Finally, XH n peaks in un-folded peptides and proteins may overlap with peaks from un-structured regions of a folded protein. In addition, CH n peaks also overlap with CH m (where n ≠ m) peaks, especially in spectra where peaks are folded. Įxperimental protein NMR spectra are typically collected in H 2O, where some protein backbone amide NH peaks overlap with side-chain NH 2 peaks. Interestingly, among all published 13C multiplicity- edited 2D experiments only the HSQC version was demonstrated on ubiquitin dissolved in D 2O. Additionally, generally, small molecules contain fewer overlapping resonances and have longer T 2 values compared to larger protein molecules.įor larger molecules like proteins, HSQC-based experiments have higher sensitivity than HMQC-based experiments, because, due to the transverse 1H magnetization during t 1, the HMQC-based experiments have additional T 2 decay. Most edited experiments were developed for and applied to small molecules at 13C natural abundance dissolved in deuterated solvents and are therefore multiple-quantum based experiments. Multiplicity- edited pulse sequences use a dedicated period of 1/ 1 J CH to edit CH 2 and CH/CH 3 peaks into the opposite phases in the same spectrum. Two-dimensional methods have become even more powerful with the advent of multiplicity- edited 1H– 13C HSQC, HMQC, HMBC and H2BC experiments. These methods are superior to homo-nuclear two-dimensional spectra because of their increased chemical shift dispersion, less homo-nuclear J-splitting, and concomitant reduced peak overlap. Two-dimensional hetero-nuclear 1H–X (X = 15N or 13C) spectral patterns are sensitive to chemical structure and therefore can detect structural change at the level of individual nuclei. There is an increasing need to fingerprint therapeutic biomolecules by solution NMR in formulated drug products at natural abundance. Furthermore we demonstrate improved water suppression using triple xyz-gradients instead of the more widely used z-gradient only water-suppression approach. In this pulse sequence, the 1/ 1 J XH editing- period is incorporated into the semi-constant time (semi-CT) X resonance chemical shift evolution period, which increases sensitivity, and importantly, the sum and the difference of the interleaved 1 J XH-active and the 1 J XH-inactive HSQC experiments yield two separate spectra for XH 2 and XH/XH 3. To meet these needs, a multiplicity- separated 1H–X HSQC (MS-HSQC) experiment was developed and tested on 500 and 700 MHz NMR spectrometers equipped with room temperature probes using RNase A (14 kDa) and retroviral capsid (26 kDa) proteins dissolved in 95% H 2O/5% D 2O. 15N), to resolve more peaks, to reduce T 2 losses and to accommodate water suppression approaches. Therefore, the existing 2D multiplicity- edited HSQC methods must be improved to acquire data on nuclei other than 13C ( i.e. However, there is an increasing need for using NMR to profile biomolecules at natural abundance dissolved in water ( e.g., protein therapeutics) where NMR experiments beyond 2D are impractical. By contrast, for larger biomolecules, peak overlap in 2D HSQC spectra is unavoidable and peaks with opposite phases cancel each other out in the edited spectra. ubiquitin) dissolved in deuterated solvents where, generally, peak overlap is minimal. Such CH n-editing experiments are useful in assignment of chemical shifts and have been successfully applied to small molecules and small proteins ( e.g. Multiplicity- edited 1H– 13C HSQC pulse sequences generate opposite signs between peaks of CH 2 and CH/CH 3 at a cost of lower signal-to-noise due to the 13C T 2 relaxation during an additional 1/ 1 J CH period. 2D NMR 1H–X (X = 15N or 13C) HSQC spectra contain cross-peaks for all XH n moieties.
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