title: Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis.
abstract: The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.
authors.affiliation: Molecular Medicine Center, Section of Nephrology, Department of Biomedical Sciences and Bioagromed, Faculty of Medicine, University of Foggia, Foggia, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Papale
authors.forename: Maria Carmela
authors.initials: MC
authors.lastname: Pedicillo
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Thatcher
authors.forename: Salvatore
authors.initials: S
authors.lastname: Di Paolo
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Lo Muzio
authors.forename: Pantaleo
authors.initials: P
authors.lastname: Bufo
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Rocchetti
authors.forename: Marta
authors.initials: M
authors.lastname: Centra
authors.forename: Elena
authors.initials: E
authors.lastname: Ranieri
authors.forename: Loreto
authors.initials: L
authors.lastname: Gesualdo
mesh_terms: D002498:Centrifugation; D005260:Female; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D016482:Urinalysis / Q000379:methods*