Clinical Nephrology – Epidemiology – Clinical Trials

Kidney International (2004) 66, 1994–2003; doi:10.1111/j.1523-1755.2004.00970.x

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

ANTHONY GW NORDEN, PETER SHARRATT, PEDRO R CUTILLAS, RAINER CRAMER, SHARON C GARDNER and ROBERT J UNWIN

Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, United Kingdom; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom; Department of Biochemistry and Molecular Biology, Ludwig Institute for Cancer Research and University College London, London, United Kingdom; and Centre for Nephrology and Departments of Medicine and Physiology, Royal Free and University College Medical School, Royal Free Campus, London, United Kingdom

Correspondence: Dr A.G.W. Norden, Department of Clinical Biochemistry, Addenbrooke's Hospital, Box 232, Hills Road, Cambridge CB2 2QR, U.K. E-mail:agwn2@cam.ac.uk

Received 1 December 2003; Revised 5 April 2004; Re-revised 3 May 2004; Accepted 20 May 2004.

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Abstract

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 plusminus 10.7 mg protein per 24 hr (mean plusminus SEM) protein defined as polypeptide >10 kD; peptide content in range 750 Da to 10 kD is 22.0 plusminus 9.6 mg. Fanconi patients excrete greatly increased amounts of protein, 1740 plusminus 660 mg/24 hr, and peptide, 446 plusminus 145 mg/24 hr. Peptides 2 to 5 kD were present in 12.9- plusminus 3.9-fold excess in Fanconi compared with normal urine. In contrast, free amino acid excretion in Fanconi was elevated only 2.14- plusminus 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.

Keywords:

MALDI-TOF, NanoLC-ESI-MS/MS, Lowe syndrome, Dent's disease, proteinuria, Fanconi syndrome

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