Collision induced ion ejection in an FTICR trapped-ion cell.

A collision algorithm was used with SimIon to evaluate collision-mediated ion ejection mechanisms in the ICR MS experiment. These mechanisms were characterized based on kinetic energy, ion mass, applied trapping potential, and collision gas mass. It was found that there are three collision-based energy regimes for ion loss from a trapped-ion cell. The first region is characterized by low initial cyclotron kinetic energy, a radial ejection mode, and a very high collision ratio (>100 collisions per ejection). The second region is characterized by a medium to high initial cyclotron kinetic energy leading to axial ejection at low collision ratio (1 to 10 collisions per ejection). The third region is characterized by a high initial cyclotron kinetic energy, a radial ejection mode, and a collision ratio of unity. It was also determined that there is a radial cyclotron mode limit, approximately 40% of the cell radius, after which an ion is ejected after a single collision. This has important consequences on the damping of the FTICR signal, various cooling techniques, ion activation techniques, and the remeasurement experiment.

Hydrogen/deuterium exchange of nucleotides in the gas phase.

Gas-phase hydrogen/deuterium exchange reactions have been performed on the 5'- and 3'-nucleotide monophosphates and on the 3'5'-cyclic nucleotides. Following negative mode electrospray ionization and transport to a Fourier transform ion cyclotron resonance cell, each nucleotide was reacted with gaseous D2O for up to 600 s. Extensive deuterium exchange was observed for the 3'- and 5'-nucleotides in negative ion mass spectra with relative rates of exchange following the trend 5'dCMP > 5'-dAMP > 5'dTMP >> 5'-dGMP and 3'-dGMP > 3'-dAMP approximately equal to 3'-dCMP approximately equal to 3'-dTMP. At least two classes of exchanging protons are observed. The more facile class is assigned to the amino protons of the bases, with a slower class attributed to the phosphate and/or hydroxyl proton. Overall, the 3'-nucleotides exchange more quickly than the 5'-oligonucleotides. The cyclic nucleotides did not undergo deuterium exchange, suggesting that a charged phosphate group proximate to the base is required to catalyze the exchange reaction. Exchange through tautomerization of the bases is no observed, although molecular modeling suggests an energy barrier of < 30 kcal.

Three-dimensional motional stabilization in the trapping field of an open-ended trapped-ion cell: application to the remeasurement experiment in Fourier transform ion cyclotron resonance mass spectrometry.

Three-dimensional motional stabilization of radial trajectories of low-mass organic ions in an open cell using only a dc trapping field is applied to the FT-ICR remeasurement experiment. More than 300 remeasurement cycles are observed with 99.59% remeasurement efficiency for benzene (m/z 78) using a high-pressure helium buffer gas. The enhancement in remeasurement efficiency is due to collisional stabilization of the guiding center of ion motion by dynamic motional averaging in the axial position-dependent radial electric field. Such dc-induced radial stabilization is in contrast to the stability produced by application of radio frequency fields characteristic of quadrupolar axialization or rf-only mode operation. The same effect is produced because ions experience a radial "pseudopotential" during axial oscillation as in time-varying fields. Trajectory simulations for ions oscillating in an open cell trapping well above a z-amplitude critical threshold energy of 0.60 eV (in a potential well of 0.84 V) indicate that radially stabilized trapping motion is achieved because the outward-directed radial electric field existing near the cell center line is compensated by an opposing inward-directed radial electric field at extended z-amplitude. Sufficient axial kinetic energy permits ion penetration into the inward-directed radial electric field regions, enabling > 50% residence time of each trapping oscillation period in regions inducing radial stability, thereby inhibiting magnetron radius growth. A high-pressure, low-mass buffer gas such as helium provides the requisite increase in the axial amplitude of the ion cloud, similar to the mechanism observed for axial excitation of low-mass ions observed in collision-induced dissociation. The result is radial stability at high pressure, even after multiple remeasurement cycles. An optimized excitation radius of 12.5% of the cell radius yields maximum remeasurement efficiency with a 500 ms relaxation delay between excitation events. Summed signal intensity decreases with increased trap potential due to the greater radial electric field and reduced axial expansion of the ion cloud and also decreases with buffer gas mass in response to greater radial scattering.

