Protein profiles, comparison with cellular

Quantitation Once protein expression profiling activities characterize qualitative features, the attention turns to delineating protein interactions and mechanistic pathways responsible for disease. These studies also require rapid sequence determination/confirmation combined with accurate and sensitive quantitative analysis. The quantitation approaches would allow for direct comparison of protein amounts (absolute or relative) from a variety of cellular states. Because of the reasons stated previously, quantitative applications are likely to be less dependent on 2-DGE and rely primarily on formats that involve specific purification and/or chromatographic separation with mass spectrometry. 

Since the major part of incorporated radioactivity was located in the 105,000 X g supernatant fraction from drug-treated cells, this fraction was separated into subtractions on a column of Sephadex G-200. The 105,000 X g supernatant fluid, which contains proteins, sRNA and soluble enzymes, as well as other cellular components, separates on a column of Sephadex G-200 into 3 peaks (Kadaya et al., 1964) when measured by absorbancy at 260 mp. (Fig. 3). Fig. 3 shows that the radioactivity of the control separated into two peaks, with the major portion of radioactivity eluted between the first and second absorbancy peak. A small portion of radioactivity was eluted in the third absorbancy peak. In contrast to the control, radioactivity of 105,000 X g supernatant fraction of drug-treated cells was eluted as a single peak, with the third absorbancy peak (Fig. 4). Radioactivity of this peak was equal to that of the control. Paper chromatography of the third peak fractions revealed the presence of nucleotides and small peptides, with edeine as the major radioactive component. When the protein content of the effluents from the column was determined by the Lowry method (Lowry 1951) all samples separated into 3 peaks as shown in Fig. 3. A comparison of protein and radioactivity profiles reveals that the inhibitors of protein synthesis affected the incorporation of tyrosine or methionine into protein fractions eluted between the first and second absorbancy peaks, leaving unaffected incorporation into the edeine containing third peak. 

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