Mass spectral analysis of neuropeptide expression and distribution in the nervous system of the lobster Homarus americanus

Abstract

The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. A total of 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.

Figure 1

Analysis of an HPLC fraction from the lobster PO tissue extract on three different MALDI instruments, including (a) MALDI TOF/TOF, (b) MALDI LTQ Orbitrap and (c) MALDI FTICR MS. (d) Venn diagram showing the number of peaks detected using each type of instrument. Each spot was analyzed three times on each instrument.

Figure 2

Comparison of multiple fragmentation reactions on the MALDI TOF/TOF and MALDI LTQ Orbitrap mass spectrometers for de novo sequencing of an unknown peptide peak at m/z 1084.6. (a) Collision induced dissociation (CID) on the LTQ Orbitrap instrument, (b) pulsed-Q dissociation (PQD) on the linear ion trap of the LTQ Orbitrap instrument, (c) high energy collisional dissociation (HCD) on the LTQ Orbitrap instrument, and (d) Collision induced dissociation on the TOF/TOF mass spectrometer. The predicted sequence is HI/LASLYKRP, and the presence of b and y ions is indicated by horizontal lines above (y ions) or below (b ions) the corresponding amino acid residues in the peptide sequence.

Figure 3

Collision-induced dissociation spectra of de novo sequenced neuropeptides from the pericardial organ of the lobster on nanoLC ESI QTOF instrument. (a) A-type allatostatin: PRNYAFGLamide; (b) FMRFamide-related peptide (FaRP): APSKNFLRFamide. Both precursor ions are doubly charged. The sequence-specific b- and y-types fragment ions and immonium ions are labeled. The presence of lysine in peptide (b) was confirmed by dimethylation of peptide via formaldehyde labeling of the crude sample extract.

Figure 4

MS/MS spectra of peptide APSKNFLRFamide acquired on two MALDI tandem MS instruments. (a) High energy collisional induced dissociation on the LTQ Orbitrap instrument, and (b) Collision induced dissociation on the TOF/TOF mass spectrometer. The presence of b and y ions is indicated by horizontal lines above (y ions) or below (b ions) the corresponding amino acid residues in the peptide sequence in each spectrum. The presence of lysine in the sequence was confirmed by formaldehyde labeling (data not shown).

Figure 5

Direct tissue profiling of two areas of the lobster pericardial organ using MALDI TOF/TOF: (a) white enlargement; (b) long fiber that projects along the muscle and into the heart. The sequences of detected peptide peaks are indicated in the spectra, and peptides from different families are distinguished by different colors.

Figure 6

Mapping the distribution of neuropeptides throughout the nervous system in H. americanus by a combination of direct tissue analysis performed on MALDI TOF/TOF and bioinformatics. Direct tissue/extract analysis was performed on eight neuronal organs: (a) brain, (b) commissural ganglion (CoG), (c) thoracic ganglion (TG), (d) subesophageal ganglion (SEG), (e) stomatogastric ganglion (STG), (f) esophageal ganglion (OG); and two major endocrine organs (g) pericardial organ (PO), and (h) sinus gland (SG). (i) The presence of multiple neuropeptide families and isoforms were compared and organized using in-house developed bioinformatics software SpecPlot. The molecular mass of each neuropeptide was listed on the top row organized in the order of families, and the first column indicates the tissue source for each row. Blue squares indicate the presence of certain neuropeptides in the specific organs, and black squares indicate absence of peptide signals.

Figure 7

MALDI imaging of neuropeptide localization in the H. americanus brain using both MALDI TOF/TOF and MALDI LTQ Orbitrap mass spectrometers. (a) Schematic drawing of the lobster brain, which contains multiple neuropils, including anterior (AMPN) and posterior medial protocerebral neuropils (PMPN), olfactory lobe (ON), accessory lobe (AL), antenna I neuropil (AnN) and lateral II antenna neuropil (LAN). Ion images of (b) VYRKPPFNGSIFamide (m/z 1423.8) and (c) NFDEIDRSGFGFN (m/z 1517.7) were obtained using MALDI TOF/TOF. Ion images of multiple known neuropeptides were acquired by MALDI LTQ Orbitrap, including: (d) VYRKPPFNGSIFamide (m/z 1423.8); (e) APSGFLGMRamide (m/z 934.5); (f) NFDEIDRSGFGFN (m/z 1517.7); and (g) overlay of the above three neuropeptides. A novel peptide HI/LASLYKPR (m/z 1084.6) in the lobster brain was also mapped using MALDI LTQ Orbitrap instrument by (h) precursor ion scanning of m/z 1084.6 and (i) selected reaction monitoring of transition between m/z 1084.6 and sequence-specific fragment ion m/z 685.4 (b₆).