13C NMR analysis of the use of alternative donors to the tetrahydrofolate-dependent one-carbon pools in Saccharomyces cerevisiae.

Serine is generally accepted as the major one-carbon donor in folate-mediated one-carbon metabolism in most cells. Previous work from our laboratory with the yeast Saccharomyces cerevisiae has demonstrated that glycine and formate can also provide one-carbon units. Under normal growth conditions, it is likely that cells utilize serine, glycine, formate, and perhaps other one-carbon donors simultaneously, but to differing degrees. In the present work, we have used 13C NMR to monitor how yeast cells distribute alternative, competing one-carbon sources into various pools. Cells were grown with [2-13C]glycine and unlabeled formate or folinic acid (leucovorin, 5-formyl-tetrahydrofolate) as competing one-carbon sources. The relative contribution of each one-carbon donor to the three oxidation states of the tetrahydrofolate-bound one-carbon pool [5-methyl-tetrahydrofolate (CH3-THF), 5,10-methylene-THF (CH2-THF), and 10-formyl-THF (10-CHO-THF)] was determined by analysis of two metabolic end products of one-carbon metabolism, choline and adenine. Glycine-derived 13C-labeled one-carbon units are incorporated into these two metabolites; dilution of the 13C indicates competition by the unlabeled one-carbon source. The results reveal that the contribution from formate, folinic acid, and glycine is different for each of the one-carbon pools. Formate competed most dramatically at the 10-CHO-THF pool, with decreasing competition into the CH2-THF and CH3-THF pools. In a mutant strain lacking cytosolic CH2-THF dehydrogenase activity, a distinct shift toward the use of glycine instead of formate as the source of one-carbon units for the more reduced pools (CH2-THF and CH3-THF) was observed, while 10-CHO-THF pools were not affected. In contrast, the formyl group of folinic acid competed almost exclusively at the 10-CHO-THF level, with barely detectable dilution of the CH2-THF and CH3-THF pools in wild-type cells. The mutant strain exhibited essentially identical results, confirming that 5-formyl-THF enters the active one-carbon pool at the level of 10-CHO-THF, presumably via 5,10-methenyl-THF. Furthermore, donation of one-carbon units by folinic acid was observed only when cells were depleted of THF by treatment with the dihydrofolate reductase inhibitor methotrexate. These results reveal that the state of equilibrium between one-carbon pools in a growing cell depends on the source of the one-carbon units. This work illustrates the power of 13C NMR for examining the in vivo utilization of alternative one-carbon donors under a variety of conditions.

Fourier transform ion cyclotron resonance mass spectrometry in a 20 T resistive magnet.

We present Fourier transform ion cyclotron resonance (FTICR) mass spectra at a magnetic field of 20 T; more than twice the highest field previously used for FTICR. Our instrument is based on a resistive magnet installed at the National High Magnetic Field Laboratory. The magnet has a 50 mm diameter bore and spatial inhomogeneity of approximately 1000 ppm over a 1 cm diameter spherical volume. However, FTICR mass resolving power far in excess of magnet homogeneity is achieved routinely for ions produced by either electron ionization (EI) or matrix-assisted laser desorption/ionization (MALDI). As examples, we show a MALDI mass spectrum of [M + H] quasimolecular ions of the peptide, human luteinizing hormone-releasing hormone (monoisotopic molecular weight, 1181.6 Da) at mass resolving power, m/delta m > 10,000; and an EI mass spectrum of molecular ions of the platinum cluster compound, Pt4(PF3)8 (average molecular weight, 1484 Da at mass resolving power, m/delta m approximately 20,000. Much better FTICR MS performance is predicted for future NHMFL resistive magnets of higher spatial and temporal homogeneity.

Real-time monitoring of the gas phase reactions of a single ion population using the remeasurement experiment in Fourier transform ion cyclotron resonance mass spectrometry.

