Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis.
BACKGROUND
Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS).


METHODS
We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day.


RESULTS
Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility.


CONCLUSIONS
We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease.
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
Di Paolo
Salvatore
S
Thatcher
Bradley John
BJ
Lo Muzio
Lorenzo
L
Bufo
Pantaleo
P
Rocchetti
Maria Teresa
MT
Centra
Marta
M
Ranieri
Elena
E
Gesualdo
Loreto
L
D015415:Biomarkers / Q000032:analysis; D005260:Female; D006801:Humans; D007834:Lasers; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D012016:Reference Values; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000032:analysis*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards; D013223:Statistics as Topic; D013696:Temperature; D013997:Time Factors
D015415:Biomarkers; D011480:Protease Inhibitors; D012471:Salivary Proteins and Peptides