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 H(N) polarisation (selected by the main transfer path) from unused H(u) 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, H(u) 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% H(u) 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 tau much shorter than optimal (tau(opt) = 1.25 x T1(H(N))). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays tau(opt) can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.
Diercks
Tammo
T
Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584, CH, Utrecht, The Netherlands. tammo@nmr.chem.uu.nl
Daniels
Mark
M
Kaptein
Robert
R
D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D011522:Protons; D012680:Sensitivity and Specificity
D011506:Proteins; D011522:Protons