Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.
Author(s): Tammen H, Schulte I, Hess R, Menzel C, Kellmann M, Mohring T, Schulz-Knappe P
Publication: Proteomics, 2005, Vol. 5, Page 3414-22
PubMed ID: 16038021 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare and contrast mass spectrometry-generated peptide profiles among serum, EDTA plasma, citrate plasma, and heparin plasma. The potential impact of centrifugation temperature during plasma processing on peptide profiles was also investigated.
Conclusion of Paper
Serum had a greater number of peptides compared to either EDTA or citrate plasma. In fact, more than 40% of peptides identified in serum were determined to be serum-specific, many of which were involved in clotting. The authors conclude that platelet poor plasma (PPP) generated by ultrafiltration or two rounds of centrifugation at room temperature is optimal for peptide profiling by mass spectrometry. Centrifugation at a lower temperature (4°C) was associated with platelet activation and subsequent contamination with platelet-related peptides. EDTA and citrate anticoagulants yielded similar peptide profiles, but heparin plasma expanded the fractionation range, altering a peptide's elution position.
Studies
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Study Purpose
The purpose of this study was to compare and contrast mass spectrometry-generated peptide profiles among serum, EDTA plasma, citrate plasma, and heparin plasma. A total of 22 samples were collected from healthy males into tubes containing clot activator (serum) or EDTA, heparin, or citrate (plasma). Prior to freezing at -80C, plasma specimens were centrifuged and filtered (0.2 m pore size). After a clot time of 1 h, serum was centrifuged and frozen at -80°C. Specimens were fractionated by reversed-phase (RP)-HPLC prior to peptide identification by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF MS/MS) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nESI-qTOF MS/MS).
Summary of Findings:
While plasma generated with EDTA and citrate displayed similar peptide profiles, plasma generated with heparin displayed a broader fractionation range. The authors postulate that the altered elution range and position for certain peptides is a result of chromatographic interference. Serum had a greater number of peptides compared to either EDTA or citrate plasma. In fact, more than 40% of peptides identified in serum were determined to be serum-specific, many of which were involved in clotting.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide HPLC Peptide MALDI-TOF MS Peptide ESI-TOF-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Citrate
Heparin
None
Biospecimen Acquisition Biospecimen location Plasma
Serum
-
Study Purpose
The purpose of this study was to compare peptide profiles produced by platelet poor plasma (PPP) processed by different methods. Plasma (EDTA and citrate) was collected from healthy male volunteers at two different sites, which also differed in their PPP preparation: filtration of centrifuged specimens through a 0.2 µm cellulose acetate filter, or two rounds of centrifugation at 4°C. A controlled experimental comparison of different PPP preparation methods was also conducted in which plasma was filtered through a 0.2 µm cellulose acetate filter, or by centrifugation at 2000 x g for 10 min, then 2500 x g for 15 min at either room temperature or 4°C. PPP was frozen at -80°C before being fractionated using RP-HPLC prior to analysis by MALDI-TOF MS/MS and nESI-qTOF MS/MS.
Summary of Findings:
Generally, peptide displays of PPP collected and processed at different sites were similar, sharing approximately 80% of MS signals. Those peptides that differed between sites were identified as platelet-related peptides, and were attributed to suboptimal processing for PPP by centrifugation at 4°C. The authors confirmed their hypothesis in a subsequent experiment. When both centrifugation steps were conducted at room temperature, peptide patterns were identical to those obtained with filtered plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide HPLC Peptide ESI-TOF-MS Peptide MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Multiple temperatures compared
Biospecimen Aliquots and Components Filtration 0.2 µm cellulose acetate filter
Centrifugation substituted
Biospecimen Aliquots and Components Blood processing method Filtration
Second centrifugation