multi-faceted-mass-spectrometric-investigation-neuropeptides

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in <em>Callinectes sapidus</em>

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

University of Wisconsin-Madison

Accurate clinical therapeutics rely on understanding the metabolic responses of individual cells. However, the high level of heterogeneity between cells means that simply sampling from large populations of cells is not necessarily a reliable approximation of an individual cell's response. As a result, there have been numerous developments in the field of single-cell analysis to address this lack of knowledge. Many of these developments have focused on the coupling of capillary electrophoresis (CE), a separation technique with low sample consumption and high resolving power, and mass spectrometry (MS), a sensitive detection method for interrogating all ions in a sample in a single analysis. In recent years, there have been many notable advancements at each step of the single-cell CE-MS analysis workflow, including sampling, manipulation, separation, and MS analysis. In each of these areas, the combined improvements in analytical instrumentation and achievements of numerous researchers have served to drive the field forward to new frontiers. Consequently, notable biological discoveries have been made possible by the implementation of these methods. Although there is still room in the field for numerous further advances, researchers have effectively minimized various limitations in detection of analytes, and it is expected that there will be many more developments in the near future.

Recent Advances and New Perspectives in Capillary Electrophoresis-Mass Spectrometry for Single Cell "Omics".

Hypoxia (i.e., low oxygen (O) levels) is a common environmental challenge for several aquatic species, including fish and invertebrates. To survive or escape these conditions, these animals have developed novel biological mechanisms, some regulated by neuropeptides. By utilizing mass spectrometry (MS), this study aims to provide a global perspective of neuropeptides in the blue crab, , and their changes over time (0, 1, 4, and 8 h) due to acute, severe hypoxia (∼10% O water saturation) stress using a 4-plex reductive dimethylation strategy to increase throughput. Using both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) MS, this study provides complementary coverage, allowing 88 neuropeptides to be identified. Interesting trends include (1) an overall decrease in neuropeptide expression due to hypoxia exposure, (2) a return to basal levels after 4 or 8 h of exposure following an initial response, (3) changes only after 4+ h exposure, and (4) an oscillating pattern. Overall, this study boosts the power of multiplexed quantitation to understand the large-scale changes due to severe hypoxia stress over time.

Temporal Study of the Perturbation of Crustacean Neuropeptides Due to Severe Hypoxia Using 4-Plex Reductive Dimethylation.

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:HO 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.

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

Oxygen (O) is a critical component of life; without proper O levels, cells are unable to respire, meaning glucose cannot be utilized. Thus, hypoxia (low O levels) is a well-documented stressor, especially in aquatic environments. Neuropeptides are a major class of regulators for stress-induced responses; however, their global expression changes during stress are not well characterized due to the natural complexity of the nervous system. Beyond being a neurological model organism, crustaceans are regularly exposed to hypoxia, making them a relevant system for this study. Several neuropeptide families, including orcokinins, RFamides, and allatostatin A-types, show dynamic dysregulation due to hypoxic stress. In particular, the brain showed the most dynamic changes with a survival mechanism "switching" (i.e., significant increase to decrease) of neuropeptide content between moderate and severe hypoxia (e.g., NFDEDRSGFA, FDAFTTGFGHS, NRNFLRFamide, and APSGFLGMRamide). Globally, neuropeptides in different tissues appeared to exhibit unique expression patterns at the various severities of hypoxia, including LSSSNSPSSTPL and NFDEIDRSSFGF. Overall, this study provides clear evidence for the benefits of globally analyzing biomolecules and that neuropeptides play a critical role in how crustaceans adapt due to hypoxic stress.

Mass Spectrometric Profiling of Neuropeptides in  during Hypoxia Stress.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Neuropeptides are an important class of endogenous peptides in the nervous system that regulate physiological functions such as feeding, glucose homeostasis, pain, memory, reproduction, and many others. In order to understand the functional role of neuropeptides in diseases or disorders, studies investigating their dysregulation in terms of changes in abundance and localization must be carried out. As multiple neuropeptides are believed to play a functional role in each physiological process, techniques capable of global profiling multiple neuropeptides simultaneously are desired. Mass spectrometry is well-suited for this goal due to its ability to perform untargeted measurements without prior comprehensive knowledge of the analytes of interest. Mass spectrometry imaging (MSI) is particularly useful because it has the capability to image a large variety of peptides in a single experiment without labeling. Like all analytical techniques, careful sample preparation is critical to successful MSI analysis. The first half of this review focuses on recent developments in MSI sample preparation and instrumentation for analyzing neuropeptides and other biomolecules in which the sample preparation technique may be directly applicable for neuropeptide analysis. The benefit offered by incorporating these techniques is shown as improvement in a number of observable neuropeptides, enhanced signal to noise, increased spatial resolution, or a combination of these aspects. The second half of this review focuses on recent biological discoveries about neuropeptides resulting from these improvements in MSI analysis. The recent progress in neuropeptide detection and analysis methods, including the incorporation of various tissue washes, matrices, instruments, ionization sources, and computation approaches combined with the advancements in understanding neuropeptide function in a variety of model organisms, indicates the potential for the utilization of MSI analysis of neuropeptides in clinical settings.

Neuropeptidomics: Improvements in Mass Spectrometry Imaging Analysis and Recent Advancements.

Neuropeptides are low abundance signaling molecules that modulate almost every physiological process, and dysregulation of neuropeptides is implicated in disease pathology. Mass spectrometry (MS) imaging is becoming increasingly useful for studying neuropeptides as new sample preparation methods for improving neuropeptide detection are developed. In particular, proper tissue washes prior to MS imaging have shown to be quick and effective strategies for increasing the number of detectable neuropeptides. Treating tissues with solvents could result in either gain or loss of detection of analytes, and characterization of these wash effects is important for studies targeting sub-classes of neuropeptides. In this communication, we apply aqueous tissue washes that contain sodium phosphate salts, including 10% neutral buffered formalin (NBF), on crustacean brain tissues. Our optimized method resulted in complementary identification of neuropeptides between washed and unwashed tissues, indicating that our wash protocol may be used to increase total neuropeptide identifications. Finally, we show that identical neuropeptides were detected between tissues treated with 10% NBF and an aqueous 1% w/v sodium phosphate solution (composition of 10% NBF without formaldehyde), suggesting that utilizing a salt solution wash affects neuropeptide detection and formaldehyde does not affect neuropeptide detection when our wash protocol is performed.

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

Due to their involvement in numerous biochemical pathways, neuropeptides have been the focus of many recent research studies. Unfortunately, classic analytical methods, such as western blots and enzyme-linked immunosorbent assays, are extremely limited in terms of global investigations, leading researchers to search for more advanced techniques capable of probing the entire neuropeptidome of an organism. With recent technological advances, mass spectrometry (MS) has provided methodology to gain global knowledge of a neuropeptidome on a spatial, temporal, and quantitative level. This review will cover key considerations for the analysis of neuropeptides by MS, including sample preparation strategies, instrumental advances for identification, structural characterization, and imaging; insightful functional studies; and newly developed absolute and relative quantitation strategies. While many discoveries have been made with MS, the methodology is still in its infancy. Many of the current challenges and areas that need development will also be highlighted in this review.

Nhu Q. Vu

Department of Chemistry

University of Wisconsin-Madison

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus