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 desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-micron diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to 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 immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed.