Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis

AIMS: To rapidly type the fengycin homologues produced by Bacillus subtilis strains with electrospray ionization/collision-induced dissociation (ESI/CID) mass spectrometry. METHODS AND RESULTS: Fengycin homologues produced by Bacillus subtilis JA were analysed. When each homologue was subjected to ESI/CID analysis, ions representing characteristic fragmentations were detected. These ions can help to identify the homologues; even homologues of the same nominal mass can be discriminated by their ESI/CID spectra. Based on the CID results, fengycin homologues can be correctly assigned. CONCLUSIONS AND SIGNIFICANCE OF THIS STUDY: ESI/CID leads to rapid detection and structural characterization of fengycin homologues or lipopeptides with similar properties. It will be very useful in studying the regulatory expression of these peptides.     

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding

Papaver alkaloids play a major role in medicine and pharmacy. In this study, [ring-(13)C(6)]-tyramine as a biogenetic precursor of these alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. Thus, based on this procedure, the structure of about 20 alkaloids displaying an incorporation of the labeled tyramine could be elucidated. These alkaloids belong to different classes, e.g. morphinan, benzylisoquinoline, protoberberine, benzo[c]phenanthridine, phthalide isoquinoline and protopine. The valuable information gained from the alkaloid profile demonstrates that the combination of these two spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products.     

Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry.

Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.     

Deuterium exchange of alpha-helices and beta-sheets as monitored by electrospray ionization mass spectrometry.

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI-MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several alpha-helical peptides and beta-sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, alpha-helical, or beta-sheet structure. For 3 isomeric 31-residue alpha-helical peptides, the number of slowly exchanging hydrogens as measured by ESI-MS in 50% CF3CD2OD (pD 9.5) provided estimates of their alpha-helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of beta-sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI-MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI-MS requires much less sample (1-50 micrograms), much shorter analysis time (10-90 min), and no chemical quenching of the exchange reaction.     

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.     

Analysis of fluorescently labeled oligosaccharides by capillary electrophoresis and electrospray ionization mass spectrometry

Neutral oligosaccharides were fluorescently conjugated with 7-amino-1, 3-naphthalenedisulfonic acid. A mixture of fluorescently labeled chitobiose, chitotriose, and chitotetrose were successfully separated by preparative capillary electrophoresis (CE) and the individual components characterized by electrospray ionization-mass spectrometry (ESI-MS). By combining fluorescent labeling with CE, the use of highly specific exoglycosidases and ESI-MS, a more structurally complex N-linked glycan was analyzed.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures.     

Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry

Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum.     

Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry

To explore the hypothesis that alterations in ethanolamine plasmalogen may be directly related to the severity of dementia in Alzheimer's disease (AD), we performed a systematic examination of plasmalogen content in cellular membranes of gray and white matter from different regions of human subjects with a spectrum of AD clinical dementia ratings (CDR) using electrospray ionization mass spectrometry (ESI/MS). The results demonstrate: (1) a dramatic decrease in plasmalogen content (up to 40 mol% of total plasmalogen) in white matter at a very early stage of AD (i.e. CDR 0.5); (2) a correlation of the deficiency in gray matter plasmalogen content with the AD CDR (i.e. approximately 10 mol% of deficiency at CDR 0.5 (very mild dementia) to approximately 30 mol% of deficiency at CDR 3 (severe dementia); (3) an absence of alterations of plasmalogen content and molecular species in cerebellar gray matter at any CDR despite dramatic alterations of plasmalogen content in cerebellar white matter. Alterations of ethanolamine plasmalogen content in two mouse models of AD, APP(V717F) and APPsw, were also examined by ESI/MS. A plasmalogen deficiency was present (up to 10 mol% of total plasmalogen at the age of 18 months) in cerebral cortices, but was absent in cerebella from both animal models. These results suggest plasmalogen deficiency may play an important role in the AD pathogenesis, particularly in the white matter, and suggest that altered plasmalogen content may contribute to neurodegeneration, synapse loss and synaptic dysfunction in AD.     

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.     

Bismuth binding studies to the human metallothionein using electrospray mass spectrometry

Bismuth compounds are currently used to treat gastric ailments and to prevent the toxic side effects of cancer treatments. The affinity of bismuth for binding to sulfur compounds has been reported and one such target biomolecule is the cysteine-rich metalloprotein metallothionein. Renal mammalian metallothionein has been shown to be induced by Bi salts, with the Bi³⁺ binding to the renal MT. However, the exact metal-to-metallothionein stoichiometric ratios for the 2-domain βα mammalian protein and the individual β and α domain fragments remain unknown. We now report that the maximum metal-to-MT stoichiometries for the individual domain fragments and the entire 2-domain protein are Bi₃-S₉-βhMT, Bi₄-S₁₁-αhMT, and Bi₇-S₂₀-βαhMT, respectively. Electrospray mass spectrometry data also unambiguously show the existence of partially metalated Bi-containing MT species during the titration of apo-MT with Bi³⁺, which demonstrates that Bi-metalation to MT occurs in a noncooperative manner.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Monitoring enzyme catalysis in the multimeric state: Direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.     

Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment

Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms.     

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.     

Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol derivatives by electrospray tandem mass spectrometry

Maltopentaose and olive pulp xylo-oligosaccharides and the correspondent alditol derivatives were analysed by ESI-MS and ESI-MS/MS. The ESI-MS spectrum of maltopentaose and maltopentaose alditols showed [M+Na]⁺and [M+H]⁺ ions. ESI-MS spectrum of xylo-oligosaccharides and their alditols showed [M+Na]⁺of neutral (Xyl_3–6) and acidic (Xyl_2–3MeGlcA and Xyl_2–3GlcA) xylo-oligosaccharides. The ESI-MS/MS spectra of maltopentaose and underivatised xylo-oligosaccharides presented fragments of glycosidic cleavages attributed to B/Z and C/Y ions. On the other hand, MS/MS spectra of the correspondent alditols showed glycosidic cleavages unambiguously identified as B-type and Y-type ions. Y-type fragment ions showed higher abundance in the MS/MS spectra of the alditol derivatives when compared to the non-reduced samples. The study of the oligoxylosyl alditols fragmentation permits to distinguish fragmentation pathways that occur both from the reducing end and from the non-reducing end of the xylan chain, allowing to obtain more information about the localization of the acidic substituent along the glucuronoxylan backbone.