Robust stability analysis and design under consideration of multiple feedback loops of the tryptophan regulatory network of Escherichia coli.

The tryptophan system present in Escherichia coli represents an important regulatory unit described by multiple feedback loops. The role of these feedback loops is crucial for the analysis of the dynamical behavior of the tryptophan synthesis. We analyze the robust stability of this system which models the dynamics of both fast state, such as transcription and synthesis of free operator, and slow state, such as translation and tryptophan synthesis under consideration of nonlinear uncertainties. In addition, we analyze the role of these feedback loops as key design components of this regulatory unit responsible for its physiological performance. The range of allowed parameter perturbations and the conditions that ensure the existence of asymptotically stable equilibria of the perturbed system are determined. We also analyze two important alternate regulatory designs for the tryptophan synthesis pathway and derive the stability conditions.

Fourier transform ion cyclotron resonance: state of the art.

This short review summarizes recent and projected advances in Fourier transform ion cyclotron resonance mass spectrometry instrumentation and applications, ranging from petroleomics to proteomics. More details are available from the cited primary literature and topical reviews.

Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptic to yield complementary sequence information.

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides.

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.

High sensitivity Fourier transform ion cyclotron resonance mass spectrometry for biological analysis with nano-LC and microelectrospray ionization.

Modifications to a 7 T nano-LC micro-ESI FT-ICR mass spectrometer, including a shorter octopole, approximately 100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 microL, 50 amol/microL) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 microL, 500 amol/microL) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with arg8-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 microL, 300 amol/microL) and a total of 100 amol in water (1 microL, 100 amol/microL). Arg8-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of FT-ICR to detect and identify a true endogenous biological analyte.

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: mass-based protein identification without liquid chromatographic separation.

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ("Edman" reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and y(n-1) fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1+ ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The basic principles and recent advances in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are reviewed. A brief history of electrospray ionization is provided, along with a complete technical description of the technique, electrospray ionization variations, and advantages. Next, the fundamental principles of Fourier transform ion cyclotron resonance mass spectrometry are covered, including ion cyclotron motion, ion cyclotron resonance excitation, and image current detection. Instrumentation and methods used to couple these techniques are then described. Topics include ion source configuration, ion transport through a strong magnetic field gradient, and ion trapping methods. The article concludes with selected applications that highlight the strengths of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca(2+)-induced conformational changes in the regulatory domain of human cardiac troponin C.

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

Blood-brain barrier penetration of 3-aminopropyl-n-butylphosphinic acid (CGP 36742) in rat brain by microdialysis/mass spectrometry.

The detection and quantitation of the novel drug 3-aminopropyl-n-butylphosphinic acid (APBP), also known as CGP 36742, was performed in vivo using microdialysis and tandem mass spectrometry. This drug is a GABA-B antagonist with high specificity for GABA-B receptors. Animals received doses of 100, 200, 500 and 1000 mg kg-1 of the drug either intravenously or per os (p.o.). Microdialysis probes, placed by stereotaxis in either the frontal cortex or third ventricle of the rat, were used to collect dialyzate samples over several hours. Samples were then analyzed by micro-electrospray tandem mass spectrometry to achieve a molecular mass and structure specific analysis. For example, animals receiving a dose of 100 mg kg-1 p.o. showed a peak concentration of approximately 10 microM in the dialyzate. For comparison, tissue and plasma samples of the drug were measured under the same conditions using gas chromatography/mass spectrometry. This work demonstrates that the microdialysis technique in combination with the molecular specificity and high sensitivity of micro-electrospray tandem mass spectrometry can be used to study the time course of the appearance of unmodified drug in the brain of a single animal.

Application of micro-electrospray liquid chromatography techniques to FT-ICR MS to enable high-sensitivity biological analysis.

