Characterization of the pH-dependent dissociation of a multimeric metalloprotein Streptomyces rubiginosus xylose isomerase by ESI FT-ICR mass spectrometry

We report an analysis of the pH-dependent dissociation of a multimeric metalloprotein, xylose isomerase from Streptomyces rubiginosus (XI), by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Xylose isomerases are industrially significant enzymes that catalyze interconversion of aldose and ketose sugars. XI is biologically active as a approximately 173-kDa tetrameric complex, comprised of four identical approximately 43-kDa subunits and eight metal cations, unequivocally identified as the Mg(2+) cations in this work. ESI FT-ICR mass spectra of XI measured in the pH range of 3.0-6.9 indicated that the dissociation of the intact holo-tetramer is initiated by the loss of all eight Mg(2+) cations at pH 相似文献   

Fragmentation study of peptides using Fourier transform ion cyclotron resonance with infrared multiphoton dissociation: experiment and simulation

In this study, the fragmentation of gas-phase protonated Angiotensin II is investigated using electrospray ionization (ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and mass spectrometry (MS) with a laser cleavage infrared multiphoton dissociation (IRMPD) technique. The experimental results show that the spectra peaks for the photoproducts are y2/b6- and y7-type ions, corresponding to the cleavage of His-Pro and Asp-Arg in the parent amino acid sequence. The fragmentation of the peptide under collision-free vacuum conditions is modeled using molecular dynamics simulations (MD). The binding energy for the peptide bonds (C'-N bond) of Angiotensin II is estimated from ab initio calculations. The calculations are directed at predicting experimental measurements of the product ions from the photodissociation of the peptide. The product distributions simulated by the MD dissociation trajectories include predominantly y7/b1 and y2/b6 pair ions.     

Encapsulation of metal cations by the PhePhe ligand: a cation-π ion cage

Structures and binding thermochemistry are investigated for protonated PhePhe and for complexes of PhePhe with the alkaline-earth ions Ba(2+) and Ca(2+), the alkali-metal ions Li(+), Na(+), K(+), and Cs(+), and the transition-metal ion Ag(+). The two neighboring aromatic side chains open the possibility of a novel encapsulation motif of the metal ion in a double cation-π configuration, which is found to be realized for the alkaline-earth complexes and, in a variant form, for the Ag(+) complex. Experimentally, complexes are formed by electrospray ionization, trapped in an FT-ICR mass spectrometer, and characterized by infrared multiple photon dissociation (IRMPD) spectroscopy using the free electron laser FELIX. Interpretation is assisted by thermochemical and IR spectral calculations using density functional theory (DFT). The IRMPD spectrum of protonated PhePhe is reproduced with good fidelity by the calculated spectrum of the most stable conformation, although the additional presence of the secondmost stable conformation is not excluded. All metal-ion complexes have charge-solvated binding modes, with zwitterion (salt bridge) forms being much less stable. The amide oxygen always coordinates to the metal ion, as well as at least one phenyl ring (cation-π interaction). At least one additional chelation site is always occupied, which may be either the amino nitrogen or the carboxy carbonyl oxygen. The alkaline-earth complexes prefer a highly compact caged structure with both phenyl rings providing cation-π stabilization in a "sandwich" configuration (OORR chelation). The alkali-metal complexes prefer open-cage structures with only one cation-π interaction, except perhaps Cs(+). The Ag(+) complex shows a unique preference for the closed-cage amino-bound NORR structure. Ligand-driven perturbations of normal-mode frequencies are generally found to correlate linearly with metal-ion binding energy.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

Top-Down Mass Spectrometry of Supercharged Native Protein-Ligand Complexes

Tandem mass spectrometry (MS/MS) of intact, noncovalently-bound protein-ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging.     

Intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to FT-ICR mass spectrometry

We report the implementation of an infrared laser onto our previously reported matrix-assisted laser desorption electrospray ionization (MALDESI) source with ESI post-ionization yielding multiply charged peptides and proteins. Infrared (IR)-MALDESI is demonstrated for atmospheric pressure desorption and ionization of biological molecules ranging in molecular weight from 1.2 to 17 kDa. High resolving power, high mass accuracy single-acquisition Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were generated from liquid- and solid-state peptide and protein samples by desorption with an infrared laser (2.94 μm) followed by ESI post-ionization. Intact and top-down analysis of equine myoglobin (17 kDa) desorbed from the solid state with ESI post-ionization demonstrates the sequencing capabilities using IR-MALDESI coupled to FT-ICR mass spectrometry. Carbohydrates and lipids were detected through direct analysis of milk and egg yolk using both UV- and IR-MALDESI with minimal sample preparation. Three of the four classes of biological macromolecules (proteins, carbohydrates, and lipids) have been ionized and detected using MALDESI with minimal sample preparation. Sequencing of O-linked glycans, cleaved from mucin using reductive β-elimination chemistry, is also demonstrated.     

