Effects of the time intervals between venipuncture and serum preparation for serum peptidome analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Author(s): Umemura H, Nezu M, Kodera Y, Satoh M, Kimura A, Tomonaga T, Nomura F

Publication: Clin Chim Acta, 2009, Vol. 406, Page 179-80

PubMed ID: 19520068 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine if blood specimen processing and handling alters serum peptide profiles.

Conclusion of Paper

The biggest effect on peptide peaks was as a result of increasing clotting time. Peaks could be divided into 3 distinct groups based on the effect of increased clotting time on peak intensity: those that decreased rapidly over the first hour and then continued a steady decline (type A), those that increased gradually over the entire 6 h (type B), and those that increased rapidly over the first 30 minutes before slowly declining or remaining constant (type C). The type of peaks observed depended on clotting time and bead type. C8 hydrophobic interaction chromatography (C8) and weak cation exchange (WCX) resulted in mostly type A peaks while immobilized metal affinity chromatography (IMAC-Cu) resulted in mostly type B peaks. No differences in relative peak intensities were observed between samples frozen at -80 degrees C and those frozen in liquid nitrogen. After 2 freeze-thaw cycles, most peaks had a coefficient of variance of less than 20%, however, additional freeze-thaw cycles significantly altered relative peak intensity. In conclusion, freezing temperature and up to 2 freeze-thaw cycles have little effect on peptide peak intensity, but clotting time and bead choice have a significant impact.

Studies

Study Purpose

The purpose of this study was to determine if clotting time, freezing temperature, freeze-thaw cycling and bead choice for peptide fractionation alter serum peptide profiles by MALDI-TOF mass spectrometry (MS).

Summary of Findings:

The biggest effect on peptide peaks was due to increasing clotting time. Peaks could be divided into 3 distinct groups (A, B, and C) based on the effect of increased clotting time on peak intensity. The type of peaks observed depended on clotting time and bead type used. C8 and WCX resulted in mostly type A peaks while IMAC-Cu resulted in mostly type B peaks. No differences in relative peak intensities were observed between samples frozen at -80 degrees C and those frozen in liquid nitrogen. After 2 freeze-thaw cycles, most peaks had a coefficient of variance of less than 20%, however, additional freeze-thaw cycles significantly altered relative peak intensity. In conclusion, freezing temperature and up to 2 freeze-thaw cycles have little effect on peptide peak intensity, but clotting time and bead choice have a significant impact.

Biospecimens

Preservative Types

Frozen

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
Peptide	MALDI-TOF MS

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Storage	Storage temperature	-80 degrees C Liquid nitrogen
Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles 7 cycles
Storage	Storage duration	1 week
Analyte Extraction and Purification	Analyte isolation method	C8 hydrophobic interaction chromatography Weak cation exchange Immobilized metal affinity chromatography
Storage	Time at room temperature	15 min 30 min 1 h 2 h 4 h 6 h
Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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