Quantitative amino acid and proteomic analysis:: Very low excretion of polypeptides >750 Da in normal urine

Background. Quantitative data on protein and polypeptide excretion in normal urine are lacking. In Fanconi syndrome, failure of proximal tubular protein reabsorption leads to 'tubular' proteinuria, but little is known about peptide excretion. Methods. Urine from normal (N=5) and Fanconi patients (Dent's disease, N=2; Lowe syndrome, N=3) was fractionated by size-exclusion chromatography into proteins (>10 kD) and smaller polypeptides. Each fraction was subjected to amino acid analysis after acid hydrolysis. In complementary proteomic approaches, urinary polypeptides were each subjected to reversed-phase high-performance liquid chromatography (HPLC) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nano-flow liquid chromatography directly coupled to electrospray ionization/tandem mass spectrometry (NanoLC-ESI-MS/MS) before and after tryptic digestion. Results. Based on amino acid composition, normal human urine, excluding Tamm-Horsfall protein, contains 33.7+/-10.7 mg protein per 24 hr (mean+/-SEM) protein defined as polypeptide >10 kD; peptide content in range 750 Da to 10 kD is 22.0+/-9.6 mg. Fanconi patients excrete greatly increased amounts of protein, 1740+/-660 mg/24 hr, and peptide, 446+/-145 mg/24 hr. Peptides 2 to 5 kD were present in 12.9-+/-3.9-fold excess in Fanconi compared with normal urine. In contrast, free amino acid excretion in Fanconi was elevated only 2.14-+/-0.73-fold. Mass spectrometric techniques determined that the major form of albumin in both normal and Fanconi urine was the full-length protein, and did not detect significant peptides of nonrenal origin. Conclusion. There is only very low excretion of polypeptides >750 Da in normal human urine. In Fanconi syndrome, excretion of unknown peptides of mass 2 to 5 kD, possibly relevant to the development of renal failure, is greatly increased.