title: A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics.
abstract: BACKGROUND
  Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.
  
  
  RESULTS
  Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.
  
  
  CONCLUSIONS
  Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Salisbury
authors.forename: Kristin J
authors.initials: KJ
authors.lastname: Boggio
authors.forename: Yun-Wei A
authors.initials: YW
authors.lastname: Hsu
authors.forename: Jeniffer
authors.initials: J
authors.lastname: Quijada
authors.forename: Anna
authors.initials: A
authors.lastname: Sivachenko
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gloeckner
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Kowalski
authors.forename: Michael L
authors.initials: ML
authors.lastname: Easterling
authors.forename: Michael
authors.initials: M
authors.lastname: Rosbash
authors.affiliation: Depts of Chemistry and Chemical Biology and Pharmaceutical Sciences and Barnett Institute of Chemical and Biological Analysis, Northeastern University, 140 The Fenway, Boston, MA 02115, USA. j.agar@neu.edu.
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Agar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016001:Confidence Intervals; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism*; D007477:Ions; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012815:Signal Processing, Computer-Assisted; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D029721:Drosophila Proteins; D007477:Ions; D009479:Neuropeptides