Correcting common errors in identifying cancer-specific serum peptide signatures.
Author(s): Villanueva J, Philip J, Chaparro CA, Li Y, Toledo-Crow R, DeNoyer L, Fleisher M, Robbins RJ, Tempst P
Publication: J Proteome Res, 2005, Vol. 4, Page 1060-72
PubMed ID: 16083255 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of collection tube, delayed centrifugation, freeze-thaw cycling, extraction method and analysis parameters on serum MALDI-TOF MS profiles from healthy controls. The improved protocol was used to identify potential markers of thyroid carcinoma. Serum was stored at -80 degrees C until analysis.
Summary of Findings:
Nonsupervised clustering and principle component analysis of the serum MALDI-TOF profiles segregated the 32 specimens by collection tube type. Delayed centrifugation resulted in progressive decreases in the height of some MALDI-TOF MS peaks and increases in the height of others. After 4 freeze-thaw cycles, the spectra were significantly changed compared to specimens thawed twice. The spectra were also significantly changed when peptides were extracted using different batches of magnetic beads, but there were no systemic day-to day variations. The most peaks were observed when extraction was completed in 40% acetonitrile/50% methanol/10% water without trifluoroacetic acid (TFA) and when spectra were acquired within 1-2 h of specimen processing. The number of laser shots and the amount of energy per shot significantly affected the spectra. Use of Entrotypical calibration during alignment instead of external calibration reduced noise and artifacts.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 5 min
1 h
5 h
Biospecimen Acquisition Type of collection container/solution Red top (glass)
SST
Storage Freeze/thaw cycling 2 cycles
4 cycles
Analyte Extraction and Purification Analyte isolation method Different batches of beads
Different concentrations of acetonitrile, methanol, and isopropanol
With 0.1% TFA
Without 0.1% TFA
MALDI-TOF MS Specific Data handling Entrotypical
External calibration
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated