Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.

Cross-platform comparison of methods for quantitative metabolomics of primary metabolism.

Quantitative metabolomics is under intense development, and no commonly accepted standard analytical technique has emerged, yet. The employed analytical methods were mostly chosen based on educated guesses. So far, there has been no systematic cross-platform comparison of different separation and detection methods for quantitative metabolomics. Generally, the chromatographic separation of metabolites followed by their selective detection in a mass spectrometer (MS) is the most promising approach in terms of sensitivity and separation power. Using a defined mixture of 91 metabolites (covering glycolysis, pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, redox metabolism, amino acids, and nucleotides), we compared six separation methods designed for the analysis of these mostly very polar primary metabolites, two methods each for gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). For analyses on a single platform, LC provides the best combination of both versatility and robustness. If a second platform can be used, it is best complemented by GC. Only liquid-phase separation systems can handle large polar metabolites, such as those containing multiple phosphate groups. As assessed by supplementing the defined mixture with (13)C-labeled yeast extracts, matrix effects are a common phenomenon on all platforms. Therefore, suitable internal standards, such as (13)C-labeled biomass extracts, are mandatory for quantitative metabolomics with any methods.