Triple Resonance Method Research Articles

Triple Resonance Methods to Improve Performance of Ion Trap Precursor and Neutral Loss Scans.

Two experiments are described that extend the capabilities of quadrupole ion trap mass spectrometers operated in the precursor and neutral loss scan mode. The first experiment, a triple resonance precursor ion scan, is used to enhance sensitivity, selectivity, and molecular coverage. This method augments the ion trap precursor ion scan with the application of a second excitation frequency to selectively activate first-generation (MS2) product ions as they are formed and produce second-generation (MS3) product ions, which are then mass-selectively ejected with a third auxiliary signal and detected. This single mass analyzer experiment can be equated to performing the sequential precursor ion scan in a multiple analyzer system (Anal. Chem. 1990, 62 (17), 1809-1818). The second capability demonstrated is "frequency tagging", a method used to differentiate between ions ejected due to inherent instability under given trapping conditions, which causes artifacts during these scans, and ions that are resonantly ejected by the product ion ejection frequency. Beat frequencies are used to modulate resonance ejection peaks but conveniently do not modulate boundary ejection peaks. Frequency tagging provides a mechanism to identify the artifact peaks that are a consequence of operating at a high trapping voltage (i.e., low mass cutoff) for optimal precursor/product ion selectivity. The experiment is demonstrated for precursor and for neutral loss scans.

NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine.

A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1H-13C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.

NMR assignments of sparsely labeled proteins using a genetic algorithm.

Sparse isotopic labeling of proteins for NMR studies using single types of amino acid (15N or 13C enriched) has several advantages. Resolution is enhanced by reducing numbers of resonances for large proteins, and isotopic labeling becomes economically feasible for glycoproteins that must be expressed in mammalian cells. However, without access to the traditional triple resonance strategies that require uniform isotopic labeling, NMR assignment of crosspeaks in heteronuclear single quantum coherence (HSQC) spectra is challenging. We present an alternative strategy which combines readily accessible NMR data with known protein domain structures. Based on the structures, chemical shifts are predicted, NOE cross-peak lists are generated, and residual dipolar couplings (RDCs) are calculated for each labeled site. Simulated data are then compared to measured values for a trial set of assignments and scored. A genetic algorithm uses the scores to search for an optimal pairing of HSQC crosspeaks with labeled sites. While none of the individual data types can give a definitive assignment for a particular site, their combination can in most cases. Four test proteins previously assigned using triple resonance methods and a sparsely labeled glycosylated protein, Robo1, previously assigned by manual analysis, are used to validate the method and develop a criterion for identifying sites assigned with high confidence.

Successive Resonances for Ion Ejection at Arbitrary Frequencies in an Ion Trap.

The use of successive resonances for ion ejection is demonstrated here as a method of scanning quadrupole ion traps with improvement in both resolution and sensitivity compared with single frequency resonance ejection. The conventional single frequency resonance ejection waveform is replaced with a dual-frequency waveform. The two included frequencies are spaced very closely and their relative amplitudes are adjusted so that the first frequency that ions encounter excites them to higher amplitudes where space charge effects are less prominent, thereby giving faster and more efficient ejection when the ions come into resonance with the second frequency. The method is applicable at any arbitrary frequency, unlike double and triple resonance methods. However, like double and triple resonance ejection, ejection using successive resonances requires the rf and AC waveforms to be phase-locked in order to retain mass accuracy and mass precision. The improved performance is seen in mass spectra acquired by rf amplitude scans (resonance ejection) as well as by secular frequency scans. Graphical Abstract ᅟ.

Towards high precision measurements of nuclear g-factors for the Be isotopes

We describe the present status of future high-precision measurements of nuclear g-factors utilizing laser-microwave double and laser-microwave-rf triple resonance methods for online-trapped, laser-cooled radioactive beryllium isotope ions. These methods have applicability to other suitably chosen isotopes and for beryllium show promise in deducing the hyperfine anomaly of 11Be with a sufficiently high precision to study the nuclear magnetization distribution of this one-neutron halo nucleus in a nuclear-model-independent manner.

Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy.

Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solubilize the protein in a low-viscosity solvent in which the rapid tumbling of the resulting particle allows for use of standard triple-resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system.

Determining the antiproton magnetic moment from measurements of the hyperfine structure of antiprotonic helium

Recent progress in the spectroscopy of antiprotonic helium has allowed for measuring the separation between components of the hyperfine structure (hfs) of the (37,35) metastable states with an accuracy of $300\phantom{\rule{0.3em}{0ex}}\mathrm{kHz}$, equivalent to a relative accuracy of $3\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}5}$. The analysis of the uncertainties of the available theoretical results on the antiprotonic helium hfs shows that the accuracy of the value of the dipole magnetic moment of the antiproton (currently known to only 0.3%) may be improved by up to two orders of magnitude by measuring the splitting of appropriately selected components of the hfs of any of the known metastable states. The feasibility of the proposed measurement by means of an analog of the triple resonance method is also discussed.

