Chemical Exchange Saturation Transfer Experiments Research Articles

Measurement of Proton Chemical Shifts in Invisible States of Slowly Exchanging Protein Systems by Chemical Exchange Saturation Transfer

Chemical exchange saturation transfer (CEST) NMR spectroscopy has emerged as a powerful technique for studies of transiently formed, sparsely populated (excited) conformational states of protein molecules in slow exchange with a dominant structure. The most popular form of the experiment, and the version originally developed, uses a weak (1)H radio frequency field to perturb longitudinal magnetization of one state with the effect transferred to magnetization in the second conformation via chemical exchange. A significant limitation of the method for protein applications emerges from (1)H magnetization transfer via dipolar relaxation (NOE effect) that can severely complicate analysis of the resulting CEST profile. This is particularly an issue since the (1)H chemical shifts of the excited state, critical for structural studies of these elusive conformers, become difficult to extract. Here we present a method for measurement of these shifts via CEST experiments in which the NOE effect is not an issue. The methodology is illustrated through applications to a pair of exchanging systems where the results are cross-validated.

Evaluating the use of a continuous approximation for model-based quantification of pulsed chemical exchange saturation transfer (CEST)

Many potential clinical applications of chemical exchange saturation transfer (CEST) have been studied in recent years. However, due to various limitations such as specific absorption rate guidelines and scanner hardware constraints, most of the proposed applications have yet to be translated into routine diagnostic tools. Currently, pulsed CEST which uses multiple short pulses to perform the saturation is the only viable irradiation scheme for clinical translation. However, performing quantitative model-based analysis on pulsed CEST is time consuming because it is necessary to account for the time dependent amplitude of the saturation pulses. As a result, pulsed CEST is generally treated as continuous CEST by finding its equivalent average field or power. Nevertheless, theoretical analysis and simulations reveal that the resulting magnetization is different when the different irradiation schemes are applied. In this study, the quantification of important model parameters such as the amine proton exchange rate from a pulsed CEST experiment using quantitative model-based analyses were examined. Two model-based approaches were considered – discretized and continuous approximation to the time dependent RF irradiation pulses. The results showed that the discretized method was able to fit the experimental data substantially better than its continuous counterpart, but the smaller fitted error of the former did not translate to significantly better fit for the important model parameters. For quantification of the endogenous CEST effect, such as in amide proton transfer imaging, a model-based approach using the average power equivalent saturation can thus be used in place of the discretized approximation.

Studying “Invisible” Excited Protein States in Slow Exchange with a Major State Conformation

Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and characterize "invisible" excited protein states in slow exchange with a visible ground-state conformation (excited-state lifetimes ranging from ∼5 to 50 ms) is presented. This method, which is an adaptation of the chemical exchange saturation transfer (CEST) magnetic resonance imaging experiment, involves irradiating various regions of the spectrum with a weak B(1) field while monitoring the effect on the visible major-state peaks. The variation in major-state peak intensities as a function of frequency offset and B(1) field strength is quantified to obtain the minor-state population, its lifetime, and excited-state chemical shifts and line widths. The methodology was validated with (15)N CEST experiments recorded on an SH3 domain-ligand exchanging system and subsequently used to study the folding transition of the A39G FF domain, where the invisible unfolded state has a lifetime of ∼20 ms. Far more accurate exchange parameters and chemical shifts were obtained than via analysis of Carr-Purcell-Meiboom-Gill relaxation dispersion data.

PCEST: Positive contrast using Chemical Exchange Saturation Transfer

Chemical Exchange Saturation Transfer (CEST) contrast utilizes selective pre-saturation of a small pool of exchanging protons and subsequent detection of the decrease in bulk water signal. The CEST contrast is negative and requires detection of small signal change in the presence of a strong background signal. Here we develop a Positive CEST (pCEST) detection scheme utilizing the analogous nature of the CEST and off-resonance T1ρ experiments and exploring increased apparent relaxation rates in the presence of the selective pre-saturation. pCEST leads to the positive contrast, i.e., increased signal intensity as the result of the presence of the agent and RF pre-saturation. Simultaneously substantial background suppression is achieved. The contrast can be switched “ON” and “OFF”, similar to the original CEST.

