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Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption
Visith Thongboonkerd1, Theptida Semangoen, Somchai Chutipongtanate
1Medical Molecular Biology Unit, Office for Research and Development, and Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. vthongbo@yahoo.com
Journal of Proteome Research
|January 26, 2007
Summary
Researchers developed a new method to isolate basic proteins in urine, a previously understudied area. Batch adsorption with SP Sepharose beads at pH 6.0 proved most effective for enriching the cationic urinary proteome.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- The human urinary proteome is primarily characterized by anionic proteins, leaving basic urinary proteins largely unstudied.
- Existing methods for urinary proteome analysis have focused on anionic components, creating a knowledge gap regarding cationic proteins.
Purpose of the Study:
- To develop and compare methods for enriching the cationic urinary proteome.
- To identify basic proteins in normal human urine and optimize enrichment techniques.
Main Methods:
- Comparison of cation exchange chromatography and batch adsorption techniques for protein enrichment.
- Utilized SP Sepharose 4 Fast Flow beads and RESOURCE-S columns for cation enrichment.
- Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS for protein identification.
Main Results:
- Batch adsorption using SP Sepharose 4 Fast Flow beads at pH 4.8 yielded the highest protein recovery.
- Batch adsorption at pH 6.0 was particularly effective for enriching interferon alpha-3 (IFNalpha3) and eosinophil-derived neurotoxin (EDN).
- Identified immunoglobulin isoforms, IFNalpha3, and EDN in the enriched cationic urinary proteome.
Conclusions:
- Batch adsorption with SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the optimal method for examining the basic/cationic urinary proteome.
- This method provides satisfactory yield and identifies maximal isoforms of IFNalpha3 and EDN.
- The described techniques can be applied to enrich cationic proteomes in other biological samples.
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