Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis.
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.
Papale
Massimo
M
Molecular Medicine Center, Section of Nephrology, Department of Biomedical Sciences and Bioagromed, Faculty of Medicine, University of Foggia, Foggia, Italy.
Pedicillo
Maria Carmela
MC
Thatcher
Bradley J
BJ
Di Paolo
Salvatore
S
Lo Muzio
Lorenzo
L
Bufo
Pantaleo
P
Rocchetti
Maria Teresa
MT
Centra
Marta
M
Ranieri
Elena
E
Gesualdo
Loreto
L
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*