An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Recent advances in mass spectrometry analysis of neuropeptides.

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.

Exploring the Sexual Dimorphism of Crustacean Neuropeptide Expression Using Callinectes sapidus as a Model Organism.

The impact of numerous diseases has been linked to differences in sex between organisms, including various neurological diseases. As neuropeptides are known to be key players in the nervous system, studying the variation of neuropeptidomic profiles between males and females in a crustacean model organism is of interest. By using high-resolution mass spectrometry with two complementary ionization sources in conjunction with quantitative chemical labeling (isotopic reductive dimethylation), differences were observed in five key neural tissues and hemolymph. Interestingly, while males and females possess numerous neuropeptide isoforms that are unique to their sex, the represented families of each sex remain largely consistent. However, some differences in familial isoforms were also observed, such as the relative numbers of neuropeptides belonging to RFamide and allatostatin A-type families. Additionally, >100 neuropeptides detected across five neural tissues and hemolymph were found to have statistically significant differences in abundance between male and female blue crab samples. Also, hundreds of putative peptide sequences were identified by de novo sequencing that may be indicative of previously undiscovered neuropeptides, highlighting the power of using a multifaceted MS approach.

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

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.

Highly multiplexed quantitative proteomic and phosphoproteomic analyses in vascular smooth muscle cell dedifferentiation.

Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFß (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFß/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFß/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFß/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFß/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFß/Smad3 activated cell proliferation and TGFß signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFß regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.

Discovery and validation of surface N-glycoproteins in MM cell lines and patient samples uncovers immunotherapy targets.

BACKGROUND: Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow. While recent advances in treatment for MM have improved patient outcomes, the 5-year survival rate remains ~50%. A better understanding of the MM cell surface proteome could facilitate development of new directed therapies and assist in stratification and monitoring of patient outcomes. METHODS: In this study, we first used a mass spectrometry (MS)-based discovery-driven cell surface capture (CSC) approach to map the cell surface N-glycoproteome of MM cell lines. Next, we developed targeted MS assays, and applied these to cell lines and primary patient samples to refine the list of candidate tumor markers. Candidates of interest detected by MS on MM patient samples were further validated using flow cytometry (FCM). RESULTS: We identified 696 MM cell surface N-glycoproteins by CSC, and developed 73 targeted MS detection assays. MS-based validation using primary specimens detected 30 proteins with significantly higher abundance in patient MM cells than controls. Nine of these proteins were identified as potential immunotherapeutic targets, including five that were validated by FCM, confirming their expression on the cell surface of primary MM patient cells. CONCLUSIONS: This MM surface N-glycoproteome will be a valuable resource in the development of biomarkers and therapeutics. Further, we anticipate that our targeted MS assays will have clinical benefit for the diagnosis, stratification, and treatment of MM patients.

Mass Spectrometric Profiling of Neuropeptides in Callinectes sapidus during Hypoxia Stress.

Oxygen (O2) is a critical component of life; without proper O2 levels, cells are unable to respire, meaning glucose cannot be utilized. Thus, hypoxia (low O2 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.

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

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.

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

Hypoxia (i.e., low oxygen (O2) 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, Callinectes sapidus, and their changes over time (0, 1, 4, and 8 h) due to acute, severe hypoxia (∼10% O2 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.

Multifaceted Mass Spectrometric Investigation of Neuropeptide Changes in Atlantic Blue Crab, Callinectes sapidus, in Response to Low pH Stress.

The decrease of pH level in the water affects animals living in aquatic habitat, such as crustaceans. The molecular mechanisms enabling these animals to survive this environmental stress remain unknown. To understand the modulatory function of neuropeptides in crustaceans when encountering drops in pH level, we developed and implemented a multifaceted mass spectrometric platform to investigate the global neuropeptide changes in response to water acidification in the Atlantic blue crab, Callinectes sapidus. Neural tissues were collected at different incubation periods to monitor dynamic changes of neuropeptides under different stress conditions occurring in the animal. Neuropeptide families were found to exhibit distinct expression patterns in different tissues and even each isoform had its specific response to the stress. Circulating fluid in the crabs (hemolymph) was also analyzed after 2-h exposure to acidification, and together with results from tissue analysis, enabled the discovery of neuropeptides participating in the stress accommodation process as putative hormones. Two novel peptide sequences were detected in the hemolymph that appeared to be involved in the stress-related regulation in the crabs.

