tryptophan (N-acetyl tryptophan methyl ester), and an unusual dissociation product, a lanthanum amidate. These spectra are compared with those predicted using density functional theory. The best structures are those of the lowest energies that differ by details in the π-interaction between La(3+) and the indole rings. Other binding sites on the tryptophan derivative are the carbonyl oxygens. In the lanthanum amidate, La(3+) replaces an H(+) in the amide bond of the tryptophan derivative.

Structure of the [M + H - H2O]+ ion from tetraglycine: a revisit by means of density functional theory and isotope labeling.

Collision-induced dissociations of protonated (18)O-labeled tetraglycines labeled separately at either the first or the second amide bond established that water loss from the backbone occurs from the N-terminal residue. Density functional theory at B3LYP/6-311++G(d,p) predicted that the low-energy [G(4) + H - H(2)O](+) product ion is an N(1)-protonated 3,5-dihydro-4H-imidazol-4-one. The ion at the lowest energy, III, is 24.8 kcal mol(-1) lower than the protonated oxazole structure, II, proposed by Bythell et al. (J. Phys. Chem A2010, 114, 5076-5082). In addition, structure III has a predicted IR spectrum that provides a better match with the published experimental IRMPD spectrum than that of structure II.

Performance and attributes of liquid chromatography-mass spectrometry with targeted charge separation in quantitative analysis of therapeutic peptides.

Herein we describe a new method, targeted enhanced multiply charged scans (tEMC), for the quantification of therapeutic peptides in tandem mass spectrometry on the linear ion trap mass spectrometer. Therapeutic peptides with chain lengths between eight and 39 amino acid residues and charge states from 2+ to 6+ were used to evaluate and illustrate the method which relies on the ability to separate ions trapped in a linear ion trap according to their charges. In particular, interference from singly charged ions on multiply charged ions can be effectively minimized. The method requires optimization of relatively few parameters, the most important of which being the exit lens barrier (EXB) voltage, thereby offering substantial time saving in a high-throughput quantification environment that currently relies on selected reaction monitoring.

Infrared irradiation in the collision cell of a hybrid tandem quadrupole/time-of-flight mass spectrometer for declustering and cleaning of nanoelectrosprayed protein complex ions.

Herein we report the performance of a hybrid quadrupole time-of-flight tandem mass spectrometer with an improved designed for coaxial infrared laser introduction for the characterization and dissociation of large protein complex ions and their aggregates formed under nanoelectrospray ionization. The major improvement from the original design (Raspopov, S. A.; El-Faramawy, A.; Thomson, B. A.; Siu, K. W. M. Anal. Chem. 2006, 78, 4572-4577) involves the use of a hollow silica waveguide and physical isolation of the infrared laser. Large model protein complex ions and their aggregates examined include alcohol dehydrogenase, avidin, GroEL, and others. Gentle heating of these complexes with the infrared laser facilitated declustering and resulted in better resolved mass spectral peaks and more accurate molecular-weight measurements.

Ultraviolet photodissociation of protonated pharmaceuticals in a pressurized linear quadrupole ion trap.

Ultraviolet photodissociation (UVPD) was evaluated as a technique for generating ion fragmentation information that is alternative and/or complementary to the information obtained by collision-induced dissociation (CID). Ions trapped in a pressurized linear ion trap were dissociated using a 355 nm or a 266 nm pulsed laser. Comparisons of UVPD and CID spectra using a set of aromatic chromophore-containing compounds (desmethyl bosentan, haloperidol, nelfinavir) demonstrated distinct characteristic fragmentation patterns resulting from photodissociation. The wavelength of light and the pressure of the buffer gas in the UVPD cell are important parameters that control fragmentation pathways. The wavelength effect is related to the absorption cross section, location of the chromophore and the energy carried by one photon. Thus, UV irradiation wavelength affects fragmentation pathways as well as the fragmentation rate. The pressure effect can be explained by collisional quenching of 'slow' fragmentation pathways. We observed that higher pressure of the buffer gas during UVPD experiments highlights unique fragment ions by suppressing slow fragmentation pathways responsible for CID-like fragmentation patterns.

Bond dissociation energies of solvated silver(I)-amide complexes: competitive threshold collision-induced dissociations and calculations.

Using competitive threshold collision-induced dissociation (TCID) measurements, experimental bond dissociation energies have been evaluated for the water, methanol, and acetonitrile adducts of silver(I)-amide complexes. The influence of the solvent molecules on the binding energy of silver(I) to acetamide, N-methylacetamide, and N,N-dimethylacetamide was investigated. Experimental results show that solvents decrease the amide binding energy by 4-6 kcal mol(-1). Using density functional theory (DFT), binding energies were evaluated using nine functionals, after full geometry optimizations with the ECP28MWB basis set for silver and the 6-311++G(2df,2pd) basis set for the other atomic constituents of the ligands. In addition, calculations employing the DZVP basis set for Ag and DZVP2 for C, H, N, and O atoms at the B3LYP and MP2 levels of theory were used to investigate the influence of the basis set on the theoretical bond energies. A comparison of the experimental and theoretical silver(I)-ligand bond dissociation energies enables an assessment of the limitations in the basis sets and functionals in describing the energetics of the metal-solvent interaction and the metal-amide interaction. No single functional/basis set combination was found capable of predicting binding energies with a sufficiently high level of accuracy for the silver(I)-amide solvent complexes.

Threshold collision-induced dissociation measurements using a ring ion guide as the collision cell in a triple-quadrupole mass spectrometer.

A triple-quadrupole mass spectrometer has been modified for bond-dissociation energy measurements via threshold collision-induced dissociations (TCIDs) by replacing the conventional collision cell with a ring ion guide. Optimal operating conditions for the ring ion guide were determined or derived, and validated using a set of complexes for which bond dissociation energies are known. A comparison with reference data (within a range of 16-57 kcal/mol) indicates an accuracy approaching that of TCID determined on a guided ion-beam mass spectrometer. Complexes for which bond-dissociation energies were measured include metal ion complexes of simple ligands, amino acids and peptides, as well as of carbonic acid. There is excellent agreement between our experimental data and literature data, as well as theoretical data determined using a high-level computational method.

Conformation switching in gas-phase complexes of histidine with alkaline earth ions.

Infrared multiple photon dissociation spectroscopy of gas-phase doubly charged alkaline earth complexes of histidine reveals a transition from dominance of the zwitterion (salt bridge, SB) conformation with Ba2+ to substantial presence of the canonical (charge-solvated, CS) conformation with Ca2+. This result is a clear illustration of the importance of metal-ion size in governing the delicate balance between these two modes of complexation of gas-phase amino acids. The two conformational motifs are clearly distinguished by characteristic spectral features, confirmed by density functional theory simulated IR spectra of the low-energy conformers. As a further illustration of histidine complexation possibilities, the spectrum of the Na+His complex shows purely CS character and emphasizes the greater tendency toward SB character induced by the higher charge in the alkaline earth complexes. Calculation of the complete series of alkaline earth/histidine complexes confirms the increasing stability of the SB conformations relative to CS with increasing metal ion size, as well as showing that among SB conformations the most highly chelated conformation (SB3) is favored for small metals, whereas the most extended conformation (SB1) is favored for large metals. A decomposition of the binding thermochemistry shows that these thermochemical trends versus metal-ion size are due to differences in electrostatic binding energies, with relatively little contribution from the deformation and rearrangement energy costs of distorting the ligand framework.

Electrospray: from ions in solution to ions in the gas phase, what we know now.

There is an advantage for users of electrospray and nanospray mass spectrometry to have an understanding of the processes involved in the conversion of the ions present in the solution to ions in the gas phase. The following processes are considered: Creation of charge droplets at the capillary tip; Electrical potentials required and possibility of gas discharges; Evolution of charged droplets, due to solvent evaporation and Coulomb explosions, to very small droplets that are the precursors of the gas phase ions; Production of gas phase ions from these droplets via the Ion Evaporation and Charge residue models; Analytical uses of ESIMS of small ions, qualitative and quantitative analysis; Effects of the ESI mechanism on the analysis of proteins and protein complexes; Determination of stability constants of protein complexes; Role of additives such as ammonium acetate on the observed mass spectra.

Binding energies of the silver ion to alcohols and amides: a theoretical and experimental study.

The silver ion binding energies to alcohols (methanol, ethanol, n-propanol, i-propanol, and n-butanol) and to amides (acetamide, N-methylacetamide, N, N-dimethylacetamide, formamide, N-methylformamide, and N, N-dimethylformamide) have been calculated using density functional theory (DFT) and measured using the threshold collision-induced dissociation (TCID) method. For DFT, the combined basis sets of ECP28MWB for silver and 6-311++G(2df,2pd) for the other atoms were found to be optimal using a series of test calculations on Ag (+) binding to methanol and to formamide. In addition, the Ag (+) binding energies of all ligands were evaluated with nine functionals after full geometric optimizations. TCID binding energies were measured using a triple quadrupole mass spectrometer. Reasonable to good agreements were obtained between the calculated and experimental silver(I) binding energies. Ligation of Ag (+) to the alcohols was primarily via the oxygen, although n-propanol and n-butanol exhibited additional, bidentate coordination via the CH hydrogens. By contrast, silver(I) binding to the amides was all monodentate via the carbonyl oxygen. There appears to be strong correlations between the binding energies and the polarizabilities of the ligands.

Histidine, lysine, and arginine radical cations: isomer control via the choice of auxiliary ligand (L) in the dissociation of [CuII(L)amino acid]*2+ complexes.

Histidine, lysine, and arginine radical cations have been generated through collision-induced dissociation (CID) of complexes [CuII(auxiliary ligand)namino acid]*2+, using tri-, bi-, as well as monodentate auxiliary ligands. On the basis of the observed CID products, the existence of two isomeric amino-acid populations is postulated. The Type 1 radical cations of histidine and lysine, stable on the mass spectrometer time scale, were found to lose water, followed by the loss of carbon monoxide under more energetic CID conditions. The arginine Type 1 radical cation behaved differently, losing dehydroalanine. The Type 2 radical cations were metastable and easily fragmented by the loss of carbon dioxide, effectively preventing direct observation. Type 1 radical cations are proposed to result from neutral (canonical) amino-acid coordination, whereas Type 2 radical cations are from zwitterionic amino-acid coordination to copper in the complex. The ratio of Type 1/Type 2 ions was found to be dependent on the auxiliary ligand, providing a method of controlling which radical cation would be formed primarily. Density functional calculations at B3LYP/6-311++G(d,p) have been used to determine the relative energies of five His*+ isomers. Barriers against interconversion between the isomers and against fragmentation have been calculated, giving insight as to why the Type 1 ions are stable, while only fragmentation products of the Type 2 ions are observable under CID conditions.

Gas-phase fragmentation of protonated benzodiazepines.

Protonated 1,4-benzodiazepines dissociate in the gas phase by the common pathway of CO elimination and by unique pathways dictated by the substituents; the latter typically differentiate one benzodiazepine from another. Protonated 3-dihydro-5-phenyl-1,4-benzodiazepin-2-one, the base diazepam devoid of substituents, dissociates by eliminating CO, HNCO, benzene, and benzonitrile. Mechanisms of these reactions are proposed with ionic products being resonance stabilized. The abundant [MH-CO]+ ion dissociates to secondary products via elimination of benzene, benzonitrile, the NH2 radical, and ammonia, yielding again ionic products that are stabilized by resonance.

Stable gas-phase radical cations of dimeric tryptophan and tyrosine derivatives.

Stable radical cations of dimeric amino acid derivatives of tryptophan and tyrosine were generated by collision-induced dissociation of [Cu(II)(diethylenetriamine)(amino acid derivative)2]*2+. The yields of the dimer radical cations were dependent on both the auxiliary ligand and the tryptophan or tyrosine derivatives used. Amino acid derivatives with an unmodified carboxylic acid group did not generate dimer radical cations. For the amino acid derivatives Ac-Trp-OMe and Ac-Trp-NH2 (Ac is N-acetyl; OMe and NH2 are the methyl ester and amide modifications of the C-terminal carboxylic group), no auxiliary ligand was required for generating the dimer radical cations. Collision-induced dissociation of the [Cu(II)(amino acid derivative)4]*2+ precursor generated the dimer radical cation [(amino acid derivative)2]*+. Stabilizing interactions, most likely involving hydrogen bonding, between the two amino acid derivatives are proposed to account for observation of the dimer radical cations. Dissociation of these ions yields protonated or radical cationic amino acid derivatives; these observations are consistent with the expectation of proton competition between monomeric units, whose proton affinities were calculated using density functional theory.

Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues.

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.

Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method.

Several factors, attributable to the ESIMS mechanism, that can affect the assumptions of the titration method are examined: (1) The assumption that the concentrations in solution of the protein P, the ligand L, and the complex PL are proportional to the respective ion intensities observed with ESIMS, is examined with experiments in which ion intensities of two non-interacting proteins are compared with the respective concentrations. The intensities are found to be approximately proportional to the concentrations. The proportionality factors are found to increase as the mass of the protein is decreased. Very small proteins have much higher intensities. The results suggest that it is preferable to use only the intensity ratio of PL and P, whose masses are very close to each other when L is small, to determine the association constant KA in solution. (2) From the charge residue model (CRM) one expects that the solution will experience a very large increase of concentration due to evaporation of the precursor droplets, before the proteins P and PL are produced in the gas phase. This can shift the equilibrium in the droplets: P + L = PL, towards PL. Analysis of the droplet evaporation history shows that such a shift is not likely, because the time of droplet evolution is very short, only several micros, and the equilibrium relaxation time is much longer. (3) The droplet history shows that unreacted P and L can be often present together in the same droplet. On complete evaporation of such droplets L will land on P leading to PL and this effect will lead to values of KA that are too high. However, it is argued that mostly accidental, weakly bonded, complexes will form and these will dissociate in the clean up stages (heated transfer capillary and CAD region). Thus only very small errors are expected due to this cause. (4) Some PL complexes may have bonding that is too weak in the gas phase even though they have KA values in solution that predict high solution PL yields. In this case the PL complexes may decompose in the clean up stages and not be observed with sufficient intensity in the mass spectrum. This will lead to KA values that are too low. The effect is expected for complexes that involve significant hydrophobic interaction that leads to high stability of the complex in solution but low stability in the gas phase. The titration method is not suited for such systems.