Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network.
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.
Jiménez
C R
CR
Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Biology, The Netherlands.
Li
K W
KW
Dreisewerd
K
K
Spijker
S
S
Kingston
R
R
Bateman
R H
RH
Burlingame
A L
AL
Smit
A B
AB
van Minnen
J
J
Geraerts
W P
WP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D017403:In Situ Hybridization; D007447:Invertebrate Hormones / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D008195:Lymnaea; D008969:Molecular Sequence Data; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism; D009474:Neurons / Q000737:chemistry* / Q000502:physiology; D009479:Neuropeptides / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D007447:Invertebrate Hormones; D009479:Neuropeptides; D011498:Protein Precursors; D012333:RNA, Messenger; C082583:VD1-RPD2 alpha preprohormone, Lymnaea; C041307:small cardioactive peptide B; C045659:small cardioactive peptide A