Electrospray ionization-Fourier Transform Ion Cyclotron Research Articles

Structural Analysis of Bikunin Glycosaminoglycan

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

Studies of the intermolecular DNA triplexes of C+·GC and T·AT triplets by electrospray ionization Fourier‐transform ion cyclotron resonance mass spectrometry

Formation and stabilities of four 14-mer intermolecular DNA triplexes, consisting of third strands with repeating sequence CTCT, CCTT, CTT, or TTT, were studied by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the gas phase. The gas-phase stabilities of the triplexes were compared with their CD spectra and melting behaviors in solution, and parallel correlation between two phases were obtained. In the presence of 20 mM NH(4) (+) (pH 5.5), the formation of the TTT triplex was not detected in both solution and the gas phase. Other triplexes showed the same order, CTCT > CCTT > CTT, of ion abundances in mass spectra and T(m) values in solution. The more stable triplexes are those that contained higher percentage of C(+).GC triplets and an alternating CT sequence. However, the CCTT with the same C(+).GC triplets as the CTCT showed a higher stability than the latter during the gas-phase dissociation. Furthermore, a biphasic triplex-to-duplex-to-single transition was detected in the gas phase, while a monophasic triplex-to-single dissociation was observed in solution. The present results reveal that hydrogen bonds and electrostatic interactions dominate in the gas phase, while base stacking and hydrophobic interactions dominate in solution to stabilize the triplexes. Moreover, weak acidic conditions (pH 5-6) promote the formation of the parallel triplexes.

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC–hinge interactions were observed in constructs including at least one unpaired guanine at the 5′-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3′-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5′-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

Characterization of hepatic glutathione S-transferases in coho salmon ( Oncorhynchus kisutch)

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon ( Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24–26 kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS–MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Focused Proteomics in Post-Mortem Human Spinal Cord

With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified (p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and beta chain, myelin basic protein, thioredoxin, alpha enolase, and choline acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

Conversions of Formaldehyde-modified 2′-Deoxyadenosine 5′-Monophosphate in Conditions Modeling Formalin-fixed Tissue Dehydration

Formalin-fixed, paraffin-embedded specimens typically provide molecular biologists with low yields of extractable nucleic acids that exhibit extensive strand cleavage and covalent modification of nucleic acid bases. This study supports the idea that these deleterious effects are promoted by the first step in formalin-fixed tissue processing--i.e., tissue dehydration with a graded series of alcohols. We analyzed the conversions of formaldehyde-modified 2'-deoxyadenosine 5'-monophosphate (dAMP) by reverse-phase ion-pair, high-performance liquid chromatography and found that dehydration does not stabilize N-methylol groups in the modified nucleotide. Furthermore, spontaneous demodification in a dry state or in anhydrous ethanol can be as fast as it is in aqueous solutions if the preparation is contaminated with salts of orthophosphoric acid. In ethanol, orthophosphates also catalyze formation of abundant N6-ethoxymethyl-dAMP, as well as cross-linking and depurination of nucleotides present in the mixture. Identification of the products was performed using ultraviolet absorbance spectroscopy and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry. Alternatives to the traditional processing of formalin-fixed tissues are discussed.

The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica.

Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

Charge-state selective fragmentation analysis for protonated peptides in infrared multiphoton dissociation

We investigate the charge-state selective cleavage in gas-phase protonated peptides by using electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer (MS). The singly and multiply protonated peptides (Angiotensin II, Angiotensin I, Urotensin II, Bradykinin, Substance P) at the selected charge state were cleaved with the techniques of infrared multiphoton dissociation (IRMPD) in FTICR MS. Systematically changing IR laser power, the fragment ions were assessed to determine the cleaved amino bonds in the peptides at the selected charge state, then to quantitatively obtain the dissociation efficiency for each fragment. The results show that the fragment ions are observed at the selective cleavage of Asp-Xaa in singly charged Angiotensin I, Angiotensin II and Urotensin II ions that contain an acidic residue (Asp) and a basic residue (Arg or Lys), while the fragment ions arising from cleavage at Xaa-Pro dominate in those doubly charged peptides. It is observed that the dissociation channel of Asp-Xaa requires higher energy than that of Xaa-Pro. The charge selective fragmentation is not seen for Bradykinin and Substance P containing strong basic amino residues without an acidic residue.

Characterization of a Novel Gastropod Toxin (6-Bromo-2-mercaptotryptamine) That Inhibits Shaker K Channel Activity

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.

Multitarget affinity/specificity screening of natural products: finding and characterizing high-affinity ligands from complex mixtures by using high-performance mass spectrometry.

In this work we describe a high-throughput screening approach based on electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) that rapidly interrogates the noncovalent interaction between RNA-based drug targets and components derived from a bacterial natural product library. The screening process detects molecules present in the natural product library that bind to a synthetic RNA target that mimics the prokaryotic 16S rRNA A-site, while simultaneously measuring specificity for the synthetic A-site target using a control RNA target that lacks the critical structural element of the A-site construct. This screening approach known as multitarget affinity/specificity screening (MASS) demonstrated the expected binding of paromomycin from a fractionated natural product library derived from Streptomyces rimosus sp. paromomycinus. A new molecule was observed to bind with specificity to the 16S A-site RNA construct. MS/MS characterization of this species yielded partial structural information suggesting it is an aminoglycoside consisting of a paromomycin core with one or more modified rings. This work demonstrates the tremendous utility of MASS for screening natural product fractions against macromolecular targets.

Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis.

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.

Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance.

When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a approximately 10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods.

Influence of charge state on product ion mass spectra and the determination of 4S/6S sulfation sequence of chondroitin sulfate oligosaccharides.

