Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Author(s): Fiedler GM, Baumann S, Leichtle A, Oltmann A, Kase J, Thiery J, Ceglarek U
Publication: Clin Chem, 2007, Vol. 53, Page 421-8
PubMed ID: 17272489 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of preanalytical variables on urine peptide profiles.
Conclusion of Paper
Peptide profiles were significantly changed when comparing fresh specimens to those that had been frozen, and in specimens collected on different days or in subsequent collections on the same day. Specimens contaminated with either blood or bacteria also had significantly different protein profiles. Once frozen, specimens showed stable peptide profiles, when stored at 4 degrees C, for up to 24 hours but were less stable at higher temperatures. Peptide patterns obtained using hydrophobic interaction chromatography beads were not pH dependent. However, weak cation-exchange chromatography beads and immobilized metal ion affinity chromatography beads gave the best yields and intensities at pH 7. Normalization of protein content by altering sample volume rather than dilution served to reduce inter-individual variation.
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage, freeze-thaw cycling, pH, contamination, dilution, and collection time on urine peptide profiles.
Summary of Findings:
Freezing pooled urine specimens decreased relative peak intensities of some proteins while increasing others when compared to fresh pooled urine specimens, but further freeze-thaw cycling had little additional effects. Thawed frozen specimens were stable at 4 degrees C for 24 hours; however, these specimens showed significant changes in peptide patterns after 6 hours at 25 degrees C, or 3 hours at 40 degrees C. First morning urine had significantly higher total protein concentration and a distinct peptide profile when compared to second morning urine. Variations in peptide profile were also found when comparing specimens collected on different days. The addition of sodium chloride or urea up to a concentration of 1000 mmol/L did not alter specimen peptide patterns, but dilution with urea in order to normalize creatinine or protein concentrations resulted in a loss of mass signals and did not reduce variation. Normalization of protein content by altering sample volume rather than dilution served to reduce inter-individual variation. Blood contamination led to some new mass signals, while bacterial contamination led to decreases in signal intensities when incubated for at least 30 minutes. Peptide patterns obtained using hydrophobic interaction chromatography beads were not pH dependent, but weak cation-exchange chromatography beads and immobilized metal ion affinity chromatography beads gave the best yields and intensities at pH 7.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Protein Colorimetric assay Small molecule Colorimetric assay Carbohydrate Colorimetric assay Electrolyte/Metal Clinical chemistry/auto analyzer Protein Clinical chemistry/auto analyzer Small molecule Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) MALDI-TOF MS Specific Resin Hydrophobic interaction chromatography
Weak cation-exchange chromatography
Immobilized metal ion affinity chromatography
Storage Storage duration 0 h
1 h
3 h
6 h
24 h
72 h
168 h
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
Biospecimen Aliquots and Components pH 6
7
8
9
Biospecimen Aliquots and Components Biospecimen components Blood contamination
Bacterial contamination
Urea
Sodium chloride
Water
Storage Storage temperature -80 degrees C
4 degrees C
25 degrees C
40 degrees C
Biospecimen Aliquots and Components Aliquot sequential collection 1st collection
2nd collection
Biospecimen Acquisition Time of biospecimen collection Day 1
Day 2
Day 3
Day 4
Day 5
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)