Detecting acetylated aminoacids in blood serum using hyperpolarized ¹³C-¹Η-2D-NMR

Highlights

• Applicability of dissolution DNP for metabolomics applications.

• Using naturally long-lived hyperpolarized tags for dissolution DNP.

• Acetylation Tags eliminate scaling of intensities based on T₁ in DNP spectra.

Abstract

Dynamic Nuclear Polarization (DNP) can substantially enhance the sensitivity of NMR experiments. Among the implementations of DNP, ex-situ dissolution DNP (dDNP) achieves high signal enhancement levels owing to a combination of a large temperature factor between 1.4 and 300 K with the actual DNP effect in the solid state at 1.4 K. For sufficiently long T₁ relaxation times much of the polarization can be preserved during dissolution with hot solvent, thus enabling fast experiments during the life time of the polarization. Unfortunately, for many metabolites found in biological samples such as blood, relaxation times are too short to achieve a significant enhancement. We have therefore introduced ¹³C-carbonyl labeled acetyl groups as probes into amino acid metabolites using a simple reaction protocol. The advantage of such tags is a sufficiently long T₁ relaxation time, the possibility to enhance signal intensity by introducing ¹³C, and the possibility to identify tagged metabolites in NMR spectra. We demonstrate feasibility for mixtures of amino acids and for blood serum. In two-dimensional dDNP-enhanced HMQC experiments of these samples acquired in 8 s we can identify acetylated amino acids and other metabolites based on small differences in chemical shifts.

Introduction

The dissolution Dynamic Nuclear Polarization (dDNP) experiment pioneered by Ardenkjær-Larsen et al. [1] can achieve signal enhancements at the order of magnitude of 10,000-fold, for nuclei with low gyromagnetic ratios such as ¹³C and ¹⁵N. These large enhancements arise from a combination of a temperature factor (for polarizations at temperatures <1.5 K and dissolution to room temperature) and the actual DNP effect [2], [3], [4]. The requirements of freezing the sample in a glass state and dissolving with hot solvent in order to transfer it to an NMR magnet for spectrum acquisition limits the selection of molecules amenable to this approach to those with sufficiently long T₁ relaxation times. Dissolution DNP has gained considerable importance as it forms the basis for what is now often termed chemical shift metabolic imaging [5], mainly using pyruvate as the polarization carrier. The limiting factors for dissolution DNP are the relatively long polarisation time and the need to carry out the experiment within the short time frame of the T₁ relaxation time of the analyte. As a consequence, a variety of systems were implemented with the ultimate goal of eliminating sample transfer time, either by using a pressurised sample delivery system [6], [7], by using dual-centre magnets [8] or recently by a solid pellet driven sample transfer approach [9]. Such implementations have enabled applications in protein folding and reaction monitoring [10], [11], [12], [13].

There have been several approaches to design probes with long life times which can preserve polarization over an extended period of time. For example, very long life-times are achieved for some ¹⁵N-bearing probes. Considerable research has been carried out to identify molecules with long-lived spin states, some have life-times greater than 15 min [14], [15], [16], [17]. However, only few probes can be attached to other molecules and are suitable to identify different molecules as part of a mixture in NMR experiments. This is where formyl and acetyl tags possess a significant advantage. As shown by Raftery and co-workers [18] the combined methyl-proton and ¹³CO chemical shifts of N-acetyl-tags have sufficient dispersion to distinguish 20 amino acids in a ‘long-range’ HSQC spectrum utilizing the small ²J_CH coupling constant between the methyl protons and the ¹³CO. Wilson et al. showed the utility of ¹³C-labeled acetyl probes in conjunction with dDNP for one-dimensional spectra of glycine, serine, valine and alanine [19]. The life-time of acetyl groups is sufficiently long for dDNP experiments (T₁ ≈ 40 s), especially when fast transfer systems such as high-pressure dissolution devices are used [15]. The T₁ of the acetyl group is longer than that of the other ¹³C-nuclei in amino acids, although shorter than typical long-lived tags [14], [15], [16], [17]. Here we show how acetyl probes can be used to obtain two-dimensional ¹³C-observed HMQC spectra after polarization of mixtures of acetylated amino acids. We also show that the dDNP approach yields sufficient sensitivity to identify a range of common amino acids in bovine blood serum after acetylating NH- and OH-bearing molecules in serum using ¹³C-labeled acetyl tags.