Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Research Articles

Mechanistic Insights into the Biodegradation of Carbon Dots by Fungal Laccase.

While carbon dots (CDs) have the potential to support the agricultural revolution, it remains obscure about their environmental fate and bioavailability by plants. Fungal laccase-mediated biotransformation of carbon nanomaterials has received little attention despite its known capacity to eliminate recalcitrant contaminants. Herein, we presented the initial investigation into the transformation of CDs by fungal laccase. The degradation rates of CDs were determined to be first-order in both substrate and enzyme. Computational docking studies showed that CDs preferentially bonded to the pocket of laccase on the basal plane rather than the edge through hydrogen bonds and hydrophobic interactions. Electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS) and other characterizations revealed that the phenolic/amino lignins and tannins portions in CDs are susceptible to laccase transformation, resulting in graphitic structure damage and smaller-sized fragments. By using the 13C stable isotope labeling technique, we quantified the uptake and translocation of 13C-CDs by mung bean plants. 13C-CDs (10 mg L-1) accumulated in the root, stem, and leaf were estimated to be 291, 239, and 152 μg g-1 at day 5. We also evidenced that laccase treatment alters the particle size and surface chemistry of CDs, which could facilitate the uptake of CDs by plants and reduce their nanotoxicity to plants.

Detection of Polycyclic Aromatic Hydrocarbons in High Organic Carbon Ultrafine Particle Extracts by Electrospray Ionization Ultrahigh-Resolution Mass Spectrometry.

The detection of polycyclic aromatic hydrocarbons (PAHs) by electrospray ionization (ESI) without additional reagents or targeted setup changes to the ionization source was observed in ultrafine particle (UFP) extracts, with high organic carbon (OC) concentrations, generated by a combustion aerosol standard (CAST) soot generator. Particulate matter (PM) was collected on filters, extracted with methanol, and analyzed by ESI Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Next to oxygen-containing species, pure hydrocarbons were found to be one of the most abundant compound classes, detected as [M + Na]+ or [M + H]+ in ESI+ and mostly as [M - H]- in ESI-. The assigned hydrocarbon elemental compositions are identified as PAHs due to their high aromaticity index (AI > 0.67) and were additionally confirmed by MS/MS experiments as well as laser desorption ionization (LDI). Thus, despite the relatively low polarity, PAHs have to be considered in the molecular attribution of these model aerosols and/or fresh emissions with low salt content investigated by ESI.

Unsupervised Structural Classification of Dissolved Organic Matter Based on Fragmentation Pathways.

Dissolved organic matter (DOM) is considered an essential component of the Earth's ecological and biogeochemical processes. Structural information of DOM components at the molecular level remains one of the most extraordinary analytical challenges. Advances in determination of chemical formulas from the molecular studies of DOM have provided limited indications on structural signatures and potential reaction pathways. In this work, we extend the structural characterization of a wetland DOM sample using precursor and fragment molecular ions obtained by a sequential electrospray ionization-Fourier transform-ion cyclotron resonance tandem mass spectrometry (ESI-FT-ICR CASI-CID MS/MS) approach. The DOM chemical complexity resulted in near 900 precursors (P) and 24 000 fragment (F) molecular ions over a small m/z 261-477 range. The DOM structural content was dissected into families of structurally connected precursors based on neutral mass loss patterns (Pn-1 + F1:n + C) across the two-dimensional (2D) MS/MS space. This workflow identified over 1900 structural families of DOM compounds based on a precursor and neutral loss (H2O, CH4O, and CO2). The inspection of structural families showed a high degree of isomeric content (numerous identical fragmentation pathways), not discriminable with sole precursor ion analysis. The connectivity map of structural families allows for the visualization of potential biogeochemical processes that DOM undergoes throughout its lifetime. This study illustrates that integrating effective computational tools on a comprehensive high-resolution mass fragmentation strategy further enables the DOM structural characterization.

