Ultra-high sensitivity multi-photon detection imaging in proteomics analyses

We report on the use of 125I and 131I labeling and of new, multicolor, multi‐photon detection (MPD) methods to routinely and quantitatively detect protein spots on two‐dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two‐dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2‐D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co‐incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.