A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics

Abstract

BackgroundNeuropeptides 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.ResultsOver 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.ConclusionsUsing 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.

Peptidomics for the discovery and characterization of neuropeptides and hormones

“…Mass spectrometers with standard or interchangeable MALDI ion sources usually have MS/MS capabilities, which allow automatic or manual sequencing of peptides in solid tissue samples [39, 40]. MALDI MSI has an added benefit of being capable of mapping peptides to specific loci within the tissue section [34, 38].…”

“…Recently, differential comparisons have been used to probe unknown mechanisms in the physiological response to environmental [51], developmental [60] or pharmacological perturbation [31, 61, 62], disease states [36, 63–65], phenotypes [39, 66], or even various sample preparation methods crucial for successful neuropeptide detection [67, 68]. It is safe to say that relative or differential MS quantitation has already become routine due to the availability of commercial reagents, sensitive analytical instrumentation, and software.…”

Advances in Mass Spectrometric Tools for Probing Neuropeptides

“…Myriad software solutions for label-free experiments, most of which are designed for proteomic studies, are currently available on the market (83–86). Total ion current normalization (87, 88) as well as normalization to internal standards [e.g., bovine serum albumin (BSA) peptides] (3, 89) are two widely accepted and simple approaches. Several statistical, mathematical methods have been evaluated on endogenous peptide samples (90, 91).…”

Neuropeptidomics of the Rat Habenular Nuclei

“…The development of modern mass spectrometry (MS) techniques, aided by improvements in sample preparation methods, have changed the way peptide detection, identification, and quantitation are performed, allowing characterization of a fairly complete set of neuropeptides present in a biological structure. − In the past decade, hundreds of neuropeptides have been discovered and identified from a range of animals using matrix-assisted laser desorption/ionization (MALDI) MS and electrospray ionization (ESI) MS. ,,− …”