Abnormal serum concentrations of proteins in Parkinson's disease.

Blood serum was used to identify protein biomarkers for diagnosis of Parkinson's disease (PD) using analytically validated quantitative 2D-gel electrophoresis, and single variable and multivariate statistics. Using banked samples from a first medical center, we identified 57 specific protein spot biomarkers with disease-specific abnormal levels in serum of patients with PD, Alzheimer's disease, amyotrophic lateral sclerosis and similar neurodegenerative conditions (337 samples), when compared to age-matched normal controls (132 samples). To further assess their clinical usefulness in Parkinson's disease, we obtained prospective newly drawn blood serum samples from a second (56 PD, 30 controls) and third (6 PD, 48 controls) medical center. The protein concentrations of the 57 biomarkers were assessed by 2D-gel electrophoresis. Stepwise linear discriminant analysis selected a combination of 21 of the 57 as optimal to distinguish PD patients from controls. When applied to the samples from the second site, the 21 proteins had sensitivity of 93.3% (52 of 56 PD correctly classified), specificity of 92.9% (28 of 30 controls correctly classified); 15 of 15 patients with mild, 28 of 30 with moderate to severe symptoms, and all of the 6 PD patients from the third site were correctly classified. Eleven of the 21 proteins showed statistically significant abnormal concentrations in patients with mild symptoms, and 14 in patients with moderate-severe symptoms. The protein identities reflect the heterogeneity of Parkinson's disease, and thus may provide the capability of monitoring the blood for a diverse range of PD pathophysiological mechanisms: cellular degeneration, oxidative stress, inflammation, and transport.

Alterations in Barrett's-related adenocarcinomas: a proteomic approach.

In this study, we applied high-resolution, two-dimensional, gel electrophoresis and matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry analysis (MALDI TOF MS) to identify novel proteins that are involved in Barrett's tumorigenesis. We analyzed 12 primary tissue samples that included 8 Barrett's-related adenocarcinomas (BA) and 4 normal mucosae samples. Twenty-three spots were consistently altered (>or=2-fold) in at least half of the tumors when compared with all normal samples and thus subjected to further analysis. The MALDI TOF MS analysis demonstrated biologically interesting upregulated proteins such as ErbB3, Dr5 and Cyclin D1 as well as several members of the zinc finger proteins (Znf146, Znf212 and Znf363). Examples of downregulated proteins included Lgi1 and Klf6. We selected four proteins (ErbB3, Dr5, Znf146 and Lgi1) that are novel for BAs for validation using quantitative real-time reverse-transcription PCR on 39 BA tissue samples when compared with normal samples. We demonstrated mRNA upregulation of ERBB3 (51.3%), DR5 (41%) and ZNF146 (30.7%) and downregulation of LGI1 (100%) in BA. We have further validated the protein overexpression of ErbB3, Dr5 and Znf146, using immunohistochemical (IHC) analysis on a tissue microarray that contained 75 BAs and normal gastric and esophageal mucosae samples. BA tissue samples demonstrated overexpression of ErbB3 (42%), Dr5 (90%) and Znf146 (30%) when compared with normal tissues. In conclusion, we have identified and validated several novel proteins that are involved in Barrett's carcinogenesis.

Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson's disease.

We have used quantitative 2D gel electrophoresis to analyze serum proteins from 422 patients with neurodegenerative diseases and normal individuals in an unbiased approach to identify biomarkers. Differences in abnormal serum levels were found between amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and related disorders for 34 protein biomarker spots, nine of which were related to the complement system. Of these nine, four spots originated from the Complement C3b-alpha-chain (C3c(1), C3c(2a), C3c(2b), and C3dg). The C3c spots (C3c(1), C3c(2a), and C3c(2b)) had the same amino acid sequence and glycosylation, though only C3c(1) was phosphorylated. In addition, Complement Factors H, Bb, and Pre-Serum amyloid protein displayed different serum concentrations in ALS, PD, and normal sera, whereas Complement C4b gamma-chain and Complement Factor I did not. The differential expression of the complement proteins provides potentially useful biomarkers as well as evidence for the involvement of inflammatory processes in the pathogenesis of ALS and PD.

2D gel blood serum biomarkers reveal differential clinical proteomics of the neurodegenerative diseases.

This review addresses the challenges of neuroproteomics and recent progress in biomarkers and tests for neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The review will discuss how the application of quantitative 2D gel electrophoresis, combined with appropriate single-variable and multivariate biostatistics, allows for selection of disease-specific serum biomarkers. It will also address how the use of large cohorts of specifically targeted patient blood serum samples and complimentary age-matched controls, in parallel with the use of selected panels of these biomarkers, are being applied to the development of blood tests to specifically address unmet pressing needs in the differential diagnosis of these diseases, and to provide potential avenues for mechanism-based drug targeting and treatment monitoring. While exploring recent findings in this area, the review discusses differences in critical pathways of immune/inflammation and amyloid formation between Parkinson's disease and amyotrophic lateral sclerosis, as well as discernable synergistic relationships between these pathways that are revealed by this approach. The potential for pathway measurement in blood tests for differential diagnosis, disease burden and therapeutic monitoring is also outlined.

Proteomic analysis of S-nitrosylated proteins in mesangial cells.

NO participates in numerous biological events in a variety of cell types including activated glomerular mesangial cells. Many of these events appear to be independent of the known effects of NO on soluble guanylyl cyclase. NO derived from all major isoforms of NO synthase can S-nitrosylate cysteine residues in target proteins, potentially altering their functional activities. Recent evidence suggests that S-nitrosylation is specific, is regulated, and may play an important regulatory role akin to phosphorylation. In the present study, the "biotin-switch" method of isolating S-nitrosylated proteins was coupled with two-dimensional PAGE protein separation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to identify target proteins for S-nitrosylation in murine mesangial cells treated with NO donors or appropriate controls. This approach resolved 790 protein spots. We analyzed the most abundant spots and identified 34 known proteins. Of these, 31 are unique S-nitrosylated proteins not previously identified, including signaling proteins, receptors and membrane proteins, cytoskeletal or cell matrix proteins, and cytoplasmic proteins. Prominent among these were peroxisome proliferator activated receptor gamma, uroguanylin, GTP-binding protein alpha, protein 14-3-3, NADPH-cytochrome P450 oxidoreductase, transcription factor IIA, melusin, mitosin, phospholipase A2-activating protein, and protein-tyrosine phosphatase. The in vivo induction of S-nitrosylation was assayed by treating mesangial cells with interleukin-1beta followed by the biotin-switch and Western blot of selected targets. These results broaden our knowledge of potential signal transduction pathways and other cell functions mediated by NO S-nitrosylation.

Identification of distinct protein expression patterns in bilateral matched pair breast ductal fluid specimens from women with unilateral invasive breast carcinoma. High-throughput biomarker discovery.

BACKGROUND: Analysis of the biochemical and cellular contents of breast ductal fluid has recently gained attention as a potential noninvasive method for studying the local microenvironment associated with the development and progression of breast carcinoma. METHODS: Patients with unilateral primary invasive breast carcinoma were eligible for the current prospective pilot study. Nipple aspiration fluid (NAF) was obtained from the breast with cancer and the normal contralateral breast and subjected to two-dimensional electrophoresis. Computer-assisted image analysis was used to analyze NAF protein expression profiles. RESULTS: The number of separate protein spots detected in NAF samples ranged from 1280 to 1649. Substantial qualitative differences were identified between NAF protein expression patterns in the breast with cancer compared with the breast without cancer. Protein spots detected in the breast with cancer and not in the breast without cancer from the same patient varied from 30 to 202 different proteins. In addition, the number of protein spots detected in the breast without cancer and not in the breast with cancer of the same patient varied from 14 to 73 different proteins. Conversely, in an individual without breast carcinoma, only three protein spots were detected in the left breast but not the right breast, and only two were detected in the right breast but not the left breast. CONCLUSIONS: The breast is a unique organ in that its microenvironment can be readily accessed and evaluated by aspiration of fluid from the nipple. Breast ductal fluid contains a large number of proteins. As breasts are paired organs, comparisons of ductal fluid from a breast with cancer and the same patient's normal contralateral breast may reveal significant differences in protein expression associated with breast carcinoma. Recent advances in image analysis, automated mass spectrometry, and bioinformatics have provided the tools necessary to use ductal fluids from breast carcinoma patients for high-throughput biomarker discovery.