A Simple and Effective Sample Preparation Strategy for MALDI-MS Imaging of Neuropeptide Changes in the Crustacean Brain Due to Hypoxia and Hypercapnia Stress

Abstract

Matrix-assisted laser desorption/ionization (MALDI)-MS imaging has been utilized to image a variety of biomolecules, including neuropeptides. Washing a tissue section is an effective way to eliminate interfering background and improve detection of low concentration target analyte molecules; however, many previous methods have not been tested for neuropeptide analysis via MALDI-MS imaging. Using crustaceans as a neurological model organism, we developed a new, simple washing procedure and applied this method to characterize neuropeptide changes due to hypoxia stress. With a 10 s 50:50 EtOH:H₂O wash, neuropeptide coverage was improved by 1.15-fold, while normalized signal intensities were increased by 5.28-fold. Specifically, hypoxia and hypercapnia stress conditions were investigated due to their environmental relevance to marine invertebrates. Many neuropeptides, including RFamides, pyrokinin, and cardioactive peptides, showed distinct up- and down-regulation for specific neuropeptide isoforms. Since crustacean neuropeptides are homologous to those found in humans, results from these studies can be applied to understand potential roles of neuropeptides involved in medical hypoxia and hypercapnia.

Figure 1.

A pictorial schematic of the proposed wash method for crustacean neuropeptides. After collecting the brain from the control or exposed crab, the tissue is embedded in gelatin for sectioning using a cryostat. Next, sections are washed in the optimal wash solution and dried under vacuum. Matrix is then applied prior to be analyzed by MALDI-MS imaging.

Figure 2.

Comparison of the top three washing protocols with number of neuropeptides identified (blue bars) and the normalized signal intensity (purple dots) (n=3 technical replicates). The number of neuropeptides varied slightly between these conditions and a control, but the intensity spiked for the 50:50 H₂O:EtOH washes.

Figure 3.

Examples of neuropeptide image changes due to the wash step in serial sections of a crustacean brain. (a) An optical image of the crustacean brain, outlined in white. The elongated tubes point towards the commissural ganglion of the crustacean nervous system. (b)-(f) Crustacean neuropeptides that were removed due to the washing step. (g) A neuropeptide image example that did not have a change in intensity or localization due to the washing step. (h)-(m) Neuropeptides that had a clear signal increase due to the washing step. All washed-control comparisons are generated at the same TIC normalized signal intensity level (0 to (b) 1.82×10⁻³, (c) 2.40×10⁻³, (d) 3.00×10⁻³, (e) 2.07×10⁻³, (f) 1.15×10⁻³, (g) 2.38×10⁻³, (h) 1.96×10⁻³, (i) 2.55×10⁻³, (j) 1.27×10⁻³, (k) 2.05×10⁻³, (l) 1.18×10⁻³, (m) 3.34×10⁻³). The white line represents a 1 mm scale bar.

Figure 4.

Examples of neuropeptides and their changes due to hypoxia and hypercapnia stress compared to a control. (a) Optical image of crab brain under each washed condition, outlined in white. The elongated tubes point towards the commissural ganglion of the crustacean nervous system. (b)-(g) Statistically significant (p<0.0125) neuropeptides between either severe hypoxia vs. control, moderate hypoxia vs. control, hypercapnia vs. control, severe hypoxia vs. moderate hypoxia, and/or moderate hypoxia vs. hypercapnia, with a table of these results provided in Table S2. All control-hypoxia-hypercapnia comparisons are generated at the same TIC normalized signal intensity level (0 to (b) 2.22×10⁻⁴, (c) 5.31×10⁻⁴, (d) 5.65×10⁻⁴, (e) 6.03×10⁻⁴, (f) 6.73×10⁻⁴, (g) 8.29×10⁻⁴). The white line represented a 1 mm scale bar. All biological replicates of each condition for the m/z values shown are available in Figures S3–8.