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

1. 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

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   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

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