New techniques, applications and perspectives in neuropeptide research.

Neuropeptides are one of the most diverse classes of signaling molecules and have attracted great interest over the years owing to their roles in regulation of a wide range of physiological processes. However, there are unique challenges associated with neuropeptide studies stemming from the highly variable molecular sizes of the peptides, low in vivo concentrations, high degree of structural diversity and large number of isoforms. As a result, much effort has been focused on developing new techniques for studying neuropeptides, as well as novel applications directed towards learning more about these endogenous peptides. The areas of importance for neuropeptide studies include structure, localization within tissues, interaction with their receptors, including ion channels, and physiological function. Here, we discuss these aspects and the associated techniques, focusing on technologies that have demonstrated potential in advancing the field in recent years. Most identification and structural information has been gained by mass spectrometry, either alone or with confirmations from other techniques, such as nuclear magnetic resonance spectroscopy and other spectroscopic tools. While mass spectrometry and bioinformatic tools have proven to be the most powerful for large-scale analyses, they still rely heavily on complementary methods for confirmation. Localization within tissues, for example, can be probed by mass spectrometry imaging, immunohistochemistry and radioimmunoassays. Functional information has been gained primarily from behavioral studies coupled with tissue-specific assays, electrophysiology, mass spectrometry and optogenetic tools. Concerning the receptors for neuropeptides, the discovery of ion channels that are directly gated by neuropeptides opens up the possibility of developing a new generation of tools for neuroscience, which could be used to monitor neuropeptide release or to specifically change the membrane potential of neurons. It is expected that future neuropeptide research will involve the integration of complementary bioanalytical technologies and functional assays.

Mass Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags for Multiplex Quantitative Proteomics.

We have developed a novel amine-reactive mass defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), that is compact in size, is suitable for various biological samples, and enables highly multiplexed quantification of peptides at the MS1 level without increasing mass spectral complexity. The DiPyrO tag structure incorporates heavy isotopes in a variety of configurations to impart as much as 45.3 mDa or as little as 5.8 mDa per tag between labeled peptides. Notably, peptides containing lysine are labeled with two tags, doubling the imparted mass defect to up to 90.6 mDa for the duplex tags and effectively reducing the resolving power requirement compared to previously reported mass defect-based quantification approaches. This permits current and previous generation LTQ-Orbitrap platforms to perform confident quantitative analyses of two DiPyrO-labeled samples at 100K resolving power, whereas 3-plex and 6-plex quantifications are possible at 240K and 480K resolving powers, respectively. In this work, we discuss the design and synthesis of the DiPyrO tag, characterize its effect on labeled proteome analysis by nanoLC-MS2, and demonstrate proof-of-principle applications of the duplex and triplex tags for quantitative proteomics using high-resolution MS acquisition on the Orbitrap Elite and Orbitrap Fusion Lumos.

Condensed-phase effects on the structural properties of FCH2CN-BF3 and ClCH2CN-BF3: a matrix-isolation and computational study.

We have measured several IR bands of FCH2CN-BF3 and ClCH2CN-BF3 in solid nitrogen, argon, and neon. These bands include the B-F asymmetric stretch (νBF(a)), the B-F symmetric stretch (νBF(s)), the BF3 symmetric deformation or "umbrella" mode (δBF(s)), and the CN stretch (νCN). For both complexes, the frequencies of these modes shift across the various media, particularly the B-F asymmetric stretching band, and thus they indicate that the inert gas matrix environments significantly alter the structural properties of FCH2CN-BF3 and ClCH2CN-BF3. Furthermore, the frequencies shift in a manner that parallels the dielectric constant of these media, which suggests a progressive contraction of the B-N distances in these systems and also that it parallels the ability of the medium to stabilize the increase in polarity that accompanies the bond contraction. We have also mapped the B-N distance potentials for FCH2CN-BF3 and ClCH2CN-BF3 using several density functional and post-Hartree-Fock methods, all of which reveal a flat, shelflike region that extends from the gas-phase minimum (near 2.4 Å) toward the inner wall (to about 1.7 Å). Furthermore, we were able to rationalize the medium effects on the structure by constructing hybrid bond potentials composed of the electrostatic component of the solvation free energy and the gas-phase electronic energy. These curves indicate that the solvation energies are greatest at short B-N distances (at which the complex is more polar), and ultimately, the potential minima shift inward as the dielectric constant of the medium increases.