Fractionation of cytosolic proteins on an immobilized heparin column.
Currently there is great interest in the development of methods to simplify complex protein mixtures for analysis by proteomic strategies. The objective of this study was to develop and evaluate immobilized heparin chromatography to simplify such mixtures and to enrich minor proteins. The method is evaluated with cytosol from human breast cancer MCF-7 cells. This protein mixture was fractionated into three portions and eluted with a stepwise salt gradient. These were characterized by protein analysis, two-dimensional gel electrophoresis, and mass spectrometry, with attention to reproducibility, overlap between fractions, simplification of protein mixtures, and enrichment of low-abundance proteins. It was possible to identify proteins enriched in the fractionated mixtures that were not even detectable in gel arrays of the total cytosol. The method was shown to be suitable for integration with other proteomics strategies.
Shefcheck
Kevin
K
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Yao
Xudong
X
Fenselau
Catherine
C
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D003600:Cytosol / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D006493:Heparin