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.

Obese and overweight prepubertal children demonstrated prominent alterations in the expression of plasma apolipoproteins compared with their normal counterparts. Low ApoA-I plasma expression levels and serum concentrations in obesity might be present in childhood before any significant alterations in total or high-density lipoprotein-cholesterol concentrations are documented. We recommend that serum ApoA-I concentrations are determined in all overweight and obese children.

Application of Preparative Electrophoresis for Clinical Proteomics in Urine: Is it Feasible?Urine samples are easily attainable which makes them ideal substrates for biomarker research. Various techniques have been employed to unravel the urine proteome and identify disease biomarkers. Even though the presence of high abundance proteins in urine is not so pronounced as in the case of plasma, the presence of proteolytic products, many of which at low abundance, along with numerous frequently random chemical modifications, makes the analysis of urinary proteins challenging. To facilitate the detection of low abundance urinary proteins, in the study presented herein we applied two different electrophoretic techniques, preparative Lithium Dodecyl Sulfate (LDS)-PAGE in combination with 2-DE for urinary protein separation and enrichment. Our results indicate the effectiveness of this approach for the enrichment of low abundance and low molecular weight proteins and peptides in urine, and contribute towards the establishment of a urinary proteomic database. The application of this technique as a biomarker discovery tool faces several challenges: these include down-scaling of the technique, possible recompensation for the consequent expected decrease in protein resolution, by optimizing steps of the experimental workflow as well as getting a good understanding of the technical variability of the technique. Under these conditions, preparative electrophoresis can become an effective tool for clinical proteomics applications.

Preparative electrophoresis is a protein fractionation approach useful for the enrichment of low-abundance gene products. Preparative electrophoresis is usually performed in the PrepCell apparatus. Proteins are separated according to their size in a cylindrical gel in the presence of an ionic detergent. The method is particularly efficient for the enrichment of low-molecular-mass gene products. Preparative electrophoresis can be followed by proteomic analysis, and the proteins eluted from the preparative gel can be separated by two-dimensional gel electrophoresis and identified by mass spectrometry.

Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.