The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Quantifying reaction kinetics of the non-enzymatic decarboxylation of pyruvate and production of peroxymonocarbonate with hyperpolarized 13C-NMR.

The transient nature of intermediate states in chemical reactions has made their detailed investigation difficult. In this study, we demonstrate the utility of hyperpolarized 13C-NMR to directly observe and quantify the kinetics of the intermediate compound in the non-enzymatic decarboxylation of pyruvate via H2O2 with time resolutions of <1 s. Reactants were sequentially added to a reaction vessel within a 9.4 T NMR magnet while continuously acquiring spectra with a low flip angle, producing the first direct observation at room temperature of the previously proposed reaction intermediate, 2-hydroperoxy-2-hydroxypropanoate. We also performed a series of NMR experiments to determine the identity of a previously unidentified peak, which was found to be peroxymonocarbonate, the product of the side reaction between HCO3-/CO2 and H2O2/OOH-. Using the information obtained from these experiments, we developed a kinetic model which fully describes the mechanism of reaction and can be fit to experimental data to simultaneously determine multiple kinetic rate constants over several orders of magnitude. We also discuss the application of this reaction to the production of hyperpolarized bicarbonate for pH imaging experiments. This study presents a template for the use of hyperpolarized 13C-NMR to study the kinetics and reaction mechanisms of innumerable organic reactions which involve polarizable substrates.

In vivo imaging of the progression of acute lung injury using hyperpolarized [1-13 C] pyruvate.

PURPOSE: To investigate pulmonary metabolic alterations during progression of acute lung injury. METHODS: Using hyperpolarized [1-13 C] pyruvate imaging, we measured pulmonary lactate and pyruvate in 15 ventilated rats 1, 2, and 4 h after initiation of mechanical ventilation. Lung compliance was used as a marker for injury progression. 5 untreated rats were used as controls; 5 rats (injured-1) received 1 ml/kg and another 5 rats (injured-2) received 2 ml/kg hydrochloric acid (pH 1.25) in the trachea at 70 min. RESULTS: The mean lactate-to-pyruvate ratio of the injured-1 cohort was 0.15 ± 0.02 and 0.15 ± 0.03 at baseline and 1 h after the injury, and significantly increased from the baseline value 3 h after the injury to 0.23 ± 0.02 (P = 0.002). The mean lactate-to-pyruvate ratio of the injured-2 cohort decreased from 0.14 ± 0.03 at baseline to 0.08 ± 0.02 1 h after the injury and further decreased to 0.07 ± 0.02 (P = 0.08) 3 h after injury. No significant change was observed in the control group. Compliance in both injured groups decreased significantly after the injury (P < 0.01). CONCLUSIONS: Our findings suggest that in severe cases of lung injury, edema and hyperperfusion in the injured lung tissue may complicate interpretation of the pulmonary lactate-to-pyruvate ratio as a marker of inflammation. However, combining the lactate-to-pyruvate ratio with pulmonary compliance provides more insight into the progression of the injury and its severity. Magn Reson Med 78:2106-2115, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

The use of hyperpolarized carbon-13 magnetic resonance for molecular imaging.

Until recently, molecular imaging using magnetic resonance (MR) has been limited by the modality's low sensitivity, especially with non-proton nuclei. The advent of hyperpolarized (HP) MR overcomes this limitation by substantially enhancing the signal of certain biologically important probes through a process known as external nuclear polarization, enabling real-time assessment of tissue function and metabolism. The metabolic information obtained by HP MR imaging holds significant promise in the clinic, where it could play a critical role in disease diagnosis and therapeutic monitoring. This review will provide a comprehensive overview of the developments made in the field of hyperpolarized MR, including advancements in polarization techniques and delivery, probe development, pulse sequence optimization, characterization of healthy and diseased tissues, and the steps made towards clinical translation.

In vivo pH mapping of injured lungs using hyperpolarized [1-13 C]pyruvate.

PURPOSE: To optimize the production of hyperpolarized 13 C-bicarbonate from the decarboxylation of hyperpolarized [1-13 C]pyruvate and use it to image pH in the lungs and heart of rats with acute lung injury. METHODS: Two forms of catalysis are compared calorimetrically to maximize the rate of decarboxylation and rapidly produce hyperpolarized bicarbonate from pyruvate while minimizing signal loss. Rats are injured using an acute lung injury model combining ventilator-induced lung injury and acid aspiration. Carbon images are obtained from both healthy (n = 4) and injured (n = 4) rats using a slice-selective chemical shift imaging sequence with low flip angle. pH is calculated from the relative HCO3- and CO2 signals using the Henderson-Hasselbalch equation. RESULTS: It is demonstrated that base catalysis is more effective than metal-ion catalysis for this decarboxylation reaction. Bicarbonate polarizations up to 17.2% are achieved using the base-catalyzed reaction. A mean pH difference between lung and heart of 0.14 pH units is measured in the acute lung injury model. A significant pH difference between injured and uninjured lungs is also observed. CONCLUSION: It is demonstrated that hyperpolarized 13 C-bicarbonate can be efficiently produced from the base-catalyzed decarboxylation of pyruvate. This method is used to obtain the first regional pH image of the lungs and heart of an animal. Magn Reson Med 78:1121-1130, 2017. © 2016 International Society for Magnetic Resonance in Medicine.