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

Coupled-channel analysis of collisional effects on HFS transitions in antiprotonic helium atoms

Collisions of metastable antiprotonic helium with atoms of a medium induce transitions between hyperfine structure sublevels as well as shifts and broadenings of the microwave M1 spectral lines. We consider these phenomena in the framework of a model with scalar and tensor interactions between and He atoms. S-matrix is obtained by solving coupled-channels equations involving 4 HFS sublevels (F = L ± 1/2, J = F ± 1/2) of the nL level and relative angular momenta up to l = 5 at the kinetic energy E ≲ 25 K. The inelastic cross sections are less than elastic ones by four orders of value for the single electron or antiproton spin-flip transitions (ΔJ = ±1, ΔF = 0, ± 1) and by seven orders of value for the double spin-flip transitions (ΔJ = 0, ± 2, ΔF = ±1). The cross sections show a resonance behaviour at very low energy (E ∼ 1–4 K). The results for the relaxation rate λ(F− → F+), the frequency shift and broadening of the M1 spectral lines for the favoured transitions (ΔF = ±1, ΔJ = ±1) are compatible with the recent experimental data obtained by a triple (laser–microwave–laser) resonance method at T = 6 K. The calculated values are lowered by a factor of 1.5–2 with the temperature rising up to 25 K.

Stark shift of the A2Π1∕2 state in YbF174

We have measured the Stark shift of the A2Π1∕2–X2Σ+ transition in YbF. We use a molecular beam triple resonance method, with two laser transitions acting as pump and probe, assisted by an rf transition that tags a single hyperfine transition of the X state. After subtracting the known ground state Stark shift, we obtain a value of 70.3(1.5)Hz∕(V∕cm)2 for the static electric polarizability of the state A2Π1∕2(J=12,f) by fitting our data to a purely quadratic Stark shift in the interval 0–5kV∕cm. A more exact analysis that does not assume a perfectly quadratic Stark effect yields the value μe=2.48(3)D for the electric dipole moment of the A2Π1∕2(v=0) state.

Triple-Resonance Methods for Complete Resonance Assignment of Aromatic Protons and Directly Bound Heteronuclei in Histidine and Tryptophan Residues

A set of three experiments is described which correlate aromatic resonances of histidine and tryptophan residues with amide resonances in 13C/15N-labelled proteins. Provided that backbone 1H and 15N positions of the sequentially following residues are known, this results in sequence-specific assignment of histidine 1H(delta2)/13C(delta2) and 1H(epsilon1)/13C(epsilon1) as well as tryptophan 1H(delta1)/13C(delta1), 1H(zeta2)/13C(zeta2), 1H(eta2)/13C(eta2), 1H(epsilon3)/13C(epsilon3), 1H(zeta3)/13C(zeta3) and 1H(epsilon1)/15N(epsilon1) chemical shifts. In the reverse situation, these residues can be located in the 1H-(15)N correlation map to facilitate backbone assignments. It may be chosen between selective versions for either of the two amino acid types or simultaneous detection of both with complete discrimination against phenylalanine or tyrosine residues in each case. The linkages between delta-proton/carbon and the remaining aromatic as well as backbone resonances do not rely on through-space interactions, which may be ambiguous, but exclusively employ one-bond scalar couplings for magnetization transfer instead. Knowledge of these aromatic chemical shifts is the prerequisite for the analysis of NOESY spectra, the study of protein-ligand interactions involving histidine and tryptophan residues and the monitoring of imidazole protonation states during pH titrations. The new methods are demonstrated with five different proteins with molecular weights ranging from 11 to 28 kDa.

Peptides and the development of double‐ and triple‐resonance solid‐state NMR of aligned samples*

Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.

Using Trimethylphosphine as a Probe Molecule to Study the Acid Sites in Al−MCM-41 Materials by Solid-State NMR Spectroscopy

