Optimization of 1 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 13 C( t 1 ) dimension with inverted phase corresponding to 1 H N resonance frequencies, with approximately 5% intensity of the parent 13 C crosspeaks. These artifacts originate from the modulation of the 1 H N frequency onto the resonance frequency of 13 Cα and/or 13 Cβ and are due to 180° pulses imperfections used for 1 H decoupling during the 13 C( t 1 ) evolution period. These sidebands can become severe for CA i , CA i−1 and/or CB i , CB i−1 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 1 H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.