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 13C(t1) dimension with inverted phase corresponding to 1HN resonance frequencies, with approximately 5% intensity of the parent 13C crosspeaks. These artifacts originate from the modulation of the 1HN frequency onto the resonance frequency of 13Cα and/or 13Cβ and are due to 180° pulses imperfections used for 1H decoupling during the 13C(t1) evolution period. These sidebands can become severe for CAi, CAi−1 and/or CBi, CBi−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 1H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.
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Acknowledgements This work is financially supported by the NIH grants GM 100310 to G. V. and AI094623 to C.G.K.. The experiments were carried out at the Minnesota NMR Center (MNMR) and at the National Magnetic Resonance Facility at Madison (NMRFAM) [NIH support: P41GM103399 (formerly P41RR002301); P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, S10RR029220. NSF support: DMB-8415048, OIA-9977486, BIR-9214394]. Many thanks to Prof. E. Komives and Dr. T. Kromann-Tofting at UCSD for providing testing sample.
- Backbone assignment
- Pulse Imperfection
- Sideband artifacts
- TROSY-based triple resonance