Sample handling for mass spectrometric proteomic investigations of human sera.
Author(s): West-Nielsen M, Høgdall EV, Marchiori E, Høgdall CK, Schou C, Heegaard NH
Publication: Anal Chem, 2005, Vol. 77, Page 5114-23
PubMed ID: 16097747 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of clotting time and temperature, storage and freeze-thaw cycling on proteome profiles in serum.
Conclusion of Paper
Principal component analysis (PCA) and spectral analysis were unable to distinguish between blood left to clot at 4 degrees C for 2-24 h or for 2-4 h at 24 degrees C, but blood left to clot for 8 or 24 h at 24 degrees C had distinct profiles. In sera specimens that had been stored at -20 degrees C for 4 years, no linear dependence of spectra on clotting time was observed, but specimens left to clot at room temperature for 72 h clustered separately from those clotted for 1 or 3 h in PCA. Up to 4 freeze-thaw cycles at -20 degrees C or 5 freeze-thaw cycles at -80 degrees C did not affect the serum spectra, nor were there differences between the freezing temperatures. When sera spiked with protein were stored at room temperature or 4 degrees C, there was a loss of peaks below m/z 2000 compared to baseline.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of clotting time and temperature and freeze-thaw cycling on serum proteome profiles.
Summary of Findings:
PCA and spectral analysis were unable to distinguish between blood left to clot at 4 degrees C for 2-24 h or for 2-4 h at 24 degrees C, but blood left to clot for 8 or 24 h at 24 degrees C had distinct profiles. In a second set of sera specimens that had been stored at -20 degrees C for 4 years, no linear dependence of spectra on clotting time (1, 3, or 72 h) was observed, but specimens left to clot at room temperature for 72 h clustered separately in PCA. Up to 4 freeze-thaw cycles at -20 degrees C or 5 freeze-thaw cycles at -80 degrees C did not affect the serum spectra, nor were there differences between the freezing temperatures.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Storage Storage duration 1 h
2 h
3 h
4 h
8 h
24 h
72 h
Storage Storage temperature 4 degrees C
24 degrees C
-20 degrees C
-80 degrees C
Storage Freeze/thaw cycling 0 cycles
4 cycles
5 cycles
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
-
Study Purpose
The purpose of this study was to determine the effects of storage temperature and duration on the proteomic profile of plasma and serum specimens spiked with protein.
Summary of Findings:
When spiked serum was stored at room temperature or 4 degrees C, there was a loss of peaks below m/z 2000 compared to baseline. The loss of peaks was faster in specimens stored at 4 degrees C than those stored at room temperature (24 degrees C). Interestingly added cytochrome C, substance P, ACTH clip 1-17, and myoglobin were not recovered from any of the spiked serum specimens, regardless of storage. The authors report that plasma specimens required very different processing and were not comparable in quality to serum specimens, and the differences were not attributable to the use of EDTA as an anticoagulant, or centrifugation, but could be partially attenuated by the use of three times the volume of blood.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Storage temperature 4 degrees C
24 degrees C
Storage Storage duration 0 h
1 h
4 h
8 h
24 h
48 h
Biospecimen Aliquots and Components Aliquot size/volume Standard volume
Three times the standard volume