Developing mass spectrometry for the quantitative analysis of neuropeptides.

INTRODUCTION: Neuropeptides are signaling molecules originating in the neuroendocrine system that can act as neurotransmitters and hormones in many biochemical processes. Their exact function is difficult to characterize, however, due to dependence on concentration, post-translational modifications, and the presence of other comodulating neuropeptides. Mass spectrometry enables sensitive, accurate, and global peptidomic analyses that can profile neuropeptide expression changes to understand their roles in many biological problems, such as neurodegenerative disorders and metabolic function. AREAS COVERED: We provide a brief overview of the fundamentals of neuropeptidomic research, limitations of existing methods, and recent progress in the field. This review is focused on developments in mass spectrometry and encompasses labeling strategies, post-translational modification analysis, mass spectrometry imaging, and integrated multi-omic workflows, with discussion emphasizing quantitative advancements. EXPERT OPINION: Neuropeptidomics is critical for future clinical research with impacts in biomarker discovery, receptor identification, and drug design. While advancements are being made to improve sensitivity and accuracy, there is still room for improvement. Better quantitative strategies are required for clinical analyses, and these methods also need to be amenable to mass spectrometry imaging, post-translational modification analysis, and multi-omics to facilitate understanding and future treatment of many diseases.

Mass Spectrometric Profiling of Neuropeptides in Response to Copper Toxicity via Isobaric Tagging.

Copper is a necessary nutrient but quickly becomes toxic at elevated levels. To properly handle environmental copper influxes and maintain metal homeostasis, organisms utilize various methods to chelate, excrete, and metabolize heavy metals. These mechanisms are believed to involve complex signaling pathways mediated by neuropeptides. This study incorporates custom N,N-dimethyl leucine isobaric tags to characterize the neuropeptidomic changes after different time points (1, 2, and 4 h) of copper exposure in a model organism, blue crab, Callinectes sapidus. Using a modified simplex optimization strategy, the number of identifiable and quantifiable neuropeptides was increased 3-fold to facilitate a deeper understanding of the signaling pathways involved in responding to heavy metal exposure. The time course exposure showed many interesting findings, including upregulation of inhibitory allatostatin peptides in the pericardial organs. Additionally, there was evidence of transport of a pigment dispersing hormone from the sinus glands to the brain. Overall, this study improves the multiplexing capabilities of neuropeptidomic studies to understand the temporal changes associated with copper toxicity.

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.

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

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.

Multiplexed quantitative neuropeptidomics via DiLeu isobaric tagging.

Neuropeptides are key signaling molecules in many pathways and can serve as potential biomarkers or therapeutics. Mass spectrometry has emerged as a powerful tool for studying neuropeptides with high sensitivity and accuracy. Isobaric tagging can further enhance this method by improving throughput and reducing sampling needs. In this chapter, we discuss the benefits and limitations of using isobaric tags to analyze neuropeptides. Methods for optimizing the data acquisition are also presented to enable a greater number of neuropeptides to be identified and quantified when using isobaric tags, specifically N,N-dimethyl leucine (DiLeu).