A microbore electrospray (ESI) injection system has been adapted to our 9.4-tesla ESI FT-ICR mass spectrometer, greatly enhancing the stability and sensitivity of the system. Spray was generated from micro-ESI needles made from sharply tapered, polished fused silica capillaries of 25 to 50 microns inner diameter. Micro-ESI permits low-level sample analysis by constant infusion at sub-microL/min flow rate over a wide range of solvent conditions in both positive- and negative-ion mode. The system is flexible and allows rapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. LC/MS analyses were accomplished by replacing micro-ESI needles with capillaries packed with reverse phase retention media to permit analyte concentration and purification prior to analysis (micro-ESI/LC). A unique nano-flow LC pumping system was developed, capable of producing a true unsplit solvent gradient at flow rates below 1 microL/min. The micro-ESI/LC FT-ICR system produces mass spectra from a mixture of three neuroactive peptides at a concentration of 500 amol/microL (5 fmol each total loaded) in biological salts with baseline separation, signal-to-noise ratio of > 10:1 and mass resolving power > 5000. These results represent a reduction in detection limit by a factor of approximately 2 x 10(6) over the best previously published LC/FT-ICR MS data.

Detection, number, and sequence location of sulfur-containing amino acids and disulfide bridges in peptides by ultrahigh-resolution MALDI FTICR mass spectrometry.

Here, we present several strategies for determining the number of sulfur atoms and disulfide bridges in selected biologically active peptides, based on MALDI FTICR mass spectrometry at femtomole sample consumption level. First, based on the 2-Da mass increase per disulfide bridge reduction, we show that repeated laser shots on the same sample spot can reduce (and therefore reveal the presence of) the disulfide bridge in oxytocin. Second, we show that the primary sequence positions of the disulfide-bridged cystines can be inferred from the presence/absence of MALDI-induced reduction in cystine-containing fragment ions. Third, we show that the presence and number of sulfur atoms as well as the degree of reduction in a peptide can all be determined directly from isotopic relative abundances of mass-resolved 34S, 13C2, and reduced all-12C species in a single ultrahigh-resolution MALDI FTICR mass spectrum. Methods for achieving such ultrahigh mass resolution of peptide ions of closely spaced m/z (m/delta m50% approximately 950,000 at m/z approximately 650) at modest magnetic field (3 T) are discussed.

Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry.

The specific molecular detection of the endogenous neuropeptides methionine ([Met]5) enkephalin and neurotensin released in vivo in rat brain has been accomplished using microdialysis and mass spectrometry. Microdialysis probes were implanted in specific brain regions and were used to collect samples from brain extracellular fluids in unanesthetized, freely moving animals. Microelectrospray/tandem mass spectrometry was used to achieve molecular-specific identification of the neuropeptides with a sensitivity in the amol/microliters range. Measurements of the amounts of neuropeptides in the dialysates obtained from studies of KCl-stimulated release showed that [Met]5-enkephalin from the globus pallidus/ventral pallidum region was present at a level of approximately 4-6 fmol/10 microliters of dialysate and neurotensin from the hypothalamus of approximately 500 amol in 10 microliters of dialysate. In this manuscript, we present the first data of a mass- and molecular-specific detection and quantitation of individual neuropeptides released in response to either intracerebrally or systemically administered compounds.

Involvement of granule, basket and stellate neurons but not Purkinje or Golgi cells in cerebellar cGMP increases in vivo.

Recent immunocytochemical studies of cerebellar nitric oxide synthase (NOS) and cGMP have aided dramatically in defining possible cellular sources of cGMP generation in the signal transduction cascade evoked by excitatory amino acids in the cerebellum. Using a mouse mutant deficient in cerebellar Purkinje cells ("nervous" mouse) and chemical lesions of cerebellar neurons with methylazoxymethanol (MAM), we have examined in vivo generation of cGMP to determine the roles of different cerebellar neuronal populations. In the case of "nervous" mice, our data indicate that cerebellar Purkinje cells are not required for NMDA-dependent increases in cGMP in the cerebellum. In marked contrast, MAM lesions which reduce granule but not Golgi cells in the granule cell layer and reduce basket and stellate cells in the molecular layer, dramatically reduced the ability of NMDA to increase cerebellar cGMP. These data support immunocytochemical data of cerebellar NOS pools and indicate the importance of granule, basket and possibly stellate cells in the generation of nitric oxide, which in turn activates guanylate cyclase, in a diversity of cells, to increase cerebellar cGMP levels.

Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins.

A "micro-electrospray" ionization source has been developed that markedly increases the sensitivity of the conventional electrospray source. This was achieved by optimization of the source to accommodate nanoliter flow rates from 300 to 800-nL/min spraying directly from a capillary needle that, for the analysis of peptides, contained C18 liquid chromatography packing as an integrated concentration-desalting device. Thus, a total of 1 fmol of methionine enkephalin was desorbed from the capillary column spray needle, loaded as a 10-µL injection of 100-amol/µL solution. The mass spectrum showed the [M + H](+) ion at m/z 574.2 with a signal-to-noise ratio of better than 5:1 from a chromatographic peak with a width of about 12 s. A narrow range (15-u) tandem mass spectrum was obtained for methionine enkephalin from the injection of 500 amol, and a full-scan tandem-mass spectrum was obtained from 50 fmol. For proteins, the average mass measurement accuracy was approximately 100-200 ppm for the injection of 2.5 fmol of apomyoglobin and 20-40 ppm for 200 fmol. Carbonic anhydrase B and bovine serum albumin showed similar mass measurement accuracies.

Micro-Electrospray: Zeptomole/attomole per microliter sensitivity for peptides.

The micro-electrospray ionization source has been optimized for the specific analysis of neuropeptides such as neurotensin and methionine enkephalin. The source has the option of integrating nanoliter flow-rate desalting and preconcentration techniques into the micro-electrospray spray needle, eliminating post-column dead volumes. For neurotensin, the most sensitive neuropeptide analyzed thus far in this work, the injection of 10 µL of a solution containing 320 zeptomolesy/gmL gave an [M + 3H](+3) ion at m/z 558.4 with S/N of > 8∶1. The MS/MS analysis of this peptide for the fragment ion at m/z 578.9 gave a S/N > 20∶1 for a solution containing 32 attomoles/µL.

Indole-2-carboxylates, novel antagonists of the N-methyl-D-aspartate (NMDA)-associated glycine recognition sites: in vivo characterization.

Recent in vitro receptor binding studies have indicated that indole-2-carboxylates with halogen substitutions at the position 5 or 6 are potent competitive antagonists of the NMDA (N-methyl-D-aspartate)-associated strychnine-insensitive glycine receptor (Gray N. M., Dappen M. S., Cheng B. K., Cordi A. A., Biesterfeldt J. P., Hood W. F. and Monahan J. B. (1992) J. med. Chem. 34: 1283-1292; Hood W. F., Gray N. M., Dappen M. S., Watson G. B., Compton R. P., Cordi A. A., Larthorn T. H. and Monahan J. B. (1992) J. Pharmac. exp. Ther. 262: 654-660). In the present investigation, a series of indole-2-carboxylates and two putative antagonists of glycine receptor HA-966 (3-amino-l-hydroxypyrrolidin-2-one) and 7-chlorokynurenic acid were examined for their effects on cGMP responses, mediated by the NMDA receptor complex, in vivo. Both SC-49648 (6-chloro-2-carboxyindole-3-acetic acid, intracerebellar injection, i.c.b.) and HA-966 (i.c.b. or intraperitoneal, i.p.) antagonized increases in levels of cyclic GMP in the cerebellum of the mouse, induced by the intracerebellar administration of NMDA and D-serine, agonists of the NMDA and the NMDA-associated glycine recognition sites, respectively. The drugs SC-49648 and 7-chlorokynurenic acid (i.p.) did not affect cGMP responses, suggesting poor bioavailability in brain. Following direct intracerebellar injection, SC-49648 was eliminated with a half-life of 12 min from the brain. Following intraperitoneal administration, SC-50132, the 3-ethylester analog of SC-49648, was eliminated from the brain with a half-life of 35 min and was found to be metabolized to SC-49648, in vivo. Some lipophilic analogs of SC-49648, designed as its prodrugs, were minimally active as glycine antagonists, in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of D-cycloserine at the N-methyl-D-aspartate-associated glycine receptor site in vivo.

