Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis.
Author(s): Papale M, Pedicillo MC, Di Paolo S, Thatcher BJ, Lo Muzio L, Bufo P, Rocchetti MT, Centra M, Ranieri E, Gesualdo L
Publication: Clin Chem Lab Med, 2008, Vol. 46, Page 89-99
PubMed ID: 18020972 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of different specimen collection, handling, storage, and analysis methods on protein profiles of saliva.
Conclusion of Paper
In the absence of protease inhibitors, saliva protein profiles showed reduced peak intensities after 3 hours at room temperature. However, the addition of protease inhibitor cocktail to the specimens eliminated this effect. Up to 5 freeze-thaw cycles had little effect on saliva protein profiles. Proteomic profiles of individuals were found to change significantly throughout the day. Significant differences based on gender were found with each chip array type. When one individual specimen was analyzed on all four chip arrays, only 14-25% of the peaks were shared between all four array types. Centrifugation of saliva specimens prior to analysis did not alter the number of peaks observed. Removal of glycoproteins by ethanol precipitation increased the number of peaks and improved the intensity and resolution. Denaturation increased the number of peaks for all arrays except the strong cationic exchange CM10 chip. The number of peaks also changed based on detection by alpha-cyano-hydroxycinnamic acid (CHCA) or sinapinic acid (SA).
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage duration, freeze-thaw cycling, collection time, denaturing agents, depletion of glycoproteins, centrifugation, matrix type, array type, gender, and individual on protein profiles of saliva. Specimens were stored at -80 degrees C until analyzed.
Summary of Findings:
In the absence of protease inhibitors, saliva protein profiles showed reduced peak intensities after 3 hours at room temperature indicating specimen degradation. However, the addition of protease inhibitor cocktail to the specimens resulted in similar peak intensities between 0 and 3 hours at room temperature. Up to 5 freeze-thaw cycles had little effect on saliva protein profiles, regardless of the addition of protease inhibitors. Proteomic profiles of individuals were found to change significantly throughout the day, which the authors believe may be partially attributable to food ingestion. Significant differences based on gender were found with each chip array type. When one individual specimen was analyzed on all four chip arrays, only 14-25% of the peaks were shared between all four array types. Centrifugation of saliva specimens at 1300 x g prior to analysis did not alter the number of peaks observed. Removal of glycoproteins by ethanol precipitation increased the number of peaks and improved the intensity and resolution. Denaturation increased the number of peaks for all arrays except the strong cationic exchange CM10 chip. The highest number of peak was obtained with different buffers for different arrays. Further, the number of peaks also changed based on detection by CHCA or SA.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide SELDI-TOF MS Protein SELDI-TOF MS Protein Colorimetric assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Analyte Extraction and Purification Protease inhibitor Cocktail
No protease inhibitor added
Storage Time at room temperature 0 h
3 h
4 h
48 h
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
Biospecimen Acquisition Time of biospecimen collection Upon waking
Before lunch
After lunch
Before bed
SELDI-TOF MS Specific Denaturation Urea and CHAPS
DTT
Urea, CHAPS and DTT
SELDI-TOF MS Specific Type of array Hydrophobic interaction
Strong cationic exchange
Metal affinity
Strong anionic exchange
SELDI-TOF MS Specific Detection method SPA
CHCA
Biospecimen Aliquots and Components Centrifugation Centrifuged
Not centrifuged