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Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and
Visith Thongboonkerd1, Somchai Chutipongtanate, Rattiyaporn Kanlaya
1Siriraj Proteomics Facility, Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok 10700, Thailand. thongboonkerd@dr.com
Journal of Proteome Research
|January 7, 2006
Summary
Evaluating 38 protocols for concentrating human urinary proteins revealed that 90% ethanol precipitation offers the highest recovery yield. Different methods impact protein spot visualization and variability in proteome analysis.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Human urine is a rich source of proteins for biomarker discovery.
- Standardized methods for urinary protein concentration are crucial for reproducible proteomic analysis.
- Existing protocols for urinary protein concentration vary, necessitating systematic evaluation.
Purpose of the Study:
- To systematically evaluate 38 protocols for concentrating normal human urinary proteins.
- To compare protein recovery yield and 2D-PAGE patterns across different concentration methods.
- To examine intra- and inter-individual variability in urinary protein profiles.
Main Methods:
- Systematic evaluation of 38 distinct protocols for urinary protein concentration.
- Comparison of protein recovery yield (percentage) for each method.
- Analysis of 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) for protein spot visualization.
- Assessment of intra-individual variability using first morning urine samples.
- Examination of inter-individual variability, including effects of hydration and sex.
Main Results:
- Precipitation with 90% ethanol yielded the highest protein recovery (92.99%).
- Acetonitrile precipitation at 75% resulted in the most visualized protein spots on 2D gels.
- Excessive water intake significantly altered urinary proteome profiles, unlike caffeine.
- First morning urine showed high total protein but fewer visualized spots.
- Significant inter-individual variability was observed, particularly for albumin and transferrin.
Conclusions:
- 90% ethanol precipitation is optimal for maximizing human urinary protein recovery.
- Method choice significantly impacts the number of detectable urinary proteins in 2D-PAGE.
- Hydration status is a key factor influencing urinary proteome profiles.
- Understanding variability is critical for large-scale human urine proteome projects.