Journal of Pharmaceutical and Biomedical Analysis 51 (2010) 907–914 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles C.D. Calvano a , A. Aresta a , M. Iacovone a , G.E. De Benedetto c , C.G. Zambonin a,∗ , M. Battaglia b , P. Ditonno b , M. Rutigliano b , C. Bettocchi b a Università degli Studi di Bari, Centro Interdipartimentale di Ricerca S.M.A.R.T., Dipartimento di Chimica, Via Orabona, 4, 70100 Bari, Italy b Università degli Studi di Bari, Dipartimento dell’Emergenza e dei Trapianti di Organi, Piazza Giulio Cesare, 70100 Bari, Italy c Università del Salento, Dipartimento dei Beni delle Arti e della Storia, Viale S. Nicola, 73100 Lecce, Italy article info Article history: Received 25 June 2009 Received in revised form 14 October 2009 Accepted 19 October 2009 Available online 30 October 2009 Keywords: Proteomics Urine samples MALDI-TOF-MS Sample preparation Prostate cancer abstract Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and tem- perature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic–lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cellular metabolism generates numerous waste compounds that require elimination from the bloodstream. This waste is even- tually expelled from the body with urine, a liquid waste product secreted by the kidneys through a filtration process from blood. Serum proteins are filtered based on their sizes and charges at the glomerular filtration barrier and can be subsequently reabsorbed in proximal renal tubules. Thus, normal urine contains a very low protein concentration (150 mg/24 h), constituted of those filtered from the plasma, as well as those arising from kidney and urogenital ∗ Corresponding author. Tel.: +39 080 5442506; fax: +39 080 5442026. E-mail address: zambonin@chimica.uniba.it (C.G. Zambonin). tract. An excess of serum proteins in urine is defined as proteinuria and is indicative of glomerular or reabsorption dysfunction. Urine can be collected in a non-invasive fashion and in large amounts, thus being an ideal protein research substrate, permitting the recovery of adequate amounts of material after protein pre- concentration. For instance, more than 1500 proteins have been recently identified by Adachi et al. [1] from an in-depth study in urine samples from healthy individuals. Normal urinary proteins generally reflect normal kidney tubu- lar physiology due to the presence of kidney proteins [2,3–7]. Thus, urine is a good material for the analysis of kidney failure result- ing from high blood pressure and diabetic nephropathy, which is the most frequent cause of renal failure in the Western world [8]. Urine is also an important source of information for bladder and prostate cancers. The levels of several proteins in urine are mea- 0731-7085/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2009.10.014