Peptide Profiling of Cells with Multiple Gene Products: Combining Immunochemistry and MALDI Mass Spectrometry with On-Plate microextraction

摘要

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinaiy approach combining single-cell MALDI-MS peptide proffi-ing, northern analysis, in situ hybridization, and inimu-nocytochemistry allows characterization of a more com-plete set of neurotransmitters than individual approaches in the Aplysia ealifornica Hi and B2 motor neurons. Because different results were obinined using both in situ and inimunohistochemical techniques compared to previ-ous reports, MALDI-MS assays have been used to exam-ine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of ac-etone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the Hi and B2 neurons—the SCP and CP1 prohormones—end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded ho-modimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzy-matic digestion.