Based on the first law of thermodynamics and the thermal diffusion equation, the deduced theoretical model of mitochondrial thermogenesis satisfies the Laplace equation and is a special case of the thermal diffusion equation. The model settles the long-standing question of the ability to increase cellular temperature by endogenous thermogenesis and explains the thermogenic characteristics of brown adipocytes. The model and calculations also suggest that the number of free available protons is the major limiting factor for endogenous thermogenesis and its speed.

Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography-mass spectrometry (LC-MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples-purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow-from sample preparation to LC-MS and data analysis-is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fastscanning, high-resolution, and accurate-mass LC-MS system.

Recent advances in optogenetics have established a precisely timed and cell-specific methodology for understanding the functions of brain circuits and the mechanisms underlying neuropsychiatric disorders. However, the fabrication of optrodes, a key functional element in optogenetics, remains a great challenge. Here, we report reliable and efficient fabrication strategies for chronically implantable optrode arrays. To improve the performance of the fabricated optrode arrays, surfaces of the recording sites were modified using optimized electrochemical processes. We have also demonstrated the feasibility of using the fabricated optrode arrays to detect seizures in multiple brain regions and inhibit ictal propagation in vivo. Furthermore, the results of the histology study imply that the electrodeposition of composite conducting polymers notably alleviated the inflammatory response and improved neuronal survival at the implant/neural-tissue interface. In summary, we provide reliable and efficient strategies for the fabrication and modification of customized optrode arrays that can fulfill the requirements of in vivo optogenetic applications.

A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ±218 and 2039 ±151 (mean ±SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ±23 nm (mean ±SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ±45 and 485 ±15 (mean ±SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases.

There is accumulating evidence that cysteine sulfenation (cys-SOH) in proteins plays an important role in cellular response to oxidative stress. The purpose of the present study was to identify mitochondrial proteins that undergo changes in cys-SOH during aging. Studies were conducted in rats when they were 5 or 30 months of age. Following blocking of free protein thiols with N-ethylmaleimide, protein sulfenic acids were reduced by arsenite to free thiol groups that were subsequently labeled with biotinmaleimide. Samples were then comparatively analyzed by two-dimensional Western blots, and proteins showing changes in sulfenation were selectively identified by mass spectrometry peptide sequencing. As a result, five proteins were identified. Proteins showing an age-related decrease in sulfenation include pyruvate carboxylase and pyruvate dehydrogenase; while those showing an age-related increase in sulfenation include aconitase, mitofilin, and tubulin (α-1). Results of the present study provide a general picture of mitochondrial protein sulfenation in brain oxidative stress and implicate the involvement of protein sulfenation in overall decline of mitochondrial function during brain aging.