A crustacean neuropeptide spectral library for data-independent acquisition (DIA) mass spectrometry applications.

Neuropeptides have tremendous potential for application in modern medicine, including utility as biomarkers and therapeutics. To overcome the inherent challenges associated with neuropeptide identification and characterization, data-independent acquisition (DIA) is a fitting mass spectrometry (MS) method of choice to achieve sensitive and accurate analysis. It is advantageous for preliminary neuropeptidomic studies to occur in less complex organisms, with crustacean models serving as a popular choice due to their relatively simple nervous system. With spectral libraries serving as a means to interpret DIA-MS output spectra, and Cancer borealis as a model of choice for neuropeptide analysis, we performed the first spectral library mapping of crustacean neuropeptides. Leveraging pre-existing data-dependent acquisition (DDA) spectra, a spectral library was built using PEAKS Online. The library is comprised of 333 unique neuropeptides. The identification results obtained through the use of this spectral library were compared with those achieved through library-free analysis of crustacean brain, pericardial organs (PO), and thoracic ganglia (TG) tissues. A statistically significant increase (Student's t-test, P value < 0.05) in the number of identifications achieved from the TG data was observed in the spectral library results. Furthermore, in each of the tissues, a distinctly different set of identifications was found in the library search compared to the library-free search. This work highlights the necessity for the use of spectral libraries in neuropeptide analysis, illustrating the advantage of spectral libraries for interpreting DIA spectra in a reproducible manner with greater neuropeptidomic depth.

Definitive Screening Designs to Optimize Library-Free DIA-MS Identification and Quantification of Neuropeptides.

Method optimization is crucial for successful mass spectrometry (MS) analysis. However, extensive method assessments, altering various parameters individually, are rarely performed due to practical limitations regarding time and sample quantity. To maximize sample space for optimization while maintaining reasonable instrumentation requirements, a definitive screening design (DSD) is leveraged for systematic optimization of data-independent acquisition (DIA) parameters to maximize crustacean neuropeptide identifications. While DSDs require several injections, a library-free methodology enables surrogate sample usage for comprehensive optimization of MS parameters to assess biomolecules from limited samples. We identified several parameters contributing significant first- or second-order effects to method performance, and the DSD model predicted ideal values to implement. These increased reproducibility and detection capabilities enabled the identification of 461 peptides, compared to 375 and 262 peptides identified through data-dependent acquisition (DDA) and a published DIA method for crustacean neuropeptides, respectively. Herein, we demonstrate a DSD optimization workflow, using standard material, not reliant on spectral libraries for the analysis of any low abundance molecules from previous samples of limited availability. This extends the DIA method to low abundance isoforms dysregulated or only detectable in disease samples, thus improving characterization of previously inaccessible biomolecules, such as neuropeptides. Data are available via ProteomeXchange with identifier PXD038520.

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.

Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides.

Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well-characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re-evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides in Callinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two-fold, from 18 to 36. Product ion-triggered electron-transfer/higher-energy collision dissociation enabled the site-specific detection of 55 intact N- and O-linked glycoforms, while the faster stepped collision energy higher-energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro- and micro-heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus.

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Comparing Selected-Ion Collision Induced Unfolding with All Ion Unfolding Methods for Comprehensive Protein Conformational Characterization.

Structural analysis by native ion mobility-mass spectrometry provides a direct means to characterize protein interactions, stability, and other biophysical properties of disease-associated biomolecules. Such information is often extracted from collision-induced unfolding (CIU) experiments, performed by ramping a voltage used to accelerate ions entering a trap cell prior to an ion mobility separator. Traditionally, to simplify data analysis and achieve confident ion identification, precursor ion selection with a quadrupole is performed prior to collisional activation. Only one charge state can be selected at one time, leading to an imbalance between the total time required to survey CIU data across all protein charge states and the resulting structural analysis efficiency. Furthermore, the arbitrary selection of a single charge state can inherently bias CIU analyses. We herein aim to compare two conformation sampling methods for protein gas-phase unfolding: (1) traditional quadrupole selection-based CIU and (2) nontargeted, charge selection-free and shotgun workflow, all ion unfolding (AIU). Additionally, we provide a new data interpretation method that integrates across all charge states to project collisional cross section (CCS) data acquired over a range of activation voltages to produce a single unfolding fingerprint, regardless of charge state distributions. We find that AIU in combination with CCS accumulation across all charges offers an opportunity to maximize protein conformational information with minimal time cost, where additional benefits include (1) an improved signal-to-noise ratios for unfolding fingerprints and (2) a higher tolerance to charge state shifts induced by either operating parameters or other factors that affect protein ionization efficiency.

Native Ion Mobility-Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level.

Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aß cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aß cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aß cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aß cytotoxicity.

ADVANCES IN HIGH-RESOLUTION MALDI MASS SPECTROMETRY FOR NEUROBIOLOGY.

Research in the field of neurobiology and neurochemistry has seen a rapid expansion in the last several years due to advances in technologies and instrumentation, facilitating the detection of biomolecules critical to the complex signaling of neurons. Part of this growth has been due to the development and implementation of high-resolution Fourier transform (FT) mass spectrometry (MS), as is offered by FT ion cyclotron resonance (FTICR) and Orbitrap mass analyzers, which improves the accuracy of measurements and helps resolve the complex biological mixtures often analyzed in the nervous system. The coupling of matrix-assisted laser desorption/ionization (MALDI) with high-resolution MS has drastically expanded the information that can be obtained with these complex samples. This review discusses notable technical developments in MALDI-FTICR and MALDI-Orbitrap platforms and their applications toward molecules in the nervous system, including sequence elucidation and profiling with de novo sequencing, analysis of post-translational modifications, in situ analysis, key advances in sample preparation and handling, quantitation, and imaging. Notable novel applications are also discussed to highlight key developments critical to advancing our understanding of neurobiology and providing insight into the exciting future of this field. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

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.