In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.

Nowadays, it is known that oxidative stress plays at least two roles within the cell, the generation of cellular damage and the involvement in several signaling pathways in its balanced normal state. So far, a substantial amount of time and effort has been expended in the search for a clear link between cardiovascular disease (CVD) and the effects of oxidative stress. Here, we present an overview of the different sources and types of reactive oxygen species in CVD, highlight the relationship between CVD and oxidative stress and discuss the most prominent molecules that play an important role in CVD pathophysiology. Details are given regarding common pharmacological treatments used for cardiovascular distress and how some of them are acting upon ROS-related pathways and molecules. Novel therapies, recently proposed ROS biomarkers, as well as future challenges in the field are addressed. It is apparent that the search for a better understanding of how ROS are contributing to the pathophysiology of CVD is far from over, and new approaches and more suitable biomarkers are needed for the latter to be accomplished.

Progressive CKD is generally detected at a late stage by a sustained decline in eGFR and/or the presence of significant albuminuria. With the aim of early and improved risk stratification of patients with CKD, we studied urinary peptides in a large cross-sectional multicenter cohort of 1990 individuals, including 522 with follow-up data, using proteome analysis. We validated that a previously established multipeptide urinary biomarker classifier performed significantly better in detecting and predicting progression of CKD than the current clinical standard, urinary albumin. The classifier was also more sensitive for identifying patients with rapidly progressing CKD. Compared with the combination of baseline eGFR and albuminuria (area under the curve [AUC]=0.758), the addition of the multipeptide biomarker classifier significantly improved CKD risk prediction (AUC=0.831) as assessed by the net reclassification index (0.303±−0.065; P<0.001) and integrated discrimination improvement (0.058±0.014; P<0.001). Correlation of individual urinary peptides with CKD stage and progression showed that the peptides that associated with CKD, irrespective of CKD stage or CKD progression, were either fragments of the major circulating proteins, suggesting failure of the glomerular filtration barrier sieving properties, or different collagen fragments, suggesting accumulation of intrarenal extracellular matrix. Furthermore, protein fragments associated with progression of CKD originated mostly from proteins related to inflammation and tissue repair. Results of this study suggest that urinary proteome analysis might significantly improve the current state of the art of CKD detection and outcome prediction and that identification of the urinary peptides allows insight into various ongoing pathophysiologic processes in CKD.