Urine is formed in the kidney by ultrafiltration from the plasma to eliminate waste products, for instance urea and metabolites. 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 [1
]. 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. Serum proteins are filtered based on their sizes and charges at the glomeruli [3
]. 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 [4
]. 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. This is about a factor 1000 less compared with other body fluids such as plasma. Excretion of more than 150 mg/day protein is defined as proteinuria and is indicative of glomerular or reabsorption dysfunction.
Urine can be collected in large amounts fully noninvasively. 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. This advantage of urine as a body fluid for diagnosis also allows collection of samples repeatedly over lengthy time periods. Furthermore, normal urinary proteins generally reflect normal kidney tubular physiology because the urinary proteome contains not only plasma proteins but also kidney proteins [7
]. 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 [14
Urinary proteomics has been conducted by combining various protein concentration and protein separation methods as well as mass spectrometry (MS) technology. In many studies, two-dimensional gel electrophoresis was employed for protein separation. One of these studies, that conducted by Pieper and coworkers [11
], identified 150 unique proteins using two-dimensional gel electrophoresis and both matrix-assisted laser desorption ionization time-of-flight MS and liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS2
). However, one-dimensional and two-dimensional chromatographic approaches have been used in several recent studies, resulting in further protein identifications. Pisitkun and coworkers [9
] reported identification of 295 unique proteins from the exosome fraction using one-dimensional gel electrophoresis and LC-MS/MS. Sun and colleagues [12
] identified 226 unique proteins using one-dimensional gel electrophoresis plus LC-MS/MS and multidimensional liquid chromatography (LC/LC)-MS/MS. Wang and coworkers [13
] applied concanavalin A affinity purification for the enrichment of N-glycoprotein in urine and identified 225 proteins using one-dimensional gel electrophoresis plus LC-MS/MS and LC/LC-MS/MS. Recently, Castagna and colleagues [10
] exploited beads coated with a hexametric peptide ligand library for urinary protein concentration and equalization, and identified 383 unique gene products by LC-MS/MS using a linear ion trap-Fourier transform (LTQ-FT) instrument. These researchers combined their set of urinary proteins with others derived from the literature to yield a total of about 800 proteins.
Some of these five largest urinary proteome catalogues contain proteins with single peptide identification (>30% of total identified proteins reported by Pisitkun and coworkers [9
]) and lack an assessment of false-positive ratios. Moreover, proteins identified in these studies seem to be the tip of the iceberg of the urinary proteome, because nearly 1000 protein spots separated by two-dimensional gel remain unidentified [11
]. These studies suggest that three steps are especially important for deep analysis: protein concentration from urine with minimal loss; protein separation to reduce the complexity of the protein mixture and remove abundant proteins; and peptide sequencing with high mass accuracy and rapid scanning.
In the present study, we employed a simple and straightforward method, namely ultrafiltration, for protein concentration. For protein separation, one-dimensional gel electrophoresis or reverse phase column chromatography was used. For peptide sequencing, we employed methods recently developed in our laboratory involving the LTQ-FT and linear ion trap-orbitrap (LTQ-Orbitrap), which have extremely high mass accuracy [15
]. The LTQ facilitates accumulation of a greater number of charges than is possible with traditional three-dimensional ion traps, and it is sufficiently fast to enable two consecutive stages of mass spectrometric fragmentation (MS/MS/MS or MS3
) on a chromatographic time scale. The Fourier transform-ion cyclotron resonance (FTICR) part of the instrument provides a very high resolution of 100,000 and mass accuracies in the sub-ppm (parts per million) range using selected ion monitoring (SIM) scans. For complex protein samples, the LTQ-FT was shown to increase the number of high-confidence identifications compared with an LCQ instrument [17
]. Together, high mass accuracy and MS3
result in dramatically increased confidence for peptide identification [15
] and allow 'rescue' of protein identifications by single peptides. A novel hybrid mass spectrometer, the LTQ-Orbitrap [18
] also provides a high mass resolving power of 60,000 and high-accuracy mass measurements (sub-ppm on average) using a lock mass strategy, even without SIM scans [15
These techniques enabled us to identify 1543 proteins in urine from an in-depth study from a single individual and pooled urine obtained from nine individuals, while virtually eliminating false-positive identifications. In the LTQ-FTICR dataset 337 proteins (26.3% of the total identified proteins) were identified with single unique peptide using MS2 and MS3. Around a third of all characterized proteins are annotated as extracellular proteins. In the total data set we found 488 proteins to be annotated as membrane proteins (47% of all proteins with localization information). Of these proteins, 225 proteins were annotated as plasma membrane proteins (21.6%). These proteins include water, drug, sodium, potassium, and chloride transporters that are localized in the kidney and regulate homeostasis of body fluids. This high-confidence collection of proteins present in human urine can serve as a reference for future biomarker discovery.