An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum.
Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before 2-DE analysis can be used to remove high abundance proteins in order to increase the detection sensitivity of proteins present in low abundance.
Ahmed
Nuzhat
N
Gynaecological Cancer Research Centre, Royal Women's Hospital and Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Victoria, Australia. nuzhata@unimelb.edu.au
Barker
Gillian
G
Oliva
Karen
K
Garfin
David
D
Talmadge
Kenneth
K
Georgiou
Harry
H
Quinn
Michael
M
Rice
Greg
G
D015415:Biomarkers / Q000097:blood*; D001797:Blood Protein Electrophoresis; D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012709:Serum Albumin / Q000302:isolation & purification*; D013997:Time Factors; D014227:Triazines / Q000737:chemistry
D015415:Biomarkers; D001798:Blood Proteins; D020543:Proteome; D012709:Serum Albumin; D014227:Triazines; C012842:Cibacron Blue F 3GA