Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented.

Retinal pigment epithelium (RPE) are specialized, multifunctional cells in the retina that form a monolayer of cuboidal, polarized cells adjoining the photoreceptor cells. The RPE are a critical component of the blood-retinal barrier, and they play essential functional roles for maintenance of retinal homeostasis and for support and health of photoreceptors. Age-dependent, progressive dysfunction and death of RPE cells and the resultant loss of photoreceptors contribute significantly to the development and progression of age-related macular degeneration (AMD) and other retinal degenerative diseases. Several different RPE cell culture models have been developed and utilized extensively as surrogates for cellular and molecular examinations of the RPE, and a large body of knowledge on RPE function in normal and pathological scenarios has been amassed in studies with cultured RPE. Proteomics has been an integral part of research efforts aimed to advance our understanding of RPE cell biology in health and disease. This review focuses on applications of proteomics to in vitro qualitative and quantitative investigation of human RPE cell culture models. The disease context discussed focuses on AMD.

Mitochondria are complex organelles that play critical roles in diverse aspects of cellular function. Heart disease and a number of other pathologies are associated with perturbations in the molecular machinery of the mitochondria. Therefore, comprehensive, unbiased examination of the mitochondrial proteome represents a powerful approach toward system-level insights into disease mechanisms. A crucial aspect in proteomics studies is design of bioanalytical strategies that maximize coverage of the complex repertoire of mitochondrial proteins. In this study, we evaluated the performance of gel-based and gel-free multidimensional platforms for profiling of the proteome in subsarcolemmal mitochondria harvested from rat heart. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and bioinformatics for protein identification. Across all three platforms, a total of 1043 proteins were identified. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage of the mitochondrial proteome. With this platform, 890 proteins with diverse physicochemical characteristics were identified; the mitochondrial protein panel encompassed proteins with various functional roles including bioenergetics, protein import, and mitochondrial fusion. Taken together, results of this study provide a large-scale view of the proteome in subsarcolemmal mitochondria from the rat heart, and aid in the selection of optimal bioanalytical platforms for differential protein expression profiling of mitochondria in health and disease.

In the sub-retinal pigment epithelium (sub-RPE) space of the aging macula, deposits of oxidized phospholipids, oxidized derivatives of cholesterol and associated oxidized low-density lipoproteins (OxLDL) are considered contributors to the onset and development of age-related macular degeneration (AMD). We investigated the gene expression response of a human-derived RPE cell line exposed for short periods of time to non-cytotoxic levels of OxLDL or LDL. In our cell model, treatment with OxLDL, but not LDL, generated an early gene expression response which affected more than 400 genes. Gene pathway analysis unveiled gene networks involved in the regulation of various cellular functions, including acute response to oxidative stress via up-regulation of antioxidative gene transcripts controlled by nuclear factor erythroid-2 related factor 2 (NRF2), and up-regulation of aryl hydrocarbon receptor-controlled detoxifying gene transcripts. In contrast, circadian rhythm-controlling genes and genes involved in lipid metabolism were strongly down-regulated. Treatment with low-density lipoprotein (LDL) did not induce the regulation of these pathways. These findings show that RPE cells are able to selectively respond to the oxidized forms of LDL via the up-regulation of gene pathways involved in molecular mechanisms that minimize cellular oxidative damage, and the down-regulation of the expression of genes that regulate the intracellular levels of lipids and lipid derivatives. The effect on genes that control the cellular circadian rhythm suggests that OxLDL might also disrupt the circadian clock-dependent phagocytic activity of the RPE. The data reveal a complex cellular response to a highly heterogeneous oxidative stress-causing agent such as OxLDL commonly present in drusen formations.