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

Salisbury JP, Boggio KJ, Hsu YW, Quijada J, Sivachenko A, Gloeckner G, Kowalski PJ, Easterling ML, Rosbash M, Agar JN - Mol Brain (2013)

Bottom Line: Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.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.

View Article: PubMed Central - HTML - PubMed

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.

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.

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High resolution MALDI-FTICR MS resolves neuropeptides PDNFMRFamide and Drostatin-3 (Ast-A3), convoluted in MALDI-TOF mass spectra. Through the use of MALDI-FTICR MS, we were able to resolve m/z 925.481 from the MALDI-TOF MS (top) as being the convolution of two neuropeptides. PDNFMRFamide, identified by MALDI-TOF/TOF MS/MS fragmentation, was observed in MALDI-FTICR MS (bottom) as 925.43564 (theoretical monoisotopic [M + H]+ = 925.43594). A second peak at m/z 925.49004 was tentatively assigned to correspond to Drostatin-3 (Ast-A3, calculated [M + H]+ = 925.48994).
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Figure 6: High resolution MALDI-FTICR MS resolves neuropeptides PDNFMRFamide and Drostatin-3 (Ast-A3), convoluted in MALDI-TOF mass spectra. Through the use of MALDI-FTICR MS, we were able to resolve m/z 925.481 from the MALDI-TOF MS (top) as being the convolution of two neuropeptides. PDNFMRFamide, identified by MALDI-TOF/TOF MS/MS fragmentation, was observed in MALDI-FTICR MS (bottom) as 925.43564 (theoretical monoisotopic [M + H]+ = 925.43594). A second peak at m/z 925.49004 was tentatively assigned to correspond to Drostatin-3 (Ast-A3, calculated [M + H]+ = 925.48994).

Mentions: Listed are D. melanogaster neuropeptides identified in this study. The final three listings are believed to be novel. Reported observed m/z’s are the average of the m/z’s observed between the two replicate experiments MALDI-TOF MS experiments described. All observed m/z’s listed were determined to be monoisotopic [M + H]+’s of listed neuropeptides based on the calculated monoisotopic [M + H]+ (shown in parentheses below observed m/z). Truncated peptides are denoted by superscripts showing amino acids present from the annotated peptide sequence (i.e. NPLP1-3AA1-AA18 is missing the final three residues of the annotated NPLP1-3 sequence, also shown). Peptide sequences include pre- and post- cleavage residues separated from sequences by a period. C-terminal amidation is denoted by an “a” at end of the peptide sequence. Abbreviations are listed in Table 2 legend. *m/z 925.481 likely corresponds to the convolution of PDNFMRFamide (monoisotopic [M + H]+ = 925.435) and Drostatin-3 (Ast-A3, monoisotopic [M + H]+ = 925.489, see Table 2), which were detected as separate peaks with MALDI-FTICR-MS (Figure 6), hence the comparatively larger m/z error.


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

Salisbury JP, Boggio KJ, Hsu YW, Quijada J, Sivachenko A, Gloeckner G, Kowalski PJ, Easterling ML, Rosbash M, Agar JN - Mol Brain (2013)

High resolution MALDI-FTICR MS resolves neuropeptides PDNFMRFamide and Drostatin-3 (Ast-A3), convoluted in MALDI-TOF mass spectra. Through the use of MALDI-FTICR MS, we were able to resolve m/z 925.481 from the MALDI-TOF MS (top) as being the convolution of two neuropeptides. PDNFMRFamide, identified by MALDI-TOF/TOF MS/MS fragmentation, was observed in MALDI-FTICR MS (bottom) as 925.43564 (theoretical monoisotopic [M + H]+ = 925.43594). A second peak at m/z 925.49004 was tentatively assigned to correspond to Drostatin-3 (Ast-A3, calculated [M + H]+ = 925.48994).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4022047&req=5

Figure 6: High resolution MALDI-FTICR MS resolves neuropeptides PDNFMRFamide and Drostatin-3 (Ast-A3), convoluted in MALDI-TOF mass spectra. Through the use of MALDI-FTICR MS, we were able to resolve m/z 925.481 from the MALDI-TOF MS (top) as being the convolution of two neuropeptides. PDNFMRFamide, identified by MALDI-TOF/TOF MS/MS fragmentation, was observed in MALDI-FTICR MS (bottom) as 925.43564 (theoretical monoisotopic [M + H]+ = 925.43594). A second peak at m/z 925.49004 was tentatively assigned to correspond to Drostatin-3 (Ast-A3, calculated [M + H]+ = 925.48994).
Mentions: Listed are D. melanogaster neuropeptides identified in this study. The final three listings are believed to be novel. Reported observed m/z’s are the average of the m/z’s observed between the two replicate experiments MALDI-TOF MS experiments described. All observed m/z’s listed were determined to be monoisotopic [M + H]+’s of listed neuropeptides based on the calculated monoisotopic [M + H]+ (shown in parentheses below observed m/z). Truncated peptides are denoted by superscripts showing amino acids present from the annotated peptide sequence (i.e. NPLP1-3AA1-AA18 is missing the final three residues of the annotated NPLP1-3 sequence, also shown). Peptide sequences include pre- and post- cleavage residues separated from sequences by a period. C-terminal amidation is denoted by an “a” at end of the peptide sequence. Abbreviations are listed in Table 2 legend. *m/z 925.481 likely corresponds to the convolution of PDNFMRFamide (monoisotopic [M + H]+ = 925.435) and Drostatin-3 (Ast-A3, monoisotopic [M + H]+ = 925.489, see Table 2), which were detected as separate peaks with MALDI-FTICR-MS (Figure 6), hence the comparatively larger m/z error.

Bottom Line: Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.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.

View Article: PubMed Central - HTML - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus