Journal of Biomolecular NMR

, Volume 33, Issue 4, pp 243–259

Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments



In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the “longitudinal” TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898–12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014–8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of 15N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non−15N-bound protons. The underlying orthogonal separation of HN polarisation (selected by the main transfer path) from unused Hu polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, Hu polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein’s relaxation properties and the extended flip-back scheme employed, we recovered up to 60% Hu equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays τ much shorter than optimal (τopt = 1.25 · T1(HN)). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays τopt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.


extended flip-back fast pulsing regime magnetisation recovery orthogonal polarisation separation resolution enhancement sensitivity enhancement 



band-selective flip-back


continuous pulsing decoupling


extended flip-back


unselected proton magnetisation




universal flip-back


water flip-back


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Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Department of NMR Spectroscopy, Bijvoet Center for Biomolecular ResearchUtrecht UniversityUtrechtThe Netherlands

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