COMBINATION OF PEPTIDE PROFILING BY MATRIX-ASSISTED-LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY AND IMMUNODETECTION ON SINGLE GLANDS OR CELLS/

The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser de sorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS),wh ich gives informative peptide profiling on complex samples such as org ans or cells; (ii) immunological wools such as enzyme-linked immunosor bent assay (ELISA) and immunocytochemistry to probe for specific pepti des in biological extracts or cells. The cellular expression of the tw o precursors of the hyperglycemic hormone (cHH) was analyzed in neuros ecretory cells (30-mu m diameter) from the crayfish Orconectes limosus . Neurohemal organs were used to optimize the sample preparation and t o demonstrate that, after peptide fingerprinting by MALDI-TOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochem istry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dua l characterization was successfully scaled down to the level of a sing le crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cas-producing cell previously identified by immunocyto chemistry demonstrated that both procHH isoforms were expressed in eac h cell analyzed.