Introduction to Proton Detection in Biological Samples under Ultra-Fast Magic Angle Spinning

Protons are characterized by having a natural abundance of more than 99.9 %, as well as a high gyromagnetic ratio. These attributes provide much greater detection sensitivity than those of ¹³C or ¹⁵N, making ¹H-detected NMR spectroscopy very attractive, not just for solution NMR. Soluble samples benefit from the fact that unwanted dipolar couplings are cancelled out by molecular tumbling, a feature that insoluble molecules lack. As a result, a strong network of homonuclear proton dipolar couplings dramatically broadens the line width of any ¹H signal in solid-state NMR.

Two approaches especially have been shown to overcome this obstacle; sample preparation using perdeuteration-reprotonation to dilute the dense proton network, and the use of increased magic angle spinning (MAS) speeds. Depending on the sample properties, ¹H dipolar couplings can be in the order of several tens of kHz. The higher the MAS rate, the more attenuated the unwanted ¹H dipolar couplings will be, resulting in better resolved ¹H signals and allowing for the use of polarization transfer using scalar couplings.

In the past, solid-state NMR was limited to moderate MAS rates (≤25 kHz) by probe designs. Since recent developments have seen a move towards small rotor sizes – for example Bruker 1.9, 1.3 and 0.7 mm H/C/N probes –the use of fast (40 to 60 kHz) and even ultra-fast (60 to 111 kHz) MAS became possible. Under these conditions, low power irradiation for heteronuclear decoupling and selective heteronuclear cross polarization is efficient, minimizing RF irradiation-induced sample heating.

In this Application Note, Bruker BioSpin introduces aspects ¹H-detection in solid-state NMR spectroscopy and presents three basic ¹H-detected pulse programs that allow initial protein backbone assignment under fast MAS. Although the three experiments yield different results, they share general pulse sequence building blocks, which are introduced in the Application Note as well. Furthermore, a detailed description of all necessary parameter optimization steps and recommended starting conditions are explained and listed.