Optimization of H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments.

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the C(t ) dimension with inverted phase corresponding to H resonance frequencies, with approximately 5% intensity of the parent C crosspeaks. These artifacts originate from the modulation of the H frequency onto the resonance frequency of Cα and/or Cβ and are due to 180° pulses imperfections used for H decoupling during the C(t ) evolution period. These sidebands can become severe for CA, CA and/or CB, CB correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 H decoupling can also be improved by this method resulting in up to 60-80% increase in sensitivity.