Direct Mass Spectrometric Peptide Profiling and Sequencing of Single Neurons Reveals Differential Peptide Patterns in a Small Neuronal Network ² C. R. Jime ´nez, ‡,⊥ K. W. Li,* ,‡ K. Dreisewerd, ‡ S. Spijker, ‡ R. Kingston, § R. H. Bateman, § A. L. Burlingame, | A. B. Smit, ‡ J. van Minnen, ‡ and W. P. M. Geraerts ‡ Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije UniVersiteit, Department of Molecular and Cellular Neurobiology, Faculty of Biology, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, Micromass, Floats Road, Wytheshawe, Manchester M23 9LZ, England, and Department of Pharmaceutical Chemistry, Mass Spectrometric Facility, UniVersity of California, San Francisco, California ReceiVed July 29, 1997; ReVised Manuscript ReceiVed NoVember 6, 1997 ABSTRACT: Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in Vitro. Neuropeptides form the largest and most diverse class of signaling molecules in the brain (1, 2) that are involved in the integration of complex physiological processes and behaviors (3, 4). Functionally related neurons commonly express overlapping yet different sets of peptides, which enables the coordination of intricate spatiotemporal patterns of activity in target cells (5-8). Due to the high complexity in vertebrate neuronal systems, invertebrate systems have been exploited as alternative model systems to investigate the significance of the use of multiple peptide messengers for neuronal communication. We studied the two giant identifiable neurons VD1 and RPD2 in the brain of the mollusc Lymnaea stagnalis (9). VD1 and RPD2 are electronically coupled and form a simple neuronal network that is involved in the modulation of cardiorespiratory activity (10, 11). Previous cDNA studies revealed that both VD1 and RPD2 contain transcripts encoding two peptide precur- sors, named the R1 and R2 peptide precursors, that are derived from alternative splicing of a single pre-mRNA (12, 13). The mature peptides have been isolated from an extract of several hundred pooled singly dissected VD1 and RPD2 and structurally characterized by Edman sequencing. Two of the peptides, the R1 and R2 peptides, are cardioactive (12). The pharmacological and immunocytochemical data (11) together indicate that the R1 and R2 peptides are the messengers that VD1 and RPD2 use to modulate heartbeat. Recently, we demonstrated the feasibility of the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) 1 to detect peptides contained in single neurons (14, 15). Analysis of VD1 revealed the presence of molecular ion species with masses corresponding to the peptides derived from the two R peptide precursors (14). Unexpectedly, a number of molecular ion species of un- known identities were also detected (14), suggesting that the peptide complexity of these neurons is higher than that indicated by previous reports (12, 13). In analogy to VD1, the functionally related RPD2 may also have a more complex peptide profile than previously described. In the present study, we extended the MALDI-MS analysis to include both VD1 and RPD2. Comparison of the MALDI mass spectra of single VD1 and RPD2 revealed that VD1 contains, in addition to the (putative) peptides that are also present in RPD2, a subset of unique (putative) peptides. As these hitherto not reported VD1-specific molecules define the ² We acknowledge the Netherlands Organization of Research (NWO) for an apparatus grant to K.W.L. and W.P.M.G. A.L.B. received financial support from NIH NCRR Grant RR 01614. * To whom correspondence should be addressed: Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. Fax: 31-20-444-7123. ‡ Vrije Universiteit. § Micromass. | University of California. ⊥ Present address: Department of Pharmaceutical Chemistry, Mass Spectrometric Facility, University of California, San Francisco, CA. 1 Abbreviations: MALDI-MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; SCP, small cardioactive peptide; MS, mass spectrometry; CID, collision-induced dissociation. 2070 Biochemistry 1998, 37, 2070-2076 S0006-2960(97)01848-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/06/1998