Surrogate formulation and molecular characterization of sulfur species in vacuum residues using APPI and ESI FT-ICR mass spectrometry

Vacuum residues (VR) are the bottom of the barrel products left after vacuum distillation of crude oils. VR are primarily used as feedstock for production of syn-gas and hydrogen via gasification; and heavy fuel oil (HFO) for use as fuel in power generation and shipping. However, VR contain relatively large amounts of sulfur (upto 8% by mass) and require the removal of varying amounts depending on the emission norms (eg. International Maritime Organization 2020 sulfur regulations). Understanding the fuel molecular structure and, in particular, the structure of sulfur species enables the adoption and optimization of suitable desulfurization strategies. In the present work, detailed molecular characterization of the sulfur species in VR was performed using positive ion atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). Ions possessing mass to charge (m/z) in the range of 100 to 1200 were detected using the ultra-high resolution instrument and were resolved into unique chemical formulas (CcHhSsNnOo). The assigned masses were then divided into molecular classes based on the presence of heteroatoms, and plots of carbon number versus double bond equivalency (DBE) were made for each molecular class. The molecular classes were further sub-divided based on the presence of sulfur families like sulfides (Su), thiophenes (Th), benzothiophenes (BT), dibenzothiophenes (DBT) and benzonaphthothiophene (BNT) and their derivatives. A single surrogate molecule that represents the average structure of the VR sample was then designed based on the average molecular parameters (AMP) obtained from APPI and ESI FT-ICR MS. Plausible core skeletal structures of VR were drawn from the average DBE value, and then a symmetrical, alkylated, polyaromatic sulfur heterocycles (PASH) molecule was formulated as the VR surrogate. A number of physical and thermo-chemical properties of the VR surrogate were then predicted using quantitative structure property relationships (QSPR). The VR surrogate proposed here will enable high-fidelity computational studies, including chemical kinetic modeling, property estimation, and emissions modeling.

Comparison of hydrophobic and amphiphilic fractions of dissolved organic matter from a water reservoir by Fourier transform ion cyclotron resonance mass spectrometry

Purpose Dissolved organic matter (DOM) composition is influenced by and modulates biogeochemical processes, yet DOM characterization techniques are challenged by its heterogeneous properties and structures. In this paper, ultrahigh electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is used to characterize previously isolated and well-characterized four DOM fractions from a water reservoir in the southwestern part of the USA.

Photooxygenation and gas-phase reactivity of multiply threaded pseudorotaxanes.

The solution-phase photooxygenation of multiply threaded crown/ammonium pseudorotaxanes containing anthracene spacers is monitored by electrospray ionization Fourier-transform ion-cyclotron-resonance (ESI-FTICR) mass spectrometry. The oxygenated pseudorotaxanes are mass-selected and fragmented by infrared multiphoton dissociation (IRMPD) and/or collision-induced dissociation (CID) experiments and and their behavior compared to that of the non-oxygenated precursors. [4+2]Cycloreversion reactions lead to the loss of O2, when no other reaction channel with competitive energy demand is available. Thus, the release of molecular oxygen can serve as a reference reaction for the energy demand of other fragmentation reactions such as the dissociation of the crown/ammonium binding motifs. The photooxygenation induces curvature into the initially planar anthracene and thus significantly changes the geometry of the divalent, anthracene-spacered wheel. This is reflected in ion-mobility data. Coulomb repulsion in multiply charged pseudorotaxanes assists the oxygen loss as the re-planarization of the anthracene increases the distance between the two charges.

Hydroconversion Behavior of Asphaltenes under Liquid-Phase Hydrogenation Conditions

To fully utilize deoiled end-cut (DOE) from selective asphaltene extraction process, Venezuela n-pentane DOE was subjected to hydroconversion in an autoclave reactor using tetralin as hydrogen donor. Venezuela DOE and its hydroconversion products were separated into gas, n-heptane maltenes (HM), n-heptane asphaltenes (HAs), and coke. The effects of reaction conditions including reaction temperature, solvent-to-feedstock (S/F) weight ratio, and reaction time on product distribution were investigated. High temperature, large S/F ratio, and long time benefited the generation of gas, HM, and coke to some extent. Under optimal conditions, over 50 wt % HAs in DOE was converted into HM fraction, with less than 3 wt % coke yield. The elemental compositions and molecular weights of HAs and HM, along with reaction time, were also analyzed. The hydrogen-to-carbon (H/C) ratio of HAs declined from 1.115 to 0.871, indicating that HAs underwent dehydrogenation and dealkylation reactions. However, the H/C ratio of HM initially increased from 1.405 to 1.548, showing that hydrogenation reaction occurred, and then decreased to 1.374 because of the cracking of HAs into HM and/or the secondary cracking of HM. The average molecular-weight decrease both for HAs and HM confirmed disaggregation and cracking reactions. The molecular composition and transformation of nitrogen and sulfur compounds before and after hydroconversion were determined by negative- and positive-ion electrospray ionization Fourier transform ion-cyclotron–resonance mass spectrometry, respectively. N1, S1, and O2 classes were dominant in the feedstock. After hydroconversion, N1 and S2 compounds decreased in HAs, indicating that they were reactive species. N1 compounds mainly cracked into small N1 compounds and also condensed into N2 compounds, while S2 compounds generally decomposed into S1 compounds.

Transgenic petunia with the iron(III)-phytosiderophore transporter gene acquires tolerance to iron deficiency in alkaline environments.

Iron is an essential nutrient for all plants. However, terrestrial plants often suffer from iron deficiency in alkaline soil due to its extremely low solubility. Alkaline soil accounts for about 30% of all cultivated ground in the world. Plants have evolved two distinct strategies, I and II, for iron uptake from the soil. Dicots and non-graminaceous monocots use Strategy I, which is primarily based on the reduction of iron(III) to iron(II) and the uptake of iron(II) by the iron-regulated transporter, IRT1. In contrast, graminaceous plants use Strategy II to efficiently acquire insoluble iron(III). Strategy II comprises the synthesis and secretion of iron-chelating phytosiderophores, such as mugineic acids and the Yellow Stripe 1 transporter proteins of the iron(III)-phytosiderophore complex. Barley, which exhibits the highest tolerance to iron deficiency in alkaline soil among graminaceous plants, utilizes mugineic acids and the specific iron(III)-mugineic acids transporter, HvYS1. In this study, we established the transgenic plant Petunia hybrida, which originally had only Strategy I, by introducing the HvYS1 transporter gene derived from barley. When the transgenic plants were grown hydroponically in media containing the iron(III)-2′-deoxymugineic acid complex, free 2′-deoxymugineic acid and its iron(III) complex were detected in the root extract of the transgenic plant by electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry. The growth of the transgenic petunia was significantly better than that of the control host in alkaline conditions. Consequently, the transgenic plant acquired a significantly enhanced tolerance to alkaline hydroponic media in the presence of the iron(III)-2′-deoxymugineic acid complex. Furthermore, the flower color of the transgenic plant deepened. The results showed that iron-phytosiderophore complexes and their transporters can potentially be utilized to overcome the worldwide iron uptake problems to diverse plant species that are found in areas with alkaline conditions.

Salicylate‐Functionalized Bismuth Oxido Clusters: Hydrolysis Processes and Microbiological Activity

AbstractHydrolysis of either Bi(HSal4Me)3 (A) or [Bi22O26(HSal4Me)14](B) in dmso gave the 4‐methylsalicylate‐substituted bismuth oxido cluster [Bi38O45(HSal4Me)24(dmso)14(H2O)2]·4H2O (1·4H2O), which crystallizes in the triclinic space group P$\bar {1}$ with cell parameters of a = 20.7214(5), b = 20.9654(6), c = 22.2128(6) Å, α = 100.867(2), β = 114.108(2), γ = 107.895(2)° and V = 7815.1(4) Å3. The hydrolysis of A was studied by using 1H DOSY NMR spectroscopy and electrospray ionization Fourier‐transform ion‐cyclotron‐resonance (ESI‐FTICR) mass spectrometry, both of which showed the formation of several larger species under ambient conditions in the presence of moisture. Furthermore, ESI‐FTICR MS analysis of cluster B showed the formation of mainly {Bi23O26} species, which indicates the partial dissociation of cluster B in solution. In addition, the three different bismuth species containing 1 (A), 22 (B) or 38 (1·4H2O) bismuth atoms per molecular formula were tested for their microbiological activity against three strains of Helicobacter pylori (251, 26695 and B128). A minimum inhibitory concentration (MIC) of 6.25 μg mL–1 for the mononuclear bismuth complex A was obtained, whereas the bismuth oxido cluster B showed a lower activity with MIC values in the range 25–50.0 μg mL–1. The activity of 1·4H2O is comparable to commercial remedies based on antimicrobial bismuth subsalicylate (BSS; 12.5 μg mL–1).

Characterization of Birch Wood Pyrolysis Oils by Ultrahigh-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Insights into Thermochemical Conversion

A laboratory-scale reactor was used to produce pyrolysis oils from Finnish silver birch hardwood (Betula pendula). The resulting wood distillates were characterized by ultrahigh-resolution (12 T) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry coupled with negative-ion electrospray ionization (ESI). Two different pyrolysis temperatures were tested: 300 and 380 °C (the resulting oil samples were named as Oil-300 and Oil-380, respectively). The detected species were sorted on the basis of heteroatom class, carbon number, and double bond equivalent (DBE). About 1200 and 1400 unique compounds were identified from the ESI FT-ICR spectra of Oil-300 and Oil-380, respectively. These were mainly oxygen-containing compounds (Ox heteroatom classes, with x = 2–14), comprising up to 90% of all identified compounds. The compounds in the O2, O3, and O4 classes comprised of different fatty acids, hydroxy/epoxy fatty acids, and diacids. The compounds in the O5–O8 classes comprised of mainly lignin degradation products and phenolic extractives. The compounds in the O9–O14 classes comprised of both low- and high-DBE compounds. Upon increasing the temperature from 300 to 380 °C, many compounds showed an overall decrease in their DBE and carbon number. The distribution of fatty acids in Oil-300 qualitatively matches the known lipid-derived fatty acid composition of silver birch. At a higher pyrolysis temperature (380 °C), hydrogenation of unsaturated C18 fatty acids toward fully saturated compounds was observed.

The Synergetic Interplay of Weak Interactions in the Ion‐Pair Recognition of Quaternary and Diquaternary Ammonium Salts by Halogenated Resorcinarenes

AbstractThe influence of halogens on the noncovalent interactions of different upper‐rim‐substituted hexylresorcinarenes with quaternary and diquaternary ammonium iodide salts was investigated in the gas phase by electrospray ionization Fourier‐transform ion‐cyclotron‐resonance (ESI‐FTICR) mass spectrometry and in solution by 1H NMR titration studies. The electronic nature of the substituents on the upper rim of the resorcinarene was directly reflected in the order of binding strengths of the hosts towards quaternary and diquaternary ammonium cations in the gas phase. In solution, the opposite trend was observed, with generally higher binding constants for the diquaternary over the quaternary salts. This phenomenon is explained by the synergetic effect originating from the interaction of the halogenated resorcinarenes with the counteranions through enhanced hydrogen bonding, leading to ion‐pair binding in solution. The electronegativity of the halogens renders the hydrogen of the hydroxy group more acidic, and hence enforces hydrogen bonding in solution. The collective effect of several weak interactions is manifested in solution, thereby emphasizing the benefits of comparing solution and gas‐phase studies in recognition processes.

Controlled tin catalyzed hydrolysis of 3-acryloxypropyltrimethoxysilane with mono- and multi-functional mercaptans

Mechanistic investigations were initiated to further our understanding of the reaction pathways for hydrolysis and condensation of organofunctional alkoxysilanes in organic media. Previous work has shown that addition of mercaptans to solutions that contain dibutyltin dilaurate (DBTDL), which functions as a catalyst, and 3-acryloxypropyltrimethoxysilane can greatly reduce the rate of silane hydrolysis and condensation. Gas chromatography/mass spectrometry (GC/MS) hydrolysis studies were carried out to understand how mercaptan structure and concentration affected the relative hydrolysis rate of the acrylate silane in solution. Detailed nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS) were carried out on simple mixtures of DBTDL and select mercaptans, and with varying mercaptan concentrations, to elucidate which significant tin complexes can form. These studies showed that mercaptans readily undergo ligand exchange reactions with the labile carboxylate groups of DBTDL and that an excess of mercaptans reduced the amount of proposed active catalyst species. It was found that an excess of mercaptan functional group was necessary to prevent significant and rapid hydrolysis of the organofunctional silane in organic media. A minimum of 1:4 DBTDL carboxylate group:mercaptan functional group was required for most mercaptans examined. A tetramercaptan, pentaerythritol tetrakis(3-mercaptopropionate), was shown to be effective in deactivating the DBTDL catalyst, even at relatively low concentrations of tetramercaptan. The findings in this work provide further support for the proposed mechanism for catalyzed hydrolysis and condensation. The tin metal was determined to be predominately 4-fold coordinated in the various tin mercaptide compounds studied. Evidence for transient tin hydroxide reactive intermediates was detected using mass spectrometry, as upon the addition of water, oligomeric condensation products were observed. However, the lifetime of these reactive intermediates precluded their direct observation by either mass spectrometry or NMR. Oligomeric and cyclic tin mercaptide species were shown to form when multifunctional mercaptans were combined with DBTDL in solution.

