Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides. Molecular & Cellular Proteomics 9:2424 -2437, 2010.From the Departments of a Chemistry and

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O À:2 leads to formation of peroxynitrite ONOO -, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration. Keywords: Alzheimer's disease, neurodegeneration, 3-nitrotyrosine, proteomics, reactive nitrogen species. 2 leads to formation of peroxynitrate ONOO ) , which, following protonation, generates cytotoxic species that oxidize and nitrate proteins (Beckman 1996). (Formally, the NO 2 added to tyrosine is a nitrite, not nitrate, but the literature has consistently used the latter term, so we shall as well in this paper.)The more common amino acidic targets of oxidation are lysine, histidine, cysteine and methionine (Butterfield and Stadtman 1997), whereas tyrosine is the commonly nitrated amino acid (Souza et al. 2001). In particular, nitration of tyrosine residues is a formal oxidation (Butterfield and Stadtman 1997), a chemical modification that has been used to investigate the mechanism by which tyrosine residues Received November 11, 2002; revised manuscript received February 19, 2003; accepted February 20, 2003. Address correspondence and reprint requests to Professor D. Allan Butterfield, Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA. E-mail: dabcns@uky.eduAbbreviations used: AD, Alzheimer's disease; HCNP, hippocampal cholinergic neurostimulating peptide; IPL, inferior parietal lobule; 3NT, 3-nitrotyrosine; PEBP, phosphatidylethanolamine-binding protein; PBST, phosphate-buffered saline with 0.01% sodium azide and 0.2% Twe...

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Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for laborintensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L)1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme a-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.