A single population of multiply charged protein ions formed by electrospray ionization is held in a trapped ion cell and remeasured continuously by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry while undergoing multiple reactions with diethylamine. In a first example, electrosprayed horse myoglobin with an average of 16 attached protons is reacted with base at a pressure of 1.5 x 10(-8) Torr for a period of 60 s. A total of 31 spectra acquired with a duty cycle of 2 s exhibit the charge state-dependent formation of up to three diethylamine adducts and removal of up to nine protons. A realtime measuring experiment is then conducted over a 1 h period to observe charge stripping of electrosprayed myoglobin ions, leaving as few as seven charges. Realtime monitoring is used to evaluate the effect of reagent gas pressure on adduct formation and is used in conjunction with high mass resolution FTICR detection to resolve the isotopic peaks within individual charge states of adduct spectra. The attractive features of real-time reaction monitoring include a dramatic reduction in experiment time and sample consumed while concurrently observing long-lived intermediates and reaction products as they form.

Cell geometry considerations for the Fourier transform ion cyclotron resonance mass spectrometry remeasurement experiment.

Three cell geometries, closed cubic, closed elongated, and open elongated, are evaluated for optimum remeasurement performance in the Fourier transform ion cyclotron resonance mass spectrometry (FTICR) experiment. The advantages and disadvantages of each cell during normal FTICR operation are discussed. Unit remeasurement efficiency is obtained for the closed elongated cell at approximately 71% of the cell radius, whereas the open elongated cell achieves unit remeasurement at only approximately 18% of the cell radius. The closed cubic cell does not achieve unit remeasurement efficiency at a detectable signal level. A correlation between remeasurement efficiency and the characteristic radial electric field of each trapped ion cell is established. Radial dispersion of the ion cloud is considered the dominant signal loss mechanism in the remeasurement experiment, and cell geometries are examined for optimized sensitivity and unit remeasurement efficiency.

Synthesis of Linear Acetylenic Carbon: The "sp" Carbon Allotrope.

A carbon allotrope based on "sp" hybridization containing alternating triple and single bonds (an acetylenic or linear carbon allotrope) has been prepared. Studies of small (8 to 28 carbon atoms) acetylenic carbon model compounds show that such species are quite stable (130 degrees to 140 degrees C) provided that nonreactive terminal groups or end caps (such as tert-butyl or trifluoromethyl) are present to stabilize these molecules against further reactions. In the presence of end capping groups, laser-based synthetic techniques similar to those normally used to generate fullerenes, produce thermally stable acetylenic carbon species capped with trifluoromethyl or nitrile groups with chain lengths in excess of 300 carbon atoms. Under these conditions, only a negligible quantity of fullerenes is produced. Acetylenic carbon compounds are not particularly moisture or oxygen sensitive but are moderately light sensitive.

Simplified application of quadrupolar excitation in Fourier transform ion cyclotron resonance mass spectrometry.

A new method for application of quadrupolar excitation to the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is presented. Quadrupolar excitation is conventionally applied to the two pairs of opposed electrodes that normally perform the excitation and detection functions in the FTICR experiment. Symmetry arguments and numerically calculated isopotential contours within the trapped ion cell lead to the conclusion that quadrupolar excitation can be applied to a single pair of opposed side electrodes. Examples of effective quadrupolar axialization via this method include a sevenfold signal-to-noise enhancement derived from 50 remeasurements of a single population of trapped bovine insulin ions and the selective isolation of a single charge state of horse heart myoglobin after an initial measurement that revealed the presence of 14 charge states.

Remeasurement at high resolving power in fourier transform ion cyclotron resonance mass spectrometry.

The Fourier transform ion cyclotron resonance mass spectrometry remeasurement experiment is demonstrated and evaluated under high resolution conditions. Signal-to-noise enhancement is observed for isotopically resolved bovine insulin peaks at a resolution of ∼ 31,000 (full width at half height). The experiment is sensitive to spacecharge effects and resultant changes in scan-to-scan signal-to-noise and resolution. Coulombic repulsion in the ion cloud during the high resolution remeasurement experiment can cause the cyclotron frequency to shift through the duration of the experiment, which results in broadened peak shapes when individual remeasurement spectra are coadded. By either reducing the number of ions in the cell or allowing the ion cloud to diffuse during the lifetime of the experiment, high resolution remeasurement spectra can be coadded without peak broadening or degradation of signal-to-noise ratio.

