Urinary proteomics has become a topical and potentially valuable field of study in relation to normal and abnormal renal function. Filtered bioactive peptides present in high concentration in the nephron of patients with tubular proteinuria may have downstream effects on renal tubular function. In renal Fanconi syndromes, such as Dent's disease, peptides implicated in altered tubular function or injury have recently been measured in urine by immunochemical methods. However, the limited availability of antibodies means that only certain peptides can be detected in this way. We have used nanoflow liquid chromatography and tandem mass spectrometry (nanoLC-MS/MS) as a complementary technique to analyse urinary peptides. Urine was desalted by solid-phase extraction (SPE) and its peptides were then separated from neutral and acidic compounds by strong cation-exchange chromatography (SCX), which was also used to fractionate the peptide mixture. Fractions from the SCX step were separated further by reversed-phase LC and analysed on-line by MS/MS. Extraction by SPE showed a good recovery of small peptides. We detected over 100 molecular species in urine samples from three individuals with Dent's disease. In addition to plasma and known urinary proteins, we identified some novel proteins and potentially bioactive peptides in urine from these patients, which were not present in normal urine. These data show that nanoLC-MS/MS complements existing techniques for the identification of polypeptides in urine. This approach is a potentially powerful tool to discover new markers and/or causative factors in renal disease; in addition, its sensitivity may also make it applicable to the direct ultramicroanalysis of renal tubule fluid.
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May 01 2003
Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome
Pedro R. CUTILLAS;
*Bioanalytical Chemistry Laboratory, Ludwig Institute for Cancer Research, First Floor, Cruciform Building, Gower Street, London WC1E 6BT, U.K.
†Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
‡Nephrology and Physiology, Royal Free and University College Medical School, Middlesex Hospital, London W1T 3AA, U.K.
Correspondence: Pedro Cutillas, Biochemistry and Nephrology, Royal Free and University College Medical School, Middlesex Hospital, London W1T 3AA, U.K. (e-mail [email protected]).
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Anthony G.W. NORDEN;
Anthony G.W. NORDEN
§Department of Clinical Biochemistry, Box 232, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QR, U.K.
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Rainer CRAMER;
Rainer CRAMER
*Bioanalytical Chemistry Laboratory, Ludwig Institute for Cancer Research, First Floor, Cruciform Building, Gower Street, London WC1E 6BT, U.K.
†Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
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Alma L. BURLINGAME;
Alma L. BURLINGAME
*Bioanalytical Chemistry Laboratory, Ludwig Institute for Cancer Research, First Floor, Cruciform Building, Gower Street, London WC1E 6BT, U.K.
†Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
ǁMass Spectrometry Laboratory, Department of Pharmaceutical Chemistry, University of California in San Francisco, Parnasus Avenue, Box 0446, San Francisco, CA 94143-0446, U.S.A.
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Robert J. UNWIN
Robert J. UNWIN
‡Nephrology and Physiology, Royal Free and University College Medical School, Middlesex Hospital, London W1T 3AA, U.K.
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Publisher: Portland Press Ltd
Received:
November 26 2002
Revision Received:
December 17 2002
Accepted:
January 15 2003
Accepted Manuscript online:
January 15 2003
Online ISSN: 1470-8736
Print ISSN: 0143-5221
The Biochemical Society © 2003
2003
Clin Sci (Lond) (2003) 104 (5): 483–490.
Article history
Received:
November 26 2002
Revision Received:
December 17 2002
Accepted:
January 15 2003
Accepted Manuscript online:
January 15 2003
Citation
Pedro R. CUTILLAS, Anthony G.W. NORDEN, Rainer CRAMER, Alma L. BURLINGAME, Robert J. UNWIN; Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome. Clin Sci (Lond) 1 May 2003; 104 (5): 483–490. doi: https://doi.org/10.1042/CS20020342
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