Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption.
Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.
Thongboonkerd
Visith
V
Medical 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
Semangoen
Theptida
T
Chutipongtanate
Somchai
S
D000327:Adsorption; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007477:Ions; D011506:Proteins / Q000032:analysis*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea / Q000737:chemistry*
D007477:Ions; D011506:Proteins; D020543:Proteome; D014508:Urea