High performance fourier transform ion cyclotron resonance mass spectrometry via a single trap electrode.

An open-ended cylindrical cell with a single annular trap electrode located at the center of the excitation and detection region is demonstrated for Fourier transform ion cyclotron resonance mass spectrometry. A trapping well is created by applying a static potential to the trap electrode of polarity opposite the charge of the ion to be trapped, after which conventional dipolar excitation and detection are performed. The annular trap electrode is axially narrow to allow the creation of a potential well without excessively shielding excitation and detection. Trapping is limited to the region of homogeneous excitation at the cell centerline without the use of capacitive coupling. Perfluorotributylamine excitation profiles demonstrate negligible axial ejection throughout the entire excitation voltage range even at an effective centerline potential of only -0.009 V. High mass resolving power in the single-trap electrode cell is demonstrated by achievement of mass resolving power of 1.45 × 10(6) for benzene during an experiment in which ions created in a high pressure source cubic cell are transferred to the low pressure analyzer single-trap electrode cell for detection. Such high performance is attributed to the negligible radius dependent radial electric field for ions cooled to the center of the potential well and accelerated to less than 60% of the cell radius. An important distinction of the single-trap electrode geometry from all previous open and closed cell arrangements is exhibition of combined gated and accumulated trapping. Because there is no potential barrier, all ions penetrate into the trapping region regardless of their translational energy as in gated trapping, but additional ions may accumulate over time, as in accumulated trapping. Ions of low translational kinetic energy are demonstrated to be preferentially trapped in the single-trap electrode cell. In a further demonstration of the minimal radial electric field of the single-trap electrode cell, positive voltages can be applied to the annular trap electrode as well as the source cell trap electrode to achieve highly efficient transfer of ions between cells.

Ion kinetic energy modulation for improved ion trapping in electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H](+19) to [M+12H](+12) and [M+15H](+15) to [M+7H](+7), respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H](+19) to [M+7H](+7), which is more representative of the source distribution.

Whole-cell detection by 13C NMR of metabolic flux through the C1-tetrahydrofolate synthase/serine hydroxymethyltransferase enzyme system and effect of antifolate exposure in Saccharomyces cerevisiae.

Folate-mediated one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth. A potential source of one-carbon units is formate. This one-carbon unit is activated to 10-formyltetrahydrofolate via the synthetase activity of the trifunctional enzyme C1-tetrahydrofolate (THF) synthase for use in purine synthesis or can be further reduced to 5,10-methylene-THF by the dehydrogenase activity of the same enzyme. 5,10-Methylene-THF is used by serine hydroxymethyltransferase (SHMT) in the synthesis of serine. Recently, 13C NMR has been used to establish that the C1-THF synthase/SHMT enzyme system is the only route from formate to serine in vivo in the yeast Saccharomyces cerevisiae [Pasternack et al. (1992) Biochemistry 31, 8713-8719]. In vitro studies have considered the kinetics of the C1-THF synthase/SHMT enzyme system in the catalytic conversion of formate to serine [Strong et al. (1987) J. Biol. Chem. 262, 12519-12525]. In the present work, we begin to study the kinetics of this two-enzyme system in its natural environment. Provision of [13C]formate and direct detection of an intracellular accumulating pool of [3-13C]serine by 13C NMR of whole cells allow us to monitor the rate of flux through this enzyme system in vivo. The rate of accumulation of soluble [3-13C]serine under [13C]formate-saturating conditions is 13.0 +/- 1.2 microM/min relative to an external standard of serine in D2O. The extracellular formate concentration at half-maximal flux was determined to be 900 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

13C NMR analysis of intercompartmental flow of one-carbon units into choline and purines in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the three-carbon of serine is normally the major one-carbon donor, although glycine and formate can substitute for serine. The second carbon of glycine enters via the glycine cleavage system in the mitochondria and can satisfy all cellular one-carbon requirements. It remains unresolved, however, as to the route by which these mitochondrial one-carbon units supply cytosolic anabolic processes. In the present work, we have used yeast mutants blocked at selected sites and 13C NMR to trace the incorporation of glycine-derived mitochondrial 5,10-methylenetetrahydrofolate into nonmitochondrial synthesis of choline and purines. Label incorporation into choline traces the methylation pathway of choline synthesis from production of serine to methylation of phosphatidylethanolamine. The active one-carbon unit of S-adenosylmethionine involved in methylation reactions originates almost solely from C3 of serine. On the other hand, flow of mitochondrial one-carbon units to 10-formyltetrahydrofolate for purine synthesis is shown to occur via both serine and formate. Formate transport accounts for at least 25% of the total, even during growth with sufficient serine to provide for the one-carbon requirements of the cell. This work shows that the synthetase function of the cytosolic C1-tetrahydrofolate synthase plays a critical role in the processing of mitochondrial one-carbon units to 10-formyltetrahydrofolate pools. In addition, this study provides evidence of two pools of glycine within the mitochondria and establishes a system of analyzing flux into the different folate derivatives.

Selective generation of charge-cependent/independent ion energy distributions from a heated capillary electrospray source.

Retarding grid and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry variable trap potential measurements are performed to determine factors that contribute to the kinetic energy distribution of ions formed in an electrospray source that uses a heated capillary for desolvation. The control of ion kinetic energies is achieved by manipulating the skimmer position in the postcapillary expansion and by varying the potential appEed to the skimmer. The selective generation of either charge-dependent or charge-independent ion energy distributions is demonstrated. Charge-dependent energy distributions of electro-sprayed ions are created by sampling ions near the Mach disk of the supersonic expansion and by using a larger diameter skimmer orifice; the FTICR spectra acquired under these conditions exhibit mass-to-charge ratio-dependent mass discrimination determined by the potential used to trap the ions. Charge-independent energies of electrosprayed ions are created by positioning the capillary adjacent to the skimmer to sample thermal ions and by using a smaller skimmer orifice to reduce expansion cooling; under these conditions ion kinetic energy is determined primarily by the skimmer potential and no mass-to-charge ratio-dependence is observed in the selection of optimum FTICR trapping conditions. The ability to select between proteins of different conformation on the basis of kinetic energy differences is demonstrated. For example, a 0.4 V difference in trap potential is observed in the selective trapping of open and closed forms of the +10 charge state of lysozyme. Finally, it is demonstrated that by operating the source under conditions which deliver a beam of ions with charge-independent energies to the cell, it is possible to obtain precursor and product ion signal magnitudes in FTTCR spectra without charge-dependent mass discrimina-tion.

Remeasurement of electrosprayed proteins in the trapped ion cell of a Fourier transform ion cyclotron resonance mass spectrometer.

A single population of multiply charged protein ions formed by electrospray ionization (ESI) is subjected to multiple Fourier transform ion cyclotron resonance (FTICR) excitation and detection events without promoting ion loss from the trapped ion cell. This nondestructive approach to mass spectrometric detection will allow detection limits to be significantly reduced by averaging the repetitive time-domain response that follows each excitation event. Under appropriate conditions, unit remeasurement efficiency is observed for large proteins; for example, 250 consecutive remeasurements of a single population of bovine albumin dimer ions (MW 132,532) yield the expected 16-fold improvement in signal-to-noise ratio compared to a single measurement. Examples presented include a 14-fmol sample of horse myoglobin (MW 16,951), which yields a 15:1 S/N for 50 remeasurements compared to a single scan S/N of 2:1 and a 30-fmol sample of bovine albumin dimer which exhibits a S/N of 25:1 for 50 remeasurements compared to a single scan S/N of 3:1. Of both practical and theoretical interest is the observation of a rapid collision-mediated homogeneous relaxation process that can return these large ions to the center of the cell within a few hundred milliseconds after excitation.

Two-dimensional coulomb-induced frequency modulation in Fourier transform ion cyclotron resonance: A mechanism for line broadening at high mass and for large ion populations.