Using trimethylphosphine (TMP) as a probe molecule, acid sites in a series of Al-MCM-41 materials with Si/Al ratios ranging from 16 to 80 were investigated by various solid-state NMR techniques including Si-29, P-31, Al-27, H-1 magic-angle spinning (MAS), and some double- or triple-resonance methods such as P-31 --> H-1 cross polarization (CP), H-1/Al-27, and H-1/P-31/Al-27 TRAPDOR (transfer of population in double resonance). By means of the H-1/Al-27 TRAPDOR technique, which has the ability to establish a correlation between 1H and Al-27, the hydroxyl groups associated with Al could be discriminated from the silanol groups. Two signals at 3.5 and 1.9 ppm were observed in the H-1/Al-27 TRAPDOR spectra; they are likely due to the bridging hydroxyl (SiOHAl) and aluminum hydroxyl groups, respectively. P-31 MAS NMR strongly supports the formation of zeolitelike Bronsted acid sites in the mesoporous material after the incorporation of aluminum. The concentration of the Bronsted sites determined from P-31 MAS NMR is about 2 orders of magnitude lower than that of total SiOH groups, which renders their direct observation by H-1 MAS NMR spectroscopy difficult. However, these sites can be well studied by P-31 --> H-1 CP/MAS and various triple-resonance TRAPDOR NMR techniques, which provide a more detailed description of the interactions between TMP and the different hydroxyl groups in the Al-MCM-41 materials. Although the acid strength of the Bronsted sites in the Al-MCM-41 materials is lower than that of microporous zeolites such as HY, these sites can protonate TMP molecules. Some of the framework T-site aluminum atoms are not associated with the bridging hydroxyl groups and are probably present as Al-OH. No Lewis acid sites were found in our samples, though the Al-27 MAS spectra show the existence of five-coordinate extra framework aluminum.

Precision spectroscopy of the Zeeman splittings of the [formula omitted] hyperfine structure for nuclear structure studies

A precision measurement for the hyperfine Zeeman splittings of 9 Be + ions, trapped and laser cooled in a linear combined ion trap was performed by using laser–rf double and triple resonance methods. The accuracies of 10 −9 for the nuclear spin flip transitions and of 10 −8 for the electron spin flip transitions were achieved at an arbitrary value of strong magnetic field of ∼0.47 T. This enables us to determine the hyperfine constant A and the ratio of nuclear g-factor to electronic g-factor with accuracies of 10 −9 and 10 −7, respectively. To show the wider applicability of our method, the particular strength of the magnetic field where the nuclear spin flip transition is independent of the magnetic field strength was intensionally not used. The confining fields of the linear combined trap have the advantage of simple and efficient concentration of the ions into the potential minimum at the center of the trap, when laser cooling of the stored ions is performed. The feasibility of single ion spectroscopy was also demonstrated and, when fully implemented will facilitate the on-line study of short lived isotopes, which can only be produced in minute numbers.

Solution NMR Studies of a 42 KDa Escherichia Coli Maltose Binding Protein/β-Cyclodextrin Complex: Chemical Shift Assignments and Analysis

The use of deuteration in concert with uniform 15N,13C-labeling has been critical for the chemical shift assignment of several proteins and protein complexes over 30 kDa. Unfortunately, deuteration reduces the number of interproton distance restraints available for structure determination, compromising the precision and accuracy of the NMR-derived structures determined from these samples. We have recently described an isotopic labeling strategy that addresses this problem by generating proteins labeled uniformly with 15N, 13C, and extensively with 2H with high levels of protonation at exchangeable sites and the methyl groups of Val, Leu, and Ile (δ1 only) (Gardner, K. H.; Kay, L. E. J. Am. Chem. Soc. 1997, 119, 7599−7600). This labeling pattern maintains the high efficiency of triple resonance methods while retaining sufficient protons to establish long-range NOEs between secondary structure elements. We demonstrate the utility of samples labeled in this manner by presenting the chemical shift assignments of one of the largest monomeric proteins assigned to date, the 370 residue Escherichia coli maltose binding protein in complex with β-cyclodextrin (42 kDa). The high level of Cα and Cβ deuteration provided by our labeling scheme enabled the collection of triple resonance data with high sensitivity and resolution, allowing assignment of over 95% of the backbone 15N, 13Cα, 1HN, and side chain 13Cβ nuclei. By using a combination of existing experiments and a new pulse scheme described here for correlating methyl chemical shifts with 13Cβ (Val), 13Cγ (Leu), or 13Cγ1 (Ile) carbons, over 98% of methyl 13C and 1H assignments from Val, Leu, and Ile (Cδ1 only) have been obtained. Analysis of the backbone chemical shifts and qualitative HN exchange data have confirmed that the MBP/β-cyclodextrin complex has a secondary structure similar to that previously observed in a 1.8 Å crystal structure.

Triple-resonance method for the detection of hyperfine transitions in heavy-alkali metals

The triple-resonance method for the detection of hyperfine transitions in heavy-alkali metals is studied theoretically. The evolution equations of the reduced fictitious tensorial components of the dressed-atom density matrix in an extended fictitious formalism are determined and solved for the stationary state. Illustrative numerical examples of dressed-caesium are included. Previously unexplained triple-resonance experiments on rubidium and caesium are interpreted in detail.