Optimization of pulse train presaturation for CEST imaging in clinical scanners

Chemical exchange saturation transfer (CEST) imaging depends on the performance of radiofrequency saturation during the experiment. Scanner specifications, in particular limited pulse width and duty-cycle, and specific absorption rate guidelines restrict the full exploitation of CEST effects in clinical MR systems. The purpose of this study was to optimize techniques for effective pulse train presaturation for CEST imaging in a whole-body MR scanner. Theoretical analysis and simulations of the spectral properties of radiofrequency pulse trains demonstrated the significance of pulse width τ(P) and interpulse delay τ(D) for effective and selective labeling of a chemically exchanging proton pool. CEST experiments with model solutions, e.g., creatine dissolved in water, showed best performance of pulse trains with τ(P) = τ(D) = 100 msec, regarding minimum direct water saturation in z-spectra and distinct magnetization transfer ratio asymmetry that can be determined quantitatively. Saturation efficiency of trains of Gaussian-shaped radiofrequency pulses using this timing was evaluated in MR imagers with field strengths of 1.5, 3, and 7 T. The proposed saturation pulse train does not require hardware modifications, offers low specific absorption rate, and can be used in a standard clinical setup.

Spin-locking versus chemical exchange saturation transfer MRI for investigating chemical exchange process between water and labile metabolite protons

Chemical exchange saturation transfer (CEST) and spin-locking (SL) experiments were both able to probe the exchange process between protons of nonequivalent chemical environments. To compare the characteristics of the CEST and SL approaches in the study of chemical exchange effects, we performed CEST and SL experiments at varied pH and concentrated metabolite phantoms with exchangeable amide, amine, and hydroxyl protons at 9.4 T. Our results show that: (i) on-resonance SL is most sensitive to chemical exchanges in the intermediate-exchange regime and is able to detect hydroxyl and amine protons on a millimolar concentration scale. Off-resonance SL and CEST approaches are sensitive to slow-exchanging protons when an optimal SL or saturation pulse power matches the exchanging rate, respectively. (ii) Offset frequency-dependent SL and CEST spectra are very similar and can be explained well with an SL model recently developed by Trott and Palmer (J Magn Reson 2002;154:157-160). (iii) The exchange rate and population of metabolite protons can be determined from offset-dependent SL or CEST spectra or from on-resonance SL relaxation dispersion measurements. (iv) The asymmetry of the magnetization transfer ratio (MTR(asym)) is highly dependent on the choice of saturation pulse power. In the intermediate-exchange regime, MTR(asym) becomes complicated and should be interpreted with care.

Improvement in the contrast of CEST MRI via intermolecular double quantum coherences

The tremendous potential of chemical exchange saturation transfer (CEST) agents as an emerging class of magnetic resonance imaging contrast media has been demonstrated in recent years. In a CEST experiment, a high CEST contrast is always welcome. However, when the exchange rate is low, which may happen in exchangeable solute protons of low concentration, it is usually hard to obtain an excellent CEST efficiency. Recently, we noted that the intermolecular multiple quantum coherence signal is more sensitive to the changes of the magnetization magnitude than a conventional single quantum coherence signal. Consequently, it may be easier when used in obtaining a CEST contrast. In this note, a modified COSY (two-dimensional correlated spectroscopy) revamped with an asymmetric Z-gradient echo detection (CRAZED) sequence combined with an off-resonance saturation pulse followed by a standard spin-echo imaging sequence was designed to obtain a better CEST contrast image based on the intermolecular double quantum coherence method. An analytical expression was derived from a dipolar field theory. Experiments were performed on an agar–glucose phantom, and the results demonstrate the feasibility of our method.