Methods
Several related approaches using mass spectrometry proteomics have been used to investigate the composition of human urine with ever increasing accuracy and depth. Though protein-based mass spectrometry (so-called top-down MS) is far advanced, studies of human urine have been carried out using peptide (bottom-up) MS that takes advantage of peptide identification using tandem MS and automated statistical database matching; for a recent review see Mallick and Kuster.
One approach to measure the urine proteome involves physicochemical methods to capture and fractionate urinary proteins, and liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to sequence peptides for protein identification using searches of known human proteins. Using ultracentrifugation to fractionate urine proteins based on molecular weight and LC-MS/MS for protein identification, Knepper and colleagues identified 295 proteins in urinary exosomes, and more than 1000 proteins in total. Using ultrafiltration and denaturing electrophoresis for protein fractionation and LC-MS/MS using an LTQ-Orbitrap hybrid spectrometer, Mann and colleagues identified more than 1500 proteins. Zeng and colleagues used protein precipitation and ion exchange chromatography and LTQ-Orbitrap LC-MS/MS to identify 1300 proteins. By combining ultracentrifugation, protein precipitation, and ion exchange chromatography for protein capture and fractionation and LTQ-Orbitrap LC-MS/MS for peptide sequencing, Kentsis, Steen and colleagues identified more than 2300 unique proteins in routinely collected clinical urine specimens. Lee and colleagues recently reported a one-step sample concentration, purification, and albumin depletion method suitable for high throughput analysis.
Another approach involves identification of candidate markers using MS or differential electrophoresis (DIGE), and subsequent identification of candidates using LC-MS/MS sequencing. DIGE-based investigations identified hundreds of protein spots in urine, though most of them remain unidentified. Recently, Mischak and colleagues used a combination of ultrafiltration and capillary electrophoresis MS to detect several thousand peptides in clinical urine specimens. Likewise, Zucht and colleagues applied a differential display approach and LC-MS/MS to detect thousands of peptides in human urine.
The Human Proteome Organization (HUPO) has established the Human Kidney and Urine Proteome Project (HKUPP) to advance these approaches. Current efforts seek to define the size, tissue origin, chemical modifications, and physical components, such as exosomes, of the urine proteome, and its specific relationships to physiological and disease states.