Fourier transform ion cyclotron resonance (FTICR) spectra generated for large ion populations exhibit frequency shifts and line broadening, apparently due to Coulomb forces between ions. Although previous two-dimensional (2D) models of Coulomb effects in FTICR accounted for frequency shifts, they did not account for spectral line broadening. In this article, a 2D model is proposed that predicts line broadening due to Coulomb-induced frequency modulation. The model considers the case of two different-mass ions orbiting at their respective cyclotron frequencies around a common guiding center. A mutual modulation of the cyclotron frequency occurs at the difference frequency between ions. If the modulation period is much shorter than the FTICR observation time, then sidebands spaced at intervals approximately equal to the modulation frequency are predicted. However, if the modulation period is similar in duration to the FTICR observation period, the sidebands can no longer be resolved, which results in spectral line broadening. This latter case is a necessary consequence for isotopic peaks in the high mass region around m/z 2000, where deterioration in FTICR performance has been observed. Computer simulations are used to confirm the mass dependence and to demonstrate other features of the model, including a strong dependence of the modulation on ion number. In support of the model, experimental FTICR spectra for large populations of methylnaphthalene ions at m/z 141 and 142 exhibit constant frequency sidebands corresponding to multiples of the difference frequency for the two ions extending from nominal values of m/z 136 to 147.

Competitive ionization of tetraphenylporphyrin in a laser-generated metal ion plasma.

The ionization of tetraphenylporphyrin (TPP) in a laser-desorbed metal ion plasma is examined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Competitive reaction pathways observed to generate abundant molecular ion species include electron detachment, cation attachment, charge exchange, metallation, and transmetallation in the positive ion mode and electron capture, metallation, and transmetallation in the negative ion mode. In general, cation attachment reactions dominate positive ion spectra below the laser irradiance threshold for plasma ignition, although the metallation product from [TPP](+) reaction with the metal atom, M, is observed. Negative ion products are not observed in the FT-ICR spectrum when a plasma is not formed. Under plasma ignition conditions, positive ion spectra include [TPP](+) formed by charge exchange with M(+), which is also present in the spectrum. Negative ion spectra are dominated by [TPP](-); which is formed by attachment to thermal electrons generated in the plasma. Metallation reactions involving TPP and the metal substrate are examined. Positive ion metallation products are observed both in the absence of a plasma through reaction of [TPP](+) with M and by a second pathway under plasma ignition conditions through reaction of TPP with M(+). In negative ion mode, metallation is only observed under plasma ignition conditions through reaction of [TPP](-) with M. Observation of metallated products is found to be consistent with formation of stable metal oxidation states in the metallated porphyrin.

Concentric tube vacuum chamber for high magnetic field, high-pressure ionization in a fourier transform ion cyclotron resonance mass spectrometer.

A new differential pumping design for external source Fourier transform ion cyclotron resonance mass spectrometry is described. A network of concentric tubes of increasing diameter terminates at a series of conductance limits across which a pressure from atmosphere to low-10(-8) torr is achieved. This design permits high-pressure sources to be positioned within the solenoidal superconducting magnet less than 20 cm from the analyzer trapped ion cell. Ionization at high magnetic field offers the advantage of radial ion confinement and consequently delivers enhanced ion current to the trapped ion cell. Ion injection utilizing this vacuum chamber design is simpler than previously reported serial pumping stage designs because elaborate focusing optics to overcome the magnetic mirror effect are unnecessary. Two probe-mounted atmospheric pressure sources are described as evidence of the general applicability of the concentric tube vacuum chamber. An electrospray source that delivers several hundred picoamperes of ion current to the cell yields high-sensitivity spectra of proteins beyond 100 kDa. Improved pumping compared with a prototype concentric tube network configuration now permits mass resolution in excess of 20,000 for the [M + 4H](4+) ion of melittin. The resolution is sufficient to distinguish isotope peaks within a single charge state. A probe-mounted, pulsed-laser ablation source that permits cluster formation in the strong magnetic field is also demonstrated.

13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments. In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism. When yeast are incubated with [13C]formate, 13C NMR spectra establish that production of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Additional supporting evidence of reversibility of GCV in vivo is the production of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes. These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.