Comparison of different depletion strategies for improving resolution of the human urine proteome.
BACKGROUND
Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated.


METHODS
To remove the most abundant proteins from a human urine sample, GenWay Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies.


RESULTS
Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343+/-20.0 microg, 5.8%; 186.3+/-13.3 microg, 7.2%; 292+/-20.6 microg, 8.8% [mean+/- standard deviation (SD), CV%], for GenWay, Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468+/-21, 331+/-7, 368+/-22 and 304+/-7 (mean+/-SD) for GenWay, Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p<0.001 compared to the GenWay procedure.


CONCLUSIONS
The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome.
Magagnotti
Cinzia
C
Center for Genomics, Bioinformatics and Biostatistics, Genomics Unit for Human Disease Diagnosis, San Raffaele Scientific Institute, Milan, Italy.
Fermo
Isabella
I
Carletti
Rose Mary
RM
Ferrari
Maurizio
M
Bachi
Angela
A
D015415:Biomarkers / Q000652:urine*; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D020543:Proteome / Q000032:analysis*; D011933:Reagent Kits, Diagnostic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; D020543:Proteome; D011933:Reagent Kits, Diagnostic