Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Jasminka Godovac-Zimmermann from UCL, together with ProteoSys Ag optimized method for affinity isolation of phospoproteins

Further development of MPD for ultra-high sensitivity analysis of proteins.
It was decided to develop further the MultiPhotonDetection technology to perform ultra-sensitive analyses of differentially displayed phosphoproteins from HeLa cells expressing either WT-CFTR or ?F508-CFTR and, at the same time, test the new back-to-back HT-MPD imager.

By using standard proteins that were trace labelled with 125I (specific activity of 0.5 Ci/mmole tyrosine residues = 1 labelled tyrosine residue in 12000 available ones), then fractionated on 1D gels and measures for 12 hrs on the MPD imager, we reached a sensitivity limit of protein detection of 90 attomoles. Serial dilution curves showed that the MPD measurement gave excellent linear quantitation over the full concentration range. The method used for protein radiolabelling can provide specific activities of about 200 Ci/mmole tyrosine residues, which means that the sensitivity limit with our current settings is at 200 zeptomoles.

To compare MPD with phosphor imager detection, 60 ng of 125I iodinated HeLa phosphoproteins (specific activity 5 Ci/mmole tyrosine residues) were separated on narrow pH unit 2D gels (pH 5.5 - 6.7), measured for 3 days on a phosphor imager and subsequently for 12 hrs on the MPD imager. In the MPD analysis a very conservative background cut-off filter was used. The MPD measurement showed approximately 10 times as many protein spots as the phosphor imager analysis.

We were also able to set up a first multicolour MPD analysis. Total proteins isolated from HeLa pTracer cells, labelled with 131I and phosphoproteins from the same cells, labelled with 125I, were separated in the same narrow pH unit 2D gel (pH 5.5 - 6.7). The 125I-labelled phosphoproteins and the 131I-labelled total proteins were then individually detected by multicolour MPD without any cross-talk problems between the two different isotopes.

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