Urine profiling by SELDI-TOF/MS: Monitoring of the critical steps in sample collection, handling and analysis

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2007-09, Vol.856 (1), p.205-213
• Main Authors: Papale, Massimo, Pedicillo, Maria Carmela, Thatcher, Bradley J., Di Paolo, Salvatore, Muzio, Lorenzo Lo, Bufo, Pantaleo, Rocchetti, Maria Teresa, Centra, Marta, Ranieri, Elena, Gesualdo, Loreto
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Centrifugation; Female; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Male; Medical sciences; Pharmacology. Drug treatments; Reproducibility of Results; Sample collection; SELDI-TOF/MS; Specimen Handling; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Standardization; Urinalysis - methods; Urine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2007.06.001

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.