Electrospray ionization-Fourier transform ion cyclotron resonance tandem mass spectrometry is used to study the influence of charge state on the product ion spectra of chondroitin sulfate oligosaccharides for determination of the sulfate position on N-acetylgalactosamine residues. Sustained off-resonance irradiation collision-induced dissociation and infrared multiphoton dissociation are investigated for tandem mass spectrometry of chondroitin sulfate. Product ion spectra were obtained for ions of varying charge states from (4,5)-unsaturated (delta-unsaturated), reduced delta-unsaturated, and saturated oligosaccharides from chondroitin sulfate A and chondroitin sulfate C, separately. It was observed that ions in which the charge (z) is less than the number of sulfates dissociate to produce predominantly even-numbered B(n), C(n), Y(n), and Z(n) ions, and that odd-numbered fragment ions are observed for ions that have z equal to the number of sulfates. Sulfate adducted ions were observed in the product ion spectra of singly charged tetramer and hexamer oligosaccharides. This sulfate adduction was determined to result from migration of neutral sulfate during excitation.

Characterization of the p13 membrane protein of Borrelia burgdorferi by mass spectrometry

Borrelia burgdorferi sensu lato is a tick-borne pathogen that causes Lyme disease. The characterization of membrane proteins from this and other pathogens may yield a better understanding of the mechanisms of infection and information useful for vaccine design. Characterization of the highly hydrophobic Borrelia outer membrane component P13 from a mutant (OspA − OspB − OspC − and OspD −) strain was undertaken by use of a combination of mass spectrometric methods. In a previous investigation, an electrospray ionization (ESI) mass spectrum of the intact protein provided an average molecular weight that was 20 Da lower than the predicted molecular weight. The mass deviation could be explained by a modification of the N-terminus of the protein such as pyroglutamylation (−17 Da) in combination with the experimental error of measurement, however more information was required. New structural information for this membrane protein was provided by peptide mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and sequencing with ESI-quadrupole-TOF tandem MS. ESI Fourier transform ion cyclotron resonance (FT-ICR) MS at 9.4 tesla revealed that the Borrelia membrane protein is not only pyroglutamylated, but also contains an amino acid substitution near the N-terminus.

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1–13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.

Melittin-Diacylphosphatidylcholine Interaction Examined by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

The structure of melittin in the presence of dioleoylphosphatidylcholine (DOPC) was investigated using hydrogen-deuterium (H/D) exchange in conjunction with collision induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). The deuterium incorporation into backbone amide hydrogens of melittin in the presence of DOPC was analyzed at different time points examining the mass of each fragment ion produced by hexapole-CID. The percentage of deuterium incorporation into the fragments of melittin was less than 45% at initial 10 s of isotope exchange. It increased rapidly as the exchange period was prolonged. After 300 s of incubation in D2O about 85% of amide hydrogens were exchanged with deuterium. When melittin was incubated in D2O in the presence of dodecylphosphocholine (DPC), the rate of isotope exchange was reduced at every time point. In the case of melittin alone, more than 80% amide hydrogens were exchanged with deuterium within 10 s. By comparing these time courses, it seems that the contact with DOPC did not induce melittin to change its conformation. DOPC possibly just shielded the melittin molecule while DPC induced melittin to form some stable conformation, such as helical structure. It was revealed that H/D exchange and MS analysis enabled to study such structural changes of a peptide brought about by the interaction with two types of phospholipid even in the presence of 50-fold amount of lipid.

Structure of melittin bound to phospholipid micelles studied using hydrogen–deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The structure of melittin bound to dodecylphosphocholine (DPC) micelles was investigated using hydrogen–deuterium (H/D) exchange in conjunction with collision induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of melittin with or without DPC micelles was analyzed at different time points examining the mass of each fragment ion produced by hexapole CID. When melittin existed alone in aqueous solution, more than 80% of amide hydrogens was exchanged within 10 s, and the deuterium content in each fragment ion showed high values throughout the experiments. When melittin was bound to DPC micelles, the percentage of deuterium incorporation into the fragment decreased remarkably at any time point. It increased little by little as the exchange period prolonged, indicating that some stable structure was formed by the interaction with DPC. The results obtained here were consistent with the previous studies on the helical structure of melittin carried out by NMR and CD analyses. The strategy using H/D exchange and MS analysis might be useful for studying structural changes of peptides and proteins caused by phospholipid micelles. It could also be applied to membrane-bound proteins to characterize their structure.

Complexation and Molecular Speciation in the Silica Sol−Gel Process Characterized by Electrospray Ionization Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry

The sol−gel reactions of tetraethyl orthosilicate have previously been characterized by various instrumental methods, each revealing different aspects of the reactions that eventually form glass, SiO2. In this paper, we use electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry (ESI FT-ICR MS) to characterize the hydrolysis and condensation intermediates based on alkali and transition metal ion attachment to ionize the intermediates. Sodium trifluoromethanesulfonate and nickel(II) nitrate hexahydrate were added to solutions containing tetraethyl orthosilicate, electrosprayed, and the resulting species observed. Sodium ion binds nonspecifically to silicates, whereas nickel produces a tris-tetraethyl orthosilicate nickel(II) complex. Sodium-bound dimers were also observed; they could be dissociated without damage to the silicate backbone by infrared multiphoton dissociation. It is thus possible to observe all of the hydrolysis products (OH exchanged for OCH2CH3) for a given silicate oligomer as well as different silicate oligomers.

Thermostability of endo-1,4-β-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering

Endo-1,4-β-xylanase II (XYNII) from Trichoderma reesei is a 21kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566–574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10–30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50°C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.

Thermostability of endo-1,4-beta-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering.

Endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566-574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10-30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50 degrees C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.