Ion‐Pair Recognition of Tetramethylammonium Salts by Halogenated Resorcinarenes

The non-covalent interactions of different upper-rim-substituted C(2)-resorcinarenes with tetramethylammonium salts were analyzed in the gas phase in an Electrospray Ionization Fourier-transform ion-cyclotron-resonance (ESI-FTICR) mass spectrometer and by (1)H NMR titrations. The order of binding strengths of the hosts towards the tetramethylammonium cation in the gas phase reflects the electronic nature of the substituents on the upper rim of the resorcinarene. In solution, however, a different trend with particularly high binding constants for halogenated resorcinarenes has been observed. This trend can be explained by a synergetic effect originating from the interaction of the halogenated resorcinarenes with the counter anions through hydrogen bonding. This study highlights the importance of weak interactions in recognition processes and points out the benefits of comparing the gas-phase data with results obtained from solution experiments.

Noncovalent Complexation of Monoamine Neurotransmitters and Related Ammonium Ions by Tetramethoxy Tetraglucosylcalix[4]arene

The noncovalent complexation of monoamine neurotransmitters and related ammonium and quaternary ammonium ions by a conformationally flexible tetramethoxy glucosylcalix[4]arene was studied by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. The glucosylcalixarene exhibited highest binding affinity towards serotonin, norepinephrine, epinephrine, and dopamine. Structural properties of the guests, such as the number, location, and type of hydrogen bonding groups, length of the alkyl spacer between the ammonium head-group and the aromatic ring structure, and the degree of nitrogen substitution affected the complexation. Competition experiments and guest-exchange reactions indicated that the hydroxyl groups of guests participate in intermolecular hydrogen bonding with the glucocalixarene.

Oligomer Content of α-Pinene Secondary Organic Aerosol

The quantity, extraction efficiency, and molecular composition of non-volatile oligomeric species in SOA generated by the reaction of α-pinene with ozone were studied. Two different methods of determining the total particulate mass in the reaction chamber were compared and found to be in good agreement when changes in the partitioning of semi-volatile compounds to the particle phase during measurement were properly handled. Almost all of the non-volatile organic carbon formed by the reaction was collected and recovered by extraction with organic solvents; recoveries with water extraction were somewhat lower. The identities of compounds extracted by the various solvents were determined using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Over 80% of the peaks weighted by mass and intensity were the same in the spectra of samples obtained from different extraction solvents. Standard addition plots were used to determine the amounts of two commercially available monomer compounds in the SOA extracts. When the response factors for those compounds were applied to other monomers detected in the mass spectra, the weight percent of monomers was estimated to be slightly less than 50%, with the remaining mass (over 50%) assigned to oligomers. The oligomer content is sufficiently large that it should be taken into account when modeling the formation and properties of laboratory SOA.

Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme

In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient Δ pgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/Δ pgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A 1 and A 2 or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.

Characteristic fragmentation behavior of the analogs of endomorphin-2 with phenylglycine in position 3 or 4 by ESI-FT tandem mass spectrometry

A series of analogs of endomorphin-2 (EM-2) with phenylglycine (Phg) in position 3 or 4 were synthesized. In electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) MS/MS spectra of these compounds, some b, y, a, and internal ions were observed and slight mass differences between the calculated and observed results are obtained. Their sequences were derived successfully. However, the MS/MS patterns of these analogs with DPhg and LPhg were very similar. It is hard to distinguish them by MS/MS spectra. Moreover, if the third position was substituted by phenylglycine ( l or d), a rearrangement could occur in MS/MS experiment to lose proline residue.

A kinetic study of guest displacement reactions on a host–guest complex with a photoswitchable calixarene

The displacement processes of several guests, incorporated in a calixarene host system, were investigated in the gas phase by electrospray ionization-Fourier transform-ion cyclotron resonance (ESI-FT-ICR) mass spectrometry. The complexes resulting from a resorcin[4]arene host with ammonia and sec-butylamine guests were isolated in an ICR-cell, separately using both states of the photoswitch as well as two reference systems for the open and closed forms of the photoswitchable host. The isolated complexes were forced to exchange the guest by using methylamine, ethylamine and sec-butylamine, resulting in different reaction rates for all the measured systems. Especially, the reaction rates of both states of the photoswitch are dependent on the provided guest. Potential side effects like proton exchanges were examined by an H/D-exchange experiment. The results were investigated and supported by quantum chemical calculations (DFT).

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 <or=5.0, followed by step-by-step dissociation of the remaining apo-tetramer to trimers, dimers and monomers. In addition, a approximately 346-kDa protein octamer was detected at pH 6.9.