Neuropeptidomic Profiling and Localization in the Crustacean Cardiac Ganglion Using Mass Spectrometry Imaging with Multiple Platforms

Abstract

The crustacean cardiac neuromuscular system is a useful model for studying how neural circuits generate behavior, as it is comprised of a simple ganglion containing nine neurons, yet acts as a robust central pattern generator. The crustacean heart is neurogenic, receiving input from neuropeptides. However, the specific effects of neuropeptides on cardiac output is not fully understood, and the large degree of comodulation between multiple neuropeptides makes studying these effects more challenging. To address this challenge, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging was used to localize neuropeptides within the cardiac ganglion (CG), providing information about the identity and localization of neuropeptides being present. CG extracts were also profiled using liquid chromatography coupled to tandem mass spectrometry (MS/MS) with a data independent acquisition method, resulting in the confirmation of 316 neuropeptides. Two MS imaging (MSI) platforms were compared to provide comprehensive results, including a MALDI-Orbitrap instrument for high mass spectral resolution for accurate identifications and a MALDI TOF/TOF instrument for improved spatial resolution and sensitivity, providing more descriptive MS images. MS images for 235 putative neuropeptides were obtained, with the identification of 145 of these being confirmed by either complementary MS/MS data or accurate mass matching. The MSI studies demonstrate the sensitivity and power of this MALDI-based in situ analytical strategy for unraveling the chemical complexity present in a small nine-cell neuronal system. The results of this study will enable more informative assays of the functions of neuropeptides within this important neural circuit.

Keywords: cardiac neurophysiology; crustacean; mass spectrometry imaging; neuropeptides.

Figure 1.

(A) Optical anatomical images coated with DHB matrix and (B) MALDI-MS images of C. borealis brain serial sections analyzed on a RapifleX TOF/TOF instrument (top) and LTQ-Orbitrap XL instrument (bottom). The comparison shows an improvement in spatial resolution for images acquired on the RapifleX. The image intensity was in general stronger for ions obtained on the RapifleX, but several showed stronger signal on the Orbitrap, such as tachykinin TPSGFLGMRamide (m/z 964.503).

Figure 2.

(A) Optical anatomical images and (B) MALDI-MS images of C. borealis pericardial organs (POs) analyzed on a RapifleX TOF/TOF instrument (left) and LTQ-Orbitrap XL instrument (right). POs were collected from the same animal and the left was analyzed on the RapifleX and the right on the Orbitrap. The comparison shows an improvement in spatial resolution for images acquired on the RapifleX. The image intensity was stronger for some ions on the RapifleX and others on the Orbitrap.

Figure 3.

(A) Optical anatomical image and (B) MALDI-MS images of C. borealis cardiac ganglion acquired on the RapifleX TOF/TOF instrument. Images represent neuropeptides localized to distinct regions of the tissue.

Figure 4.

Representation of neuropeptides identified in the cardiac ganglion (CG), including the distribution of families neuropeptides belonged to that were identified with (A) LC-ESI-MS/MS with DIA analysis and (B) MALDI-MS with accurate mass matching. Also shown is (C) a comparison of two LC-MS/MS acquisition methods, DIA and DDA, showing the superior performance of DIA for the current analysis, and (D) a breakdown of the validation of neuropeptides detected with MSI on the RapifleX instrument, including those with confident MS/MS spectra, high-resolution accurate masses matching to the known database, and those with neither, listed as unconfirmed identifications.