EPR and Multiple Electron Resonance Spectroscopic Indication ofpara-Nitration of Aminyl Radicals

Anipso-nitration was detected at position 4 ofN-methoxy- andN-ethoxy-N-(4-carboxy-2,6-dinitrophenyl)aminyl radicals upon oxidation of the corresponding amines with lead tetraacetate in toluene and in methylene chloride. A very fast substitution by the nitro group was also observed for another capto-dative aminyl radical, namelyN-(3′,5′-di-tert-butylphenyl)-N-(4-carboxy-2,6-dinitrophenyl)aminyl. The hyperfine coupling constant of the aminyl nitrogen was established to be nearly the same for these aminyl radicals irrespective of the donor group bonded via the aminyl nitrogen to the polynitrophenyl group. The rotational correlation time of the aminyl radicals was determined. Kinetic measurements ofN-ethoxy-N-(p-carboxy-2,6-dinitrophenyl)aminyl radical at different temperatures (250–290 K) from EPR spectra provided further proof of the nitration reaction. Proton hyperfine couplings of a total of eight aminyl radicals, with their signs, were measured by ENDOR and TRIPLE resonance methods. The hyperfine coupling of aminyl nitrogen ofN-(3′,5′-di-tert-butylphenyl)-N-(2,6-dinitrophenyl)aminyl radical was also measured by ENDOR; hyperfine couplings of the nitro group nitrogens at the 2,6-positions could not be measured but had non-zero hyperfine coupling according to EPR simulation. Protons at the 2′,6′-positions of thetert-butyl-substituted phenyl ring were found to have unequal and invariant hyperfine coupling at temperatures from 200 to 270 K, and the diarylaminyl radical was concluded by SYBYL calculation to have a twisted (unequal 2′,6′-positions) structure.

An NMR Study of Poly(vinylidene fluoride) Structure by 1H, 13C, and 19F Triple Resonance Method

1H, 13C, and 19F triple resonance NMR experiments on poly(vinylidene fluoride) (PVDF) in solution were carried out in order to provide a detailed primary structural analysis of PVDF. The 19F NMR signals were measured by the 1H broad-band decoupled 19F J-correlated 2D NMR and 1H–19F J-correlated two dimensional (2D) methods, and the 13C NMR signals were measured by the 1H broad-band decoupled 19F–13C J-correlated 2D method. On the basis of the spectral analysis, a complete primary-structural analysis of PVDF was carried out. From these results, it was demonstrated that the 1H, 13C, and 19F triple resonance NMR method is a very useful means for analysing primary-structure of fluoropolymers.

An Alternative Triple-Resonance Method for the Through-Bond Correlation of Intranucleotide H1' and H8 NMR Signals of Purine Nucleotides. Application to a DNA Dodecamer with Fully 13C/15N-Labeled Deoxyadenosine Residues

An Alternative Triple-Resonance Method for the Through-Bond Correlation of Intranucleotide H1' and H8 NMR Signals of Purine Nucleotides. Application to a DNA Dodecamer with Fully 13C/15N-Labeled Deoxyadenosine Residues

Accurate measurement of homonuclear coupling constants using JHH-TOCSY

The homonuclear JHH coupling constant is one of the most important NMR parameters. In complex molecules with quite long relaxation times, E.COSY (1) in combination with the DISCO procedure (2) seems to be the best homonuclear method for quantifying J couplings. Other homonuclear methods are z-filtered COSY (3)) J-6 spectroscopy (4)) and selective excitation (5). These methods fail, however, in molecules with short spin-spin relaxation times where the linewidth is in the range of coupling constants. For these situations, some triple-resonance methods have been proposed recently (6-8), which can be used for 15Nand “Ccuenriched peptides and proteins. Their pulse sequences include a heteronuclear coherence transfer from “N to 13C. Additionally the magnetization transfer using a 13C spin lock works well for completely 13C-enriched molecules (9). In these spectra, the signal is split in Fi by the ‘JCH coupling constant, whereas the 3J(HN-H~) coupling constant can be extracted from the displacement in F2. From these methods, precisely estimated coupling constants are obtained, and they are, in distinction to E.COSY, independent of the linewidth. Until now no similar method has been proposed that could be used for molecules at natural abundance. For this purpose, we have developed two new pulse sequences shown in Figs. la and 1 b, called JHH-TOCSY. Sequence 1 a is a H,H correlation and sequence 1 b is a H,X correlation. As for the methods mentioned above, these sequences provide the JHH splitting for XH groups only, whereas XH2 groups show no splitting. For overlapping XH and XH2 groups, and for spectral simplification, XH selection may be used as shown in Fig. 1 c. Figure 1 d shows the 30 extension of sequence 1 a with and without XH selection. JHH-TOCSY is well suited to molecules at natural abundance or for 13Cor “N-monolabeled compounds. In comparison with the triple-resonance methods, the sequences are much shorter, because there is no need for the long 1 /( 2 JCN) delay. The evolution of the magnetization after 2, is as follows (only the relevant operators are shown); cf. Fig. la: