Performance Characteristics of Mass Spectrometry-Based Analytical Procedures for Quantitation of Nitrosamines in Pharmaceuticals: Insights from an Inter-laboratory Study.

With the discovery of carcinogenic nitrosamine impurities in pharmaceuticals in 2018 and subsequent regulatory requirements for risk assessment for nitrosamine formation during pharmaceutical manufacturing processes, storage or from contaminated supply chains, effective testing of nitrosamines has become essential to ensure the quality of drug substances and products. Mass spectrometry has been widely applied to detect and quantify trace amounts of nitrosamines in pharmaceuticals. As part of an effort by regulatory authorities to assess the measurement variation in the determination of nitrosamines, an inter-laboratory study was performed by the laboratories from six regulatory agencies with each of the participants using their own analytical procedures to determine the amounts of nitrosamines in a set of identical samples. The results demonstrated that accurate and precise quantitation of trace level nitrosamines can be achieved across multiple analytical procedures and provided insight into the performance characteristics of mass spectrometry-based analytical procedures in terms of accuracy, repeatability and reproducibility.

A selective 15N-to-(1)H polarization transfer sequence for more sensitive detection of 15N-choline.

The sensitivity and information content of heteronuclear nuclear magnetic resonance is frequently optimized by transferring spin order of spectroscopic interest to the isotope of highest detection sensitivity prior to observation. This strategy is extended to 15N-choline using the scalar couplings to transfer polarization from 15N to choline's nine methyl 1H spins in high field. A theoretical analysis of a sequence using nonselective pulses shows that the optimal efficiency of this transfer is decreased by 62% as the result of competing 15N-(1)H couplings involving choline's four methylene protons. We have therefore incorporated a frequency-selective pulse to support evolution of only the 15N-methyl 1H coupling during the transfer period. This sequence provides a 52% sensitivity enhancement over the nonselective version in in vitro experiments on a sample of thermally polarized 15N-choline in D2O. Further, the 15N T1 of choline in D2O was measured to be 217+/-38 s, the 15N-methyl 1H coupling constant was found to be 0.817+/-0.001 Hz, and the larger of choline's two 15N-methylene 1H coupling constants was found to be 3.64+/-0.0 1Hz. Possible improvements and applications to in vivo experiments using long-lived hyperpolarized heteronuclear spin order are discussed.

Dipolar dynamic frequency shifts in multiple-quantum spectra of methyl groups in proteins: correlation with side-chain motion.

Small deviations from the expected relative positions of multiplet components in double- and zero-quantum 1H-13C methyl correlation maps have been observed in spectra recorded on a 7-kDa protein. These dynamic frequency shifts (DFS) are the result of dipolar cross-correlations that derive from fields produced by the spins within the methyl groups. The shifts have been quantified and compared with values calculated from a Redfield analysis. Good agreement is noted between the signs of the predicted and experimentally observed relative shifts of lines in both F1 and F2 dimensions of spectra, as well as between the magnitudes of the calculated and observed shifts in the F2 (1H) dimension. The experimental DFS values show a reasonable correlation with 2H relaxation-derived measures of methyl side-chain dynamics, as expected from theory. This suggests that in cases where such shifts can be quantified, they can serve as qualitative measures of motion.

Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins.

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using 13CH3, 13CH2D, and 13CHD2 methyl isotopomers, is presented. For each labeling scheme, 1H-13C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with 13CH3, 13CH2D, and 13CHD2 labeling in Ile delta1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence's performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for 13CH3-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (13CH3 HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the 13CH3 HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both 13C and 1H spins. The HSQC and HMQC spectra of 13CH2D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, 13CH2D TROSY, that exploits cross-correlation effects to record 1H-13C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of 13CHD2-labeled methyl groups can be recorded with HSQC or HMQC; a new 13CHD2 HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the 13CH3 labeling strategy is the most beneficial from the perspective of sensitivity (gains > or =2.4 relative to either 13CH2D or 13CHD2 labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.

Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: an application to an 82-kDa enzyme.

New NMR experiments for the measurement of side-chain dynamics in high molecular weight ( approximately 100 kDa) proteins are presented. The experiments quantify (2)H spin relaxation rates in (13)CH(2)D or (13)CHD(2) methyl isotopomers and, for applications to large systems, offer significant gains both in sensitivity (2-3-fold) and resolution over previously published HSQC schemes. The methodology has been applied to investigate Ile dynamics in the 723-residue, single polypeptide chain enzyme, malate synthase G. Methyl-axis order parameters, S(axis), characterizing the amplitudes of motion of the methyl groups, have been derived from both (13)CH(2)D and (13)CHD(2) probes and are in excellent agreement. The distribution of order parameters is trimodal, reflecting the range of dynamics that are available to Ile residues. A reasonable correlation is noted between and inverse temperature factors from X-ray studies of the enzyme. The proposed methodology significantly extends the range of protein systems for which side-chain dynamics can be studied.

Sparsely populated folding intermediates of the Fyn SH3 domain: matching native-centric essential dynamics and experiment.

A complete description of how a protein folds requires the characterization of intermediate conformations traversed during the folding transition. We have calculated dynamics trajectories of a simplified model of the Fyn SH3 domain with a native-centric potential energy function. Analysis of the resulting site-resolved energy trajectory identifies an ensemble of intermediate conformations for folding and another for unfolding. The model's folding intermediate is structured in the three beta-strands that make up the protein's core and is strikingly similar to intermediates detected in a recent NMR study of Fyn SH3 folding and to folding transition states elucidated in mutagenesis studies of SH3 domains. The unfolding intermediate is formed by dissociation of the folded protein's two terminal beta-strands from its core. The presence of such an intermediate is consistent with the results of a protein-engineering study on the src SH3 domain showing that these strands separate before the rate-limiting step of unfolding. Despite the presence of these conformations intermediate between the native and fully unfolded states, the computed heat capacity vs. temperature profile of the model protein indicates that its thermodynamics satisfies the usual calorimetric criterion for two-state folding. This observation highlights the fact that, if not properly interpreted, methods such as calorimetry that do not probe multiple sites in a molecule can lead to an oversimplified view of folding. The close agreement between results from this simplified model and experimental work underscores the important contributions that computational methods can make in providing insights into protein folding.

Magnetic resonance realization of decoherence-free quantum computation.

We report the realization, using nuclear magnetic resonance techniques, of the first quantum computer that reliably executes a complete algorithm in the presence of strong decoherence. The computer is based on a quantum error avoidance code that protects against a class of multiple-qubit errors. The code stores two decoherence-free logical qubits in four noisy physical qubits. The computer successfully executes Grover's search algorithm in the presence of arbitrarily strong engineered decoherence. A control computer with no decoherence protection consistently fails under the same conditions.

Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes.

A comparison of HSQC and HMQC pulse schemes for recording (1)H[bond](13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. (1)H[bond](13)C correlation spectra are recorded on U-[(15)N,(2)H], Ile delta 1-[(13)C,(1)H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5 degrees C, and of (ii) the protease ClpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5 degrees C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.