Electron capture, collision-induced, and electron capture-collision induced dissociation in Q-TOF.

Recently, we demonstrated that a radio-frequency-free electromagnetostatic (rf-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell (Voinov et al., Rapid Commun Mass Spectrom 22, 3087-3088, 2008; Voinov et al., Anal Chem 81, 1238-1243, 2009). Subsequently, we used our rf-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides (Voinov et al., Rapid Commun Mass Spectrom 23, 3028-3030, 2009). In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.

High resolution mass analysis of N- and C-terminal negative ions resulting from resonance electron capture by aliphatic amino acids.

High mass resolving power was applied to study resonance electron capture by glycine, alanine, and valine, and accurate mass measurements helped to distinguish between some negative ions having the same nominal masses. It was established that the C- and N-terminal negative ions of the same nominal masses were formed at different electron energies from different resonance states. The typical fragmentation pathways in deprotonated amino acids via loss of water initiated by collisional activation were not observed upon resonant electron capture by aliphatic amino acids. Instead, [M-18](-) negative ions in the vicinity of 5 eV were found to be associated with simultaneous loss of either ammonia and a hydrogen atom or an amino group and a hydrogen molecule.

Resonance electron capture by serine.

Formation of negative ions via dissociative electron attachment (DEA) to the amino acid serine in the gas phase was studied using two different crossed electron/molecular beam techniques and quantum chemical calculations. Resonance electron capture mass spectrum and effective ion yield curves of 16 negative ions were measured over the electron energy range from close to 0 to 11 eV. The negative ions from serine were detected from resonance states in the vicinity of 0, 1.3, 5, and 8 eV. The dominant reaction channel at low electron energies was (M-H)(-). The relative cross section for this ion exceeds more than 20 times that of any other fragment negative ions. A high-resolution experiment was applied to study fine structures in (M-H)(-) cross section. We have found that the second OH group influences some dissociative channels. Quantum chemical calculations were applied to interpret products of the DEA reaction channels.

Fragmentation of peptide negative molecular ions induced by resonance electron capture.

A simple robust method to study resonance gas-phase reactions between neutral peptides of low volatility and free electrons has been designed and implemented. Resonance electron capture (REC) experiments were performed by several neutral model peptides and two naturally occurring peptides. The assignment of negative ions (NIs) formed in these gas-phase reactions was based on high mass-resolving power experiments. From these accurate mass measurements, it was concluded that fragment NIs formed by low (1-2 eV) energy REC are of the same types as those observed in electron capture/transfer dissociation, where the positive charge is a factor. The main feature resulting from these REC experiments by peptides is the occurrence of z(n)-1 ions, which are invariably of the highest abundances in the negative ion mass spectra of larger peptides. [M-H](-) NIs presumably the carboxylate anion structure dominate the REC spectra of smaller peptides. There was no evidence for the occurrence of the complementary reaction, i.e., the formations of c(n)+1 ions. Instead, c(n) ions arose without hydrogen/proton transfer albeit with lower abundances than that observed for z(n)-1 ions. Only the amide forms of small peptides showed more abundant ion peaks for the c(n) ions than for the z(n)-1 ions. The mechanisms for the N-C(alpha) bond cleavage are discussed.

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis.

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.

Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.

The retinoid X receptor (RXR) is a ligand-activated transcription factor that plays an important role in growth and development and the maintenance of cellular homeostasis. A thermodynamic ultraviolet circular dichroism, tryptophan fluorescence and ligand binding activity with guanidine as a chemical denaturant are consistent with a two step mechanism. The dimeric LBD equilibrates with a monomeric intermediate (DeltaG(0)(H(2)O) equal to 8.3 kcal/mol) that is in equilibrium with the unfolded state (DeltaG(0)(H(2)O) equal to 2.8 kcal/mol). The intermediate was characterized by analytical ultracentrifugation, spectroscopy, and collisional fluorescence quenching, which imply that the monomeric intermediate maintains a high degree, but not all, of native secondary structure. Although intrinsic fluorescence from native and intermediate suggests little change in tryptophan environments, fluorescence intensities from fluorescein reporter groups differ significantly between the two structures. Analysis of the collisional quenching results imply that the intermediate is characterized by tryptophans with increased accessibility to small solutes and less overall compactness than the native protein.

Radio-frequency-free cell for electron capture dissociation in tandem mass spectrometry.

A radio frequency-free (RFF), analyzer-independent cell has been devised for electron-capture dissociation (ECD) of ions. The device is based on interleaving a series of electrostatic lenses with the periodic structure of magnetostatic lenses commonly found in a traveling wave tube. The RFF electrostatic/magnetostatic ECD cell was installed in a Finnigan TSQ700 ESI triple quadrupole (QqQ) spectrometer, and its performance was evaluated by recording product-ion spectra of doubly protonated substance P, doubly protonated gramicidin S, doubly protonated neurotensin, and triply protonated neurotensin. These spectra were readily obtained without recourse to a buffering gas or synchronizing electron injection with a specific phase of an RF field. The mass spectra produced with the modified instrument appear in all respects (other than resolution and mass accuracy, which were limited by the mass spectrometer used) to be at least as good for purposes of peptide identification as those recorded with Fourier transform ion cyclotron resonance (FT ICR) instruments; however, the effort and time to produce the mass spectra were much less than required to produce their FT ICR counterparts. The cell's design and compact construction should allow it to be incorporated at relatively little cost into virtually any type of tandem mass spectrometer, for example, triple quadrupole, hybrid quadrupole ion trap, hybrid quadrupole time-of-flight, or even FT-ICR.

The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry.

Comprehensive mass spectral fragmentation patterns have been established for sequencing chromatographically isolated A-type proanthocyanidins (PAs) using electrospray ionization tandem mass spectrometry (ESI-MS(n)) in the positive ion mode similar to those used for sequencing previously reported B-type PAs. Sequence-identifying fragmentations for A-type PAs include heterocyclic ring fission (HRF), retro-Diels-Alder (RDA) fission, benzofuran-forming (BFF) fission, and quinone methide (QM) fission. There is commonality in fragmentation patterns between A-type and B-type PAs, but distinguishing features in the mass spectral patterns between the two classes include 2-Da mass differences in the pseudo molecular ions, the propensity for the A-type PAs to undergo QM fissions and yield bis-quinoid ions as opposed to mono-quinoid ions in the upper unit of the sequence, and the reluctance of A-type linkages to undergo RDA, BFF, and BFF/H(2)O fissions from the upper unit. The positions of one or more A-type (C2-->O-->C7') ether linkages have been located in sequences of PAs ranging in chain lengths of two to five monomer units using ESI-MS(n) data. Using the fragmentation information from ESI-MS(n) experiments, a total of 17 PAs were structurally sequenced by systematic real time ESI-MS(n). Among them ten A-type and six B-type hop PAs are reported here for the first time.

Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRalpha LBD.

Protein amide hydrogen/deuterium (H/D) exchange was used to compare the interactions of two antagonists, UVI 2112 and UVI 3003, with that of the agonist, 9-cis-retinoic acid, upon binding to the human retinoid X receptor alpha ligand-binding domain (hRXRalpha LBD) homodimer. Analysis of the H/D content by mass spectrometry showed that in comparison to 9-cis-retinoic acid, the antagonists provide much greater protection toward deuterium exchange-in throughout the protein, suggesting that the protein-antagonist complex adopts a more restricted conformation or ensemble of conformations in which solvent accesses to amide protons are reduced. A comparison between the two antagonists shows that UVI 3003 is more protective in the C-terminal region due to the extra hydrophobic interactions derived from the atoms in the benzene ring of the carboxylic acid chain. It was less protective within regions comprising peptides 271-278 and 326-330 due to differences in conformational orientation, and/or shorter carboxylic acid chain length. Decreased deuterium exchange-in in the segment 234-239 where the residues do not involve interactions with the ligand was observed with the two antagonists, but not with 9-cis-RA. The amide protons of helix 12 of the agonist- or antagonist-occupied protein in solution have the same deuterium exchange rates as the unliganded protein, supporting a suggestion made previously that helix 12 can cover the occupied binding cavity only with the cofactor present to adjust its location.

Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry.

alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.

Solvent-free MALDI-MS for the analysis of beta-amyloid peptides via the mini-ball mill approach: qualitative and quantitative advances.

Manual and automated solvent-free mini-ball mill (MBM) matrix-assisted laser desorption/ionization (MALDI) analysis of mixtures of beta-amyloid peptides (1-11), (33-42), (1-42) and non-beta-amyloid component of Alzheimer's disease peptide yielded interpretable spectra for all of the peptides present regardless of their relative amounts in the samples. This was not the case for solvent-based MALDI analysis using traditional acidic aqueous/organic solvent conditions, which resulted in severe over-representation of hydrophilic peptide (1-11) and provided no spectra for insoluble amphiphilic peptide (1-42) even when present at 50% relative molar amount. Less accurate representation of components in mixtures by the traditional method appears to be a combination of poor dissolution of peptides in the solvent and preferential ionization of more hydrophilic peptides in the mixture. Consequently, only MBM provided a complete tryptic map of beta-amyloid (1-42) compared to 67% coverage by traditional MALDI. Acetonitrile (0.1% TFA) led to improved coverage only at a 50% molar ratio of peptide (1-42), but also to a side product of (1-42), Met oxidation (amino acid 35), a phenomenon not observed in MBM MALDI analysis. Traditional MALDI analysis resulted in over-representation of hydrophilic soluble beta-amyloid (1-11) in defined mixtures and autoproteolytic peptides of trypsin. In contrast, over-representation and under-representation were less pronounced in solvent-free MALDI in all of the investigated cases. Analysis of defined peptide and tryptic peptide mixtures showed that MBM MALDI yielded greater qualitative reliability, which also improved quantitative response relative to the solvent-based approach.

Charge-remote metastable ion decomposition of free fatty acids under FAB MS: evidence for biradical ion structures.

Classical charge-remote fragmentation (CRF) of a series of long-chain saturated and monounsaturated fatty acid anions, a well-known phenomenon under collisional activation conditions, is observed for the first time during fast atom bombardment of the analyte-matrix mixture without collisional activation. The process is efficient enough to allow collision-induced dissociation and metastable ion decomposition MS/MS spectra of any charge-remote [M-H2-(CH2)n]- fragments as well as spectra of neutral losses to be recorded. The results obtained are in contradiction to the generally accepted theory that CRF results exclusively in terminally unsaturated carboxylate anions. The new results indicate that a multistep radical mechanism is involved in CRF ion formation. The first step of the process appears to be accompanied by hydrogen elimination that occurs randomly throughout the molecule. The primary fragment radical ions formed can decompose further with the formation of the next generation of CRF ions.

Tandem mass spectrometry for sequencing proanthocyanidins.

Proanthocyanidins (PAs) are a group of bioflavonoids consisting of oligomers based on catechin monomeric units. These polyphenolic compounds are widely distributed in higher plants and are an integral part of the human diet. A sensitive LC-tandem mass spectrometric (LC/ESI-MS(n)) method in the positive ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described. The hydroxylation patterns and interflavanoid linkage for A- and B-type PAs were determined by fragment ions derived from a retro-Diels-Alder (RDA) fission, heterocyclic ring fission (HRF), a novel benzofuran-forming (BFF) fission described here for the first time, and a quinone methide (QM) fission. The subunit sequence of the PAs was determined by diagnostic ions derived from HRF/RDA fission, HRF/BFF fission, and RDA/HRF fission together with QM fission. A total of 26 PAs were reliably sequenced by the newly established tandem mass spectrometric protocol. It is shown that the protocol based on a combination of these different fragmentation patterns allows for uniquely identifying PA oligomers.

Distinguishing between cis/trans isomers of monounsaturated fatty acids by FAB MS.

Fast-atom bombardment (FAB) mass spectrometry in the negative ion mode can be used to unambiguously distinguish between cis and trans isomers of monounsaturated fatty acids by the relative signal strengths of an intense pair of ion signals. Under normal FAB ionization/desorption conditions, the deprotonated molecules (i.e., [M - H]-) of six fatty acids underwent charge remote fragmentation. A characteristic fragmentation pattern of two intense peak clusters of peaks with three weak intervening clusters of peaks are used in each case to identify the position of the double bond. The possibility of resonance electron capture occurring during the FAB process is discussed.

Resonant electron capture by some amino acids esters.

Resonant electron capture by Gly, Ala and Phe esters have shown that the most efficient negative ion (NI) fragmentations are associated with the C-termini. A new mechanism for the negative ion-forming processes at energies lower than those associated with the pi*(OO) shape resonance involves coupling between dipole-bound and valence negative ion states of the same symmetry for amino acid conformers with high permanent dipoles. The interaction avoids crossing of the NI states and instead leads to formation of two adiabatic potential energy surfaces. Underivatized amino acids most effectively fragment from the bottom adiabatic surface via generation of [M-H](-) carboxylate anions by hydrogen-atom tunneling through the barrier; fragmentation of the their esters with formation of analogues [M-X](-) NIs occurs through the upper adiabatic state without penetration of the barrier in which the energy of the valence sigma*OX resonance exceeds the bond dissociation energy of the neutral molecule. Low and high temperature resonant electron capture experiments point to the importance of conformational preferences of the amino acids for optimum dissociation of the parent NIs in the gas phase.

Solvent-free MALDI-MS for the analysis of a membrane protein via the mini ball mill approach: case study of bacteriorhodopsin.

A mini ball mill (MBM) solvent-free matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method allows for the analysis of bacteriorhodopsin (BR), an integral membrane protein that previously presented special analytical problems. For well-defined signals in the molecular ion region of the analytes, a desalting procedure of the MBM sample directly on the MALDI target plate was used to reduce adduction by sodium and other cations that are normally attendant with hydrophobic peptides and proteins as a result of the sample preparation procedure. Mass analysis of the intact hydrophobic protein and the few hydrophobic and hydrophilic tryptic peptides available in the digest is demonstrated with this robust new approach. MS and MS/MS spectra of BR tryptic peptides and intact protein were generally superior to the traditional solvent-based method using the desalted "dry" MALDI preparation procedure. The solvent-free method expands the range of peptides that can be effectively analyzed by MALDI-MS to those that are hydrophobic and solubility-limited.

Investigation of ligand interactions with human RXRalpha by hydrogen/deuterium exchange and mass spectrometry.

Several different agonists of the retinoic X receptor alpha (hRXRalpha) were examined for their effects on the amide H/D exchange kinetics of the homodimeric protein using mass spectrometry. Some agonists, LG 100268, SR11246, and DHA, bind such that slower deuterium exchange-in occurs compared with 9-cis-retinoic acid (9-cis-RA), whereas others, fenretinide and methoprenic acid, result in poorer protection during binding and hence faster exchange-in. Protection against H/D exchange by different agonists and the inhibition of H/D exchange kinetics relative to 9-cis-RA varies markedly in different regions of the protein. Agonists LG 100268, SR11246, and DHA generally inhibit faster exchange processes in the ligand binding regions of hRXRalpha than does the native ligand 9-cis-RA. In at least half of these regions, the level of protection by 9-cis-RA lags behind the agonists even after 60 min. Methoprenic acid did not significantly protect hRXRalpha against amide hydrogen exchange. An efficient method is described for comparing the effects of different agonists on the protein structure of the agonist-RXRalpha complex.

Structural identification and distribution of proanthocyanidins in 13 different hops.

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4alpha-->8)-catechin, gallocatechin-(4alpha-->6)-catechin, catechin-(4alpha-->8)-gallocatechin, catechin-(4alpha-->6)-gallocatechin, afzelechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-catechin-(4alpha-->8)-catechin, epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin, catechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, and gallocatechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4beta-->8)-catechin, epicatechin-(4beta-->8)-epicatechin, catechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-epicatechin, and epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.