Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples Daniel Lafitte a, *, Bertrand Dussol b,d , Soren Andersen c , Alain Vazi d , Patricia Dupuy d , Ole N. Jensen c , Yvon Berland b,d , Jean-Michel Verdier a,1 a UMR CNRS 6032, Faculte ´ de Pharmacie, Marseille, France b Ho ˆpital de la Conception, Service de Ne ´phrologie, Marseille, France c Protein Research Group, Department of Biochemistry & Molecular Biology, University of Southern Denmark, Odense University, Denmark d CIC INSERM-APHM, Marseille, France Received 30 April 2002; received in revised form 12 September 2002; accepted 12 September 2002 Abstract Objectives: We optimized of the preparation of urinary samples to obtain a comprehensive map of urinary proteins of healthy subjects and then compared this map with the ones obtained with patient samples to show that the pattern was specific of their kidney disease. Design and methods: The urinary protein excretion patterns of 49 healthy men and 4 patients with renal disease were obtained by 2D electrophoresis. Silver nitrate stained protein spots were identified by mass spectrometry. Results: Reproductive 2D gels identified 5 classes of proteins systematically found in healthy subjects, irrespective of age. The proteins evidenced included serum albumin and its specific proteolytic products, Tamm-Horsfall, transferrin, zinc-2-glycoprotein, 1-microglobu- lin, haptoglobin, and Ig light chains. Conclusions: This study of a large panel of healthy individual showed that 2D urinary pattern was constant over time and reveal for the first time the relative abundance of albumin fragments in healthy subjects’ urine. The profiles of the 4 patients, were specific of their renal disease. © 2002 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Proteomics; 2D electrophoresis; Urinary proteins pattern; Healthy subjects; Mass spectrometry; Kidney diseases 1. Introduction High-resolution 2-D polyacrylamide gel electrophoresis (2D-PAGE) is a powerful tool for analyzing proteins in complex mixtures. 2D-PAGE is useful for the analysis of human samples in a clinical research environment; it serves in identifying the origin of samples of body fluids (e.g., spinal, kystic, serum, pleural, ascitic, etc.), analyzing pro- tein phenotypes, monitoring disease process, and discover- ing new disease markers in body fluids or tissue biopsies [1]. The 2D patterns of normal serum and normal cerebro- spinal fluid are available on many web sites like www.ex- pasy.ch. Using this technique to compare the 2D pattern of serum from a patient with the normal profile highlighted the presence of a monoclonal immunoglobulin undetectable by the usual techniques. Previous report exits on 2D electro- phoresis of urinary proteins of healthy subjects [2 to 4], on the 2D urinary pattern for renal diseases [5 to 9] or for diseases that may secondarily affect the kidneys such as rheumatoid arthritis [11]. Some of these studies have been hampered by the low protein content of urine and by the presence of nondialyzable compounds like glycosaminogly- cans that interfere with protein migration in SDS-PAGE [10]. This study was therefore undertaken to optimize the preparation of urine samples for 2D-PAGE and to determine the normal 2D pattern of urinary protein in healthy subjects (using a significant number of individuals). The proteins recovered were subsequently identified by mass spectrom- etry and sequence database searches. Our preparation of urinary samples prevented the loss of proteins during the purification steps. We identified 5 classes of proteins sys- tematically found in healthy subjects, irrespective of age. In 1 Present address: EPHE, Universite ´ Montpellier II, Montpellier, France. * Corresponding author. Tel.: +1-33-491-83-56-16; fax: +1-33-491- 782024. E-mail address: daniel.lafitte@pharmacie.univ-mrs.fr (D. Lafitte). Clinical Biochemistry 35 (2002) 581–589 0009-9120/02/$ – see front matter © 2002 The Canadian Society of Clinical Chemists. All rights reserved. PII: S0009-9120(02)00362-4