This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.

Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted Bladder cancer (BC) 1 is the second in incidence and mortality cancer of the genitourinary system (1) and estimated to be the ninth most common malignancy (2). It is associated with a high recurrence rate underscoring the need for continuous surveillance following initial treatment. Cystoscopy still remains the gold standard for diagnosis and follow-up monitoring of bladder cancer. However, it is an invasive and unpleasant procedure, rendering particularly the regular surveillance program (e.g. cystoscopy every three months for the first year following initial diagnosis) not well accepted by the patients (3, 4). Urine Cytology is a noninvasive current detection tool for BC, suffering however from suboptimal sensitivity, especially for low grade tumors and being subjected to interobserver variability (5). The invasive nature of cystoscopy and the low effectiveness of cytology have prompted the search for novel and better ways to diagnose the disease with special emphasis on the early detection of disease recurrences and/or progression.Urine is regularly used in clinical practice and yields a wealth of information about the state of an individual's health. Because it can be collected in a noninvasive way it is more accessible than plasma or serum. In addition, there is no need for trained personnel for urine collection. Urine contains cells and cellular debris, inorganic ions (K ϩ , Na ϩ , Cl Ϫ , and Ca ϩ2 ), organic molecules (urea, uric acid, and creatinine) and proteins. If renal function is normal, urinary protein content is less

The paper describes an innovative approach to the use of gel free proteomics to identify the peptides that are present in milk during clinical mastitis, which is a major cause of loss of production to dairy farmers worldwide. The use of capillary electrophoresis, liquid chromatography and mass spectrometry has been able to identify panels of peptides which can be used for disease diagnosis and for differential diagnosis of the causative bacteria of the infections of the mammary gland. As well as contributing to our knowledge of the pathophysiology of bovine mastitis the results could be the basis of improved detection and differential diagnosis of the disease.

The protein components of urine are useful indicators of renal function and human health in general. Urine samples are easily attainable making them ideal substrates for biomarker research. Analysis of the urine proteome however, has been hindered by the great variability of the urine specimens, and the presence of various proteins in low abundance or modified forms. To alleviate some of these problems urine samples from five different individuals were pooled, concentrated and the proteome characterized by a combination of preparative electrophoresis and 2-DE, followed by PMF. A total of 778 protein spots corresponding to 141 different gene products were identified. In comparison, 171 spots corresponding to 44 unique proteins were identified in the unfractionated starting material. Among the proteins identified from the preparative electrophoresis were many of low abundance such as proteins involved in signal transduction. Furthermore, the median molecular mass of the identified proteins from the preparative electrophoresis was significantly lower in comparison to the proteins identified from the unfractionated starting material (39 886 Da versus 71 317 Da, respectively). Concluding, application of this methodology provides a coherent analysis of the urine proteome and contributes to the generation of the urine protein map in health and disease.

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We fabricated a TiO(2)-ZrO(2) affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono- and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of β-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO(2)-ZrO(2) stationary phase was characterized by X-ray diffraction and was found to be mostly TiO(2) and ZrO(2) in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of >1.4 μg (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 ± 3%, and can be used with small samples (less than 0.1 μL depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 μg and sample volumes of 1-10 μL), and to on-chip efforts based on packed bed and frit formats.

Urine is a biological fluid that is non-invasively and easily harvested, and exhibits high stability from the proteomics point of view. At the downside, the overall low protein content of urine as well as the presence of low- and high-abundance proteins underscores the need for protein enrichment. As a continuation of previous efforts towards the comprehensive characterization of the urine proteome, the current study targeted the mining of urine proteins through the combined application of different protein separation methodologies, specifically, liquid chromatography and preparative electrophoresis along with 1D gel electrophoresis and protein identification by mass spectrometry. In order to enhance comparison and integration of different experimental data sets, the "standard" urine sample developed within the European Kidney and Urine Proteomics (EuroKUP) COST Action, was employed. As a contribution to the existing knowledge, we focused on maintaining and providing information about experimental mass of the identified proteins as well as information pertaining to their relative abundance--as allowed by technical limitations--thus providing an initial view of different isoforms representation and facilitating their future characterization. The difficulties in comparing proteome mining data sets become once more evident, underscoring the need for adopting standardized ways for data reporting as well as for potential new approaches for data analysis involving a thorough investigation of received information at the peptide level.

One of the aims in the field of proteomics is the identification of a protein or polypeptide, or a range of these compounds, that could provide pre-symptomatic indication of the onset of a disease. A number of analytical techniques have been employed to try and achieve this end. These techniques have been applied to the complete range of body fluids and tissues that are readily available from clinical studies. Of these sample sources, the urinary low molecular weight peptidome has been shown to reflect changes in the health status of the individual. The alterations that occur in the polypeptide make up of urine, which reflect changes in biological status, are known as biomarkers. To be able to determine these changes no single technique has emerged that can cope with detecting the large number of peptides present and quantifying them over the wide concentration range they exist in. In this investigation, we made use of a single reflectron time of flight (RTOF)-MS analyser to which we first connected a CE system and then a nanoflow HPLC. Two pooled male and female standard urine samples were compared on these systems. Both techniques had similar results in terms of number of peptides detected and the mass range the peptides were detected over. The major differences in terms of biomarker research were the ability in CE to calibrate the migration time of the peptides to allow comparison between samples. In addition, CE was shown not to suffer from carry over from previous samples as was seen in the LC analysis.