Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.
BACKGROUND
Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins.


METHODS
Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting.


RESULTS
A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein.


CONCLUSIONS
Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.
Thongboonkerd
Visith
V
Core Proteomics Laboratory and Molecular Signaling Group, Kidney Disease Program, Department of Medicine, University of Louisville, 570 South Preston Street, Louisville, KY 40202, USA.
McLeish
Kenneth R
KR
Arthur
John M
JM
Klein
Jon B
JB
D000096:Acetone; D011232:Chemical Precipitation; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D012697:Serine Endopeptidases; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D053802:Tryptases; D014461:Ultracentrifugation; D014556:Urine / Q000737:chemistry*
D011506:Proteins; D012997:Solvents; D000096:Acetone; D012697:Serine Endopeptidases; D053802:Tryptases