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:H 2 O wash, neuropeptide coverage was improved by 1.15-fold, while normalized signal intensities were increased by 5.28fold. 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.

Complementary neuropeptide detection in crustacean brain by mass spectrometry imaging using formalin and alternative aqueous tissue washes

“…Thus, mass spectrometry (MS) is well-suited for the study of neuropeptides, especially with the invention of soft ionization methods, such as matrix-assisted laser desorption/ionization (MALDI) [11]. Utilization of MS to study neuropeptides has become increasingly popular as sample preparation methods optimized for neuropeptides are developed [12][13][14][15][16][17]. A unique advantage of MALDI-MS over predominant liquid chromatography (LC)-MS techniques is the capability to do MS imaging to obtain localization information about intact neuropeptides simultaneously without the need for antibodies.…”

“…The low abundance of neuropeptides (~pM-nM level) presents a great challenge for MS imaging analysis as micronthick tissue sections are analyzed instead of concentrated tissue homogenates. Recent advancements in sample preparation methods, such as applying an organic solvent wash to rinse the tissue section [14,18,19], development of new matrices [20], and derivatization of amine-containing compounds [21], for performing MS imaging analysis of neurochemicals, have been successfully applied to answer biological questions. However, most of these methods are not focused on neuropeptide enrichment.…”

“…Due to our interest in sample preparation strategies for enriching neuropeptides for MS imaging analysis [14], here we explore the use of chemical modifiers, specifically formalin fixation, to identify neuropeptide localization in the crustacean brain. Our strategy is similar to a previous study from Harris et al, where they treated formalin fixation as a tissue wash to enhance lipid detection [33].…”

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI

“…For example, wash protocols are indispensable for imaging of peptides and proteins in tissue sections to get rid of physiological salts and abundant lipids. Furthermore, wash protocols can also be used to enhance the subsequent ionization or extraction of the analytes into the matrix layer [17][18][19]. OTED belongs to often-used strategies for identification and visualization of proteins [2] and glycans [5].…”

Molecular Mapping of Neutral Lipids using Silicon Nanopost Arrays and TIMS Imaging Mass Spectrometry

“…Imaging mass spectrometry (IMS) enables the spatial mapping of biomolecules such as proteins 1, 2 , peptides 3,4 , lipids 5,6 and metabolites 7,8 in biological tissues with high chemical specificity and without the need for target specific reagents. IMS has been widely adopted in fields like molecular histology for differentiation of cancerous and noncancerous tissues [9][10][11] , as well as the pharmaceutical industry for analyzing the distribution of drugs and their metabolites in tissues.…”