Urinary proteomics has been conducted by combining various protein concentration and protein separation methods as well as mass spectrometry (MS) technology.
Thus, urine is good material for the analysis of disease processes that affect proximal organs, such as kidney failure resulting from high blood pressure and diabetic nephropathy, which is the most frequent cause of renal failure in the Western world. Furthermore, normal urinary proteins generally reflect normal kidney tubular physiology because the urinary proteome contains not only plasma proteins but also kidney proteins. This advantage of urine as a body fluid for diagnosis also allows collection of samples repeatedly over lengthy time periods. Therefore, despite the low protein concentration, more than adequate amounts of material (at least 0.5 mg) can be collected from a single sample, although protein in urine must be concentrated. Urine can be collected in large amounts fully noninvasively. Excretion of more than 150 mg/day protein is defined as proteinuria and is indicative of glomerular or reabsorption dysfunction. This is about a factor 1000 less compared with other body fluids such as plasma. Thus, protein concentration in normal donor urine is very low (less than 100 mg/l when urine output is 1.5 l/day), and normal protein excretion is less than 150 mg/day. After passing through glomeruli, abundant serum proteins such as albumin, immunoglobulin light chain, transferrin, vitamin D binding protein, myoglobin, and receptor-associated protein are reabsorbed, mainly by endocytic receptors, megalin, and cubilin in proximal renal tubules. Serum proteins are filtered based on their sizes and charges at the glomeruli. Components in the ultrafiltrate such as water, glucose, amino acids, and inorganic salts are selectively reabsorbed, and less than 1% of ultrafiltrate is excreted as urine. Although the kidney accounts for only 0.5% of total body mass, a large volume of plasma (350-400 ml/100 g tissue/min) flows into the kidney, generating a large amount of ultrafiltrate (150-180 l/day) under normal physiologic conditions. Urine is formed in the kidney by ultrafiltration from the plasma to eliminate waste products, for instance urea and metabolites. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future. Our analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies. Plasma membrane proteins are probably present in urine by secretion in exosomes. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation.
Here we applied these methods to the analysis of the human urinary proteome. Our laboratory has developed methods for the in-depth characterization of body fluids these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification.
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