The antibiotic, D-cycloserine has been shown to be a partial agonist at the N-methyl-D-aspartate (NMDA)-coupled, strychnine-insensitive glycine receptor by in vitro receptor binding. This partial agonism was further investigated in an in vivo system, by monitoring changes in levels of cyclic guanosine-monophosphate (cGMP), a well characterized second messenger response, mediated by the NMDA receptor complex, in the cerebellum of the mouse. Parenteral injections of D-cycloserine produced a biphasic dose-response curve which suggested partial agonism. In support of this contention, when intracerebellar injections were made together with D-serine, a glycine agonist, D-cycloserine attenuated the N-methyl-D-aspartate receptor-mediated increase in levels of cGMP. Likewise, systemic administration of D-cycloserine attenuated increases in cGMP induced by pentylenetetrazol. These data are relevant to the study of N-methyl-D-aspartate-mediated neurotransmission, since D-cycloserine is a parenterally bioavailable compound, with both agonist and depressant properties at the N-methyl-D-aspartate-associated glycine receptor.

Effects of sigma ligands on mouse cerebellar cyclic guanosine monophosphate (cGMP) levels in vivo: further evidence for a functional modulation of N-methyl-D-aspartate (NMDA) receptor complex-mediated events by sigma ligands.

In the present investigation, the effects of sigma ligands [WY-47384 [8-fluoro-2,3,4,5-tetrahydro-2[3-(3-pyridinyl)propyl)1H- pyrido(4,3b)indole], (+)-pentazocine, (+)-SFK 10,047 (N-allylnormetazocine), mafoprazine, opipramol, dextromethorphan, dextrorphan, (+)-3-PPP [3-(3-hydroxyphenyl)-N-propylpiperidine], (-)-butaclamol, DTG [1,3-di(2-tolyl)guanidine], rimcazole, ifenprodil and BMY-14802 [alpha-(fluorophenyl)-4-(5-fluoropyrimidinyl)-1-piperazine butanol]] on harmaline-, pentylenetetrazol (PTZ)-, methamphetamine (MA)- and D-serine-induced increases in mouse cerebellar levels of cGMP were determined. Ifenprodil, BMY-14802, dextromethorphan, dextrorphan, (+)-SKF 10,047, opipramol and mafoprazine reversed harmaline-, PTZ-, MA- and D-serine-induced increases in levels of cGMP. Rimcazole reversed only the harmaline-induced response. WY-47384 reversed harmaline-, MA-, D-serine-, but not PTZ- or quisqualate-induced increases in levels of cGMP. (+)-Pentazocine attenuated harmaline- and D-serine-, but not PTZ- and MA-induced cGMP responses. Haloperidol did not affect harmaline- and D-serine-induced cGMP responses. (+)-3-PPP and (-)-butaclamol did not affect any of the responses studied. Furthermore, (+)-3-PPP-induced increases in levels of cGMP were reversed by the competitive N-methyl-D-aspartate (NMDA) antagonist, CPP]3-(2-carboxypiperazin-4-yl)propyl- 1-phosphonic acid, the non-competitive NMDA antagonist, (+)-MK-801 (dizocilipine maleate), the NMDA-associated glycine receptor antagonist, HA-966 (3-amino-1-hydroxypyrrolidin-2-one), the partial glycine agonist, DCS (D-cycloserine) as well as by the sigma ligands, ifenprodil, WY-47384, (+)-pentazocine, (+)-SKF 10,047, dextromethorphan and dextrorphan but not by rimcazole.(ABSTRACT TRUNCATED AT 250 WORDS)