Peptide profiling of cells with multiple gene products: Combining immunochemistry and MALDI mass spectrometry with on-plate microextraction

Due to the intracellular chemical complexity and a wide range of transmitte r concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analys is, in si-tu hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compare d to previous reports, MALDI-MS assays have been used to examine CP1-relate d gene products in these cells. However, MALDI with standard sample prepara tion does not detect the presence of the CP1 gene products. A novel on-plat e microextraction approach using concentrated MALDI matrix 2,5-dihydroxyben zoic acid with a mixture of acetone and water as the soh ent has been devel oped to allow the detection of trace-level gene expression products. Both n europeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones -end with large peptides that have multiple cysteine residues. For SCP, MAL DI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1,a disulfide-bonded homodimer is detected and the disulfide bond ing pattern elucidated using MALDI-MS coupled with on-plate enzymatic diges tion.