Studies on Fragmentation of Peptide by Nano-electrospray Ionization Fourier Transform Ion Cyclotron Resonance Multi-stage Tandem Mass Spectrometry

The sequence analysis of peptides was performed by nano-electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry(Nano-ESI-FT-ICR-MSn) and several peptides were chosen as examples. With the aid of the collision induced dissociation(CID), FT-ICR provides not only precise mass/charge ratio, but also structure information of the selected peptides. The fragment ions were identified according to the observed molecular weights and peptide sequence was determined successfully. So Nano-ESI-FT-ICR-MSn is a useful tool for identification of the amino acid sequence of peptides with high confidence. Besides, a pathway for the dehydration of y ions without amino acids containing carboxylic acid under sustained off-resonance irradiation collision-induced dissociation(SORI-CID) condition was proposed.     

Fourier transform-ion cyclotron resonance mass spectrometric resolution,identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library

The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent.     

Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Study on Sodium Azide Cluster Ions

Introduction Mass spectrometry (MS) has been an important technique for studying size-dependent cluster properties with unparalleled sensitivity, specificity, and versatil-ity.1,2 Especially Fourier transform ion cyclotron reso-nance (FT-ICR) MS offers the benefits of ultra-high mass resolving power, superior mass accuracy, and the ability of affording useful structure information with tandem MS. The introduction of electrospray ionization (ESI) as a gentle mode of ion generation for MS…     

Fourier transform ion cyclotron resonance study of multiply charged aggregates of small singly charged peptides formed by electrospray ionization

Aggregates of singly protonated peptides formed with a nanoelectrospray ion source have been observed in the gas phase using Fourier transform ion cyclotron resonance (FT-ICR). Employment of “soft” ion sampling conditions in the source, which were developed previously to generate water clusters of biomolecules, provides significant yields of aggregates of singly protonated GGDPG ([2GGDPG + 2H]²⁺), GGEPG ([2GGEPG + 2H]²⁺), and VEPIPY (2VEPIPY + 2H]²⁺). With peptide mixtures, heteroaggregates, e.g., [GGDPG + GGEPG + 2H]²⁺ have also been observed along with the homoaggregates. These weakly bound noncovalent complexes undergo facile exothermic dissociation into the corresponding singly protonated monomer species with normal operation of the electrospray ion source. For example, the aggregates were not observed in FT-ICR experiments utilizing a conventional electrospray ionization (ESI) or fast atom bombardment source or with a quadrupolar ion trap mass spectrometer equipped with a conventional ESI source. The formation and metastability of these aggregates are dependent on highly specific intermolecular hydrogen bonding between the monomers. The amino acid sequence (DPG) of GGDPG mimics the well-known β reverse turn of proteins and semiempirical calculations show that it provides excellent hydrogen bonding sites for a protonated N-terminus amino group. Support for this conjecture is provided by the failure to observe aggregate formation of singly protonated peptides with several larger peptides, including hexaglycine and hexaalanine.     

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1‐azabicyclo[2.2.2]octane (ABCO) or 1,4‐diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI‐MS) and longer retention times on the reverse‐phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision‐induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a‐ and b‐type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision‐induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI‐MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of heavy petroleum fraction by positive-ion electrospray ionization FT-ICR mass spectrometry and collision induced dissociation: Bond dissociation behavior and aromatic ring architecture of basic nitrogen compounds

This paper examined the bond dissociation behavior and aromatic ring architecture of basic nitrogen compounds in Sudan heavy petroleum fraction. Both broadband and quadrupole isolation modes positive-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with collision induced dissociation (CID) techniques were used to characterize a low sulfur crude oil derived vacuum residuum (VR). The appropriate CID operating condition was selected by comparing the molecular weight distributions of the basic nitrogen compounds under various CID operating conditions. Both oddand even-electron fragment ions were observed from the mass spectrum, indicating that the heterolytic and homolytic bond cleavages occurred simultaneously during the CID process. The odd-electron fragment ions were predominant in each class species, indicating preferential heterolytic bond cleavages. At the optimal CID condition, the alkyl groups decomposed deeply and just left the aromatic cores of the nitrogen compounds. No significant variation in double bond equivalent (DBE) value was observed between the fragment and parent ions, revealing that the domination of single core structure.     

