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Diagnostics
Use of Urinary Proteomics for the Discovery of Biomarkers of Renal Disease
a report by
José Manuel González-Buitrago
1
and Laura Ferreira
2
1. Head, Autoimmunity Laboratory, Department of Clinical Biochemisty, Director, Research Unit, University Hospital Salamanca, and
Professor of Biochemistry, University of Salamanca; 2. Post-doctoral Fellow, Research Unit, University Hospital Salamanca
Proteomics is the study of protein expression in a tissue or biological TOF analysers that measure the mass of intact peptides, while ESI has
fluid.
1
The proteome of a tissue or a biological fluid includes not mostly been coupled with ion traps and triple quadrupole instruments
only proteins translated directly from genetic material, but also all of the and used to generate fragment ion spectra of selected precursors ions.
proteins modified during and after translation. The term ‘clinical The power of MS has been rendered even greater by the addition of
proteomics’ refers to studies in which clinical samples are used.
2
One of tandem mass spectrometers, and by coupling tandem mass
the objectives of clinical proteomics is finding biomarkers of disease. spectrometers to liquid chromatography.
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Comparison of the protein patterns in a biological fluid between patients
with a given disease and healthy individuals is the main approach used to The strategies used to discover biomarkers have been called top-down
discover disease biomarkers.
3
Urine has been defined as a fluid biopsy of and bottom-up.
7
Top-down strategies involve the separation of proteins
the kidney and genito-urinary tract, and provides much information followed by tryptic digestion of isolated proteins and analyses of tryptic
about these organs. This article will review the current situation of urinary peptides by MS. In contrast, in bottom-up strategies, proteins are first
proteomics, placing special emphasis on its application as a tool for the broken down into small peptides by trypsin digestion, and the resulting
discovery of biomarkers of renal diseases. peptides are subsequently separated by chromatography and analysed
using MS.
Proteomics Technologies
In the search for biomarkers of disease using proteomics, the main A technique also used in proteomic studies in the search for
technological approach is the combination of a protein separation biomarkers is surface-enhanced desorption/ionisation (SELDI)-TOF MS,
technique and mass spectrometry (MS) to identify the proteins which uses protein–chip arrays coupled with a mass spectrometer.
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separated.
4
The methods used for the separation of proteins and peptides Proteins in the sample (urine) are first selectively adsorbed on the chip
include 2D polyacrylamide gel electrophoresis (2D-PAGE), high- and then extracted by laser desorption/ionisation and introduced in
performance liquid chromatography (HPLC), capillary electrophoresis (CE) the TOF MS. The technique generates a map in which individual
and multidimensional systems (HPLC–HPLC, HPLC–CE). proteins are displayed as separate peaks according to their mass to
charge (m/z) ratio. Comparison of ‘mass fingerprint’ between the urine
Two main ionisation methods have been developed to volatilise of healthy controls and patients can detect biomarkers of disease.
proteins and peptides for analysis by MS. These methods are
electrospray ionisation (ESI) and matrix-assisted laser desorption/ Urine Samples
ionisation (MALDI). Time-of-flight (TOF), quadrupole, ion trap and Normal urine has a low protein concentration with a high salt content
Fourier transform ion cyclotron resonance (FT-ICR) are frequently used that interferes with proteomic separation techniques. Several protocols
mass analysers in proteomic studies.
5
MALDI is usually coupled with have been used to isolate/concentrate urine proteins. Those most
frequently employed use precipitation, lyophilisation, ultracentrifugation
and centrifugal filtration. Recently, a systematic evaluation of sample
José Manuel González-Buitrago is Head of the
Autoimmunity Laboratory in the Department of Clinical
preparation methods for human urinary proteomics has been
Biochemistry and Director of the Research Unit at the published.
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The authors indicated that there is no perfect single protocol
University Hospital of Salamanca. He is also a Professor of
to examine the entire urinary proteome, and that a combination of
Biochemistry at the University of Salamanca. Dr
González-Buitrago has published 28 books and
several sample preparation methods is required.
monographs on biochemistry, molecular pathology and
autoimmunity, and has authored or co-authored 162
articles – 76 of them in international journals. He is active
Urine Proteins in Health and Disease
in research into proteomics and autoimmunity. Blood proteins are filtered at the glomeruli according to their size and
E: buitrago@usal.es
charge. After passing through the glomeruli, a large amount of serum
proteins are reabsorbed, mainly by endocytosis in the proximal renal
Laura Ferreira is a Post-doctoral Fellow in the Research
tubules. The urinary proteome contains not only plasma proteins, but
Unit of the University Hospital of Salamanca, Spain. She
has co-authored nine articles in international journals on
also kidney proteins and proteins from the lower urinary tract. In
molecular biology and proteomics. Her research focus is
healthy people, approximately 30% of urinary proteins are plasma
clinical proteomics.
proteins; the other 70% are produced in the kidney. The excretion of
more than 150mg/day of protein is defined as proteinuria and is
indicative of dysfunction. According to the type of molecule excreted
into the urine, proteinuria may be classified as the glomerular, tubular,
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