Speciation of nitrogen containing aromatics by atmospheric pressure photoionization or electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

We determine the elemental compositions of aromatic nitrogen model compounds as well as a petroleum sample by atmospheric pressure photoionization (APPI) and electrospray Ionization (ESI) with a 9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. From the double-bond equivalents calculated for the nitrogen-containing ions from a petroleum sample, we can infer the aromatic core structure (pyridinic versus pyrrolic nitrogen heterocycle) based on the presence of M(+.) (odd-electron) versus [M+H](+) (even-electron) ions. Specifically, nitrogen speciation can be determined from either a single positive-ion APPI spectrum or two ESI (positive- and negative-ion) spectra. APPI operates at comparatively higher temperature than ESI and also produces radical cations that may fragment before detection. However, APPI fragmentation of aromatics can be eliminated by judicious choice of instrumental parameters.     

Structure of electron-capture dissociation fragments from charge-tagged peptides probed by tunable infrared multiple photon dissociation

In this study, we propose the first spectroscopic structural characterization of c-type ions produced by ECD of a peptide. The structure of c-type ions formed by electron capture dissociation and the overall mechanism leading to their formation are still a question of debate. Depending on the mechanism, c-type ions have been proposed to have either an enol-imine structure (-C(OH)NH) or an amide one (-C(O)-NH2). Since these ions are isomeric, mass spectrometry only cannot discriminate between them, but infrared spectroscopy can bring experimental evidence and help determine which scheme is operative. Using the coupling between a tunable free electron laser and a FT-ICR mass spectrometer, we show that c-type ions have an amide structure, characterized by an IR signature of the C=O stretch at 1731 cm(-1). This result is particularly interesting from the perspective of the elucidation of the ECD mechanism.     

Energetics and dynamics of peptide fragmentation from multiple-collision activation and surface-induced dissociation studies

This account summarizes the energetics and dynamics of peptide fragmentation obtained using a new approach recently developed in our laboratory. The approach involves RRKM modeling of time- and energy- resolved tandem mass spectrometry (MS/MS) data obtained using collisional activation. We demonstrate that surface-induced dissociation (SID) on a long time-scale of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is perfectly suited for studying the energetics and dynamics of peptide fragmentation. The advantages provided by SID include very fast ion activation, which eliminates possible discrimination against higher-energy dissociation pathways, and efficient "amplification" of small changes in dissociation parameters. We present a summary of results obtained for small alanine-containing peptides as well as larger peptides including angiotensin analogs and a series of peptides containing the LDIFSDF motif.     

Mass spectrometric studies of the gas phase retro-<Emphasis Type="Italic">Michael</Emphasis> type fragmentation reactions of 2-hydroxybenzyl-<Emphasis Type="Italic">N</Emphasis>-pyrimidinylamine derivatives

The gas-phase fragmentation reactions of 2-hydroxybenzyl-N-pyrimidinylamine derivatives (Compounds 1 to 6), the O-N-type acid-catalyzed Smiles rearrangement products of 2-pyrimidinyloxy-N-arylbenzylamine derivatives, have been examined via positive ion matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation (IRMPD) mass spectrometry in FT-ICR MS and via negative ion electrospray ionization (ESI) in-source collision-induced dissociation (CID) mass spectrometry, respectively. The major fragmentation pathway of protonated 1 to 6 gives the F ions under IRMPD; theoretical results show that the retro-Michael reaction channel is more favorable in both thermodynamics and kinetics. This explanation is supported by H/D exchange experiments and the MS/MS experiment of acetylated 1. Deprotonated 1 to 6 give rise to the solitary E ions (aromatic nitrogen anions) in the negative ion in-source CID; theoretical calculations show that a retro-Michael mechanism is more reasonable than a gas-phase intramolecular nucleophilic displacement (SN2) mechanism to explain this reaction process.     

The effect of the secondary structure on dissociation of peptide radical cations: fragmentation of angiotensin III and its analogues

Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of Co (III)(salen)-peptide complexes. Both the energetics and the mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable toward fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation, while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.     

ESI‐MS and MALLS analysis of quaternary structure of molluscan hemocyanins

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI)) and multi‐angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK‐trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI‐mass spectrometry (MS) and MALDI‐MS, methods gave insights into post‐translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI‐MS and those calculated by their gene sequences. Copyright © 2012 John Wiley & Sons, Ltd.     

傅立叶变换离子回旋共振质谱及其研究进展

本文综述了傅立叶变换离子回旋共振质谱的基本原理及其颇具特色的数据处理方法.以两种典型的离子化方法为代表,介绍了目前主要使用的离子源,并总结了该方法在分析科学,尤其是在大分子分析、气相离子反应动力学研究和复杂体系分析等领域中的广泛应用.