Hui Ye

N,N-Dimethyl Leucines as Novel Isobaric Tandem Mass Tags for Quantitative Proteomics and Peptidomics

Herein, we describe the development and application of a set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS(2)) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency, and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and epsilon-amino group of the lysine side chain of a peptide, a balance group, and a reporter group. A mass shift of 145.1 Da is observed for each incorporated label. Intense a(1) reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS(2). All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost. Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (approximately 43%) and quantitation accuracy (<15%) for tryptically digested protein samples. Furthermore, enhanced fragmentation of DiLeu labeling reagents offers greater confidence in protein identification and neuropeptide sequencing from complex neuroendocrine tissue extracts from a marine model organism, Callinectes sapidus.

MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen-fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula-Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen-fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique - matrix-assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) - to study metabolite distribution in roots and root nodules of M.xa0truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8-bis(dimethyl-amino) naphthalene, DMAN] with a conventional matrix 2,5-dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non-fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.

Mapping of neuropeptides in the crustacean stomatogastric nervous system by imaging mass spectrometry.

AbstractConsiderable effort has been devoted to characterizing the crustacean stomatogastric nervous system (STNS) with great emphasis on comprehensive analysis and mapping distribution of its diverse neuropeptide complement. Previously, immunohistochemistry (IHC) has been applied to this endeavor, yet with identification accuracy and throughput compromised. Therefore, molecular imaging methods are pursued to unequivocally determine the identity and location of the neuropeptides at a high spatial resolution. In this work, we developed a novel, multi-faceted mass spectrometric strategy combining profiling and imaging techniques to characterize and map neuropeptides from the blue crab Callinectes sapidus STNS at the network level. In total, 55 neuropeptides from 10 families were identified from the major ganglia in the C. sapidus STNS for the first time, including the stomatogastric ganglion (STG), the paired commissural ganglia (CoG), the esophageal ganglion (OG), and the connecting nerve stomatogastric nerve (stn) using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) and the MS/MS capability of this technique. In addition, the locations of multiple neuropeptides were documented at a spatial resolution of 25xa0μm in the STG and upstream nerve using MALDI-TOF/TOF and high-mass-resolution and high-mass-accuracy MALDI-Fourier transform ion cyclotron resonance (FT-ICR) instrument. Furthermore, distributions of neuropeptides in the whole C. sapidus STNS were examined by imaging mass spectrometry (IMS). Different isoforms from the same family were simultaneously and unambiguously mapped, facilitating the functional exploration of neuropeptides present in the crustacean STNS and exemplifying the revolutionary role of this novel platform in neuronal network studies.n Figure55 neuropeptides from 10 families were mapped in the crustacean stomatogastric nervous system with high spatial and spectral resolution via MALDI-TOF/TOF and MALDI-FT-ICR IMS

Top-down proteomics with mass spectrometry imaging: a pilot study towards discovery of biomarkers for neurodevelopmental disorders.

In the developing mammalian brain, inhibition of NMDA receptor can induce widespread neuroapoptosis, inhibit neurogenesis and cause impairment of learning and memory. Although some mechanistic insights into adverse neurological actions of these NMDA receptor antagonists exist, our understanding of the full spectrum of developmental events affected by early exposure to these chemical agents in the brain is still limited. Here we attempt to gain insights into the impact of pharmacologically induced excitatory/inhibitory imbalance in infancy on the brain proteome using mass spectrometric imaging (MSI). Our goal was to study changes in protein expression in postnatal day 10 (P10) rat brains following neonatal exposure to the NMDA receptor antagonist dizocilpine (MK801). Analysis of rat brains exposed to vehicle or MK801 and comparison of their MALDI MS images revealed differential relative abundances of several proteins. We then identified these markers such as ubiquitin, purkinje cell protein 4 (PEP-19), cytochrome c oxidase subunits and calmodulin, by a combination of reversed-phase (RP) HPLC fractionation and top-down tandem MS platform. More in-depth large scale study along with validation experiments will be carried out in the future. Overall, our findings indicate that a brief neonatal exposure to a compound that alters excitatory/inhibitory balance in the brain has a long term effect on protein expression patterns during subsequent development, highlighting the utility of MALDI-MSI as a discovery tool for potential biomarkers.

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

UNLABELLEDnThe conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without the assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems.nnnBIOLOGICAL SIGNIFICANCEnMass spectrometry (MS)-based neuropeptidomics aims to completely characterize the neuropeptides in a target organism as an important first step toward a better understanding of the structure and function of these complex signaling molecules. Although liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition is a powerful tool in peptidomic research, it often lacks the capability for de novo sequencing of mid-size and large peptides due to inefficient fragmentation of peptides larger than 4kDa. This study describes a multi-scale strategy for complete and confident sequence elucidation of various sizes of neuropeptides in the crustacean nervous system. The aim is to fill a technical gap where the conventional strategy is inefficient for comprehensive characterization of a complex neuropeptidome without assistance of genomic information. Nine novel neuropeptides in a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ of the spiny lobster, P. interruptus. The resulting molecular information extracted from such multi-scale peptidomic analysis will greatly accelerate functional studies of these novel neuropeptides.

From pixel to voxel: a deeper view of biological tissue by 3D mass spectral imaging.

Three dimensional mass spectral imaging (3D MSI) is an exciting field that grants the ability to study a broad mass range of molecular species ranging from small molecules to large proteins by creating lateral and vertical distribution maps of select compounds. Although the general premise behind 3D MSI is simple, factors such as choice of ionization method, sample handling, software considerations and many others must be taken into account for the successful design of a 3D MSI experiment. This review provides a brief overview of ionization methods, sample preparation, software types and technological advancements driving 3D MSI research of a wide range of low- to high-mass analytes. Future perspectives in this field are also provided to conclude that the outlook for 3D MSI is positive and promises ever-growing applications in the biomedical field with continuous developments of this powerful analytical tool.

Advancing Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Capillary Electrophoresis Analysis of Peptides

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.

Probing Neuropeptide Signaling at the Organ and Cellular Domains via Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.

Visualizing Neurotransmitters and Metabolites in the Central Nervous System by High Resolution and High Accuracy Mass Spectrometric Imaging

The spatial localization and molecular distribution of metabolites and neurotransmitters within biological organisms is of tremendous interest to neuroscientists. In comparison to conventional imaging techniques such as immunohistochemistry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has demonstrated its unique advantage by directly localizing the distribution of a wide range of biomolecules simultaneously from a tissue specimen. Although MALDI-MSI of metabolites and neurotransmitters is hindered by numerous matrix-derived peaks, high-resolution and high-accuracy mass spectrometers (HRMS) allow differentiation of endogenous analytes from matrix peaks, unambiguously obtaining biomolecular distributions. In this study, we present MSI of metabolites and neurotransmitters in rodent and crustacean central nervous systems acquired on HRMS. Results were compared with those obtained from a medium-resolution mass spectrometer (MRMS), tandem time-of-flight instrument, to demonstrate the power and unique advantages of HRMSI and reveal how this new tool would benefit molecular imaging applications in neuroscience.

A vision for better health: mass spectrometry imaging for clinical diagnostics.

BACKGROUNDnMass spectrometry imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules from small molecules to large proteins by creating detailed distribution maps of selected compounds. Its usefulness in biomarker discovery towards clinical applications has obtained success by correlating the molecular expression of tissues acquired from MSI with well-established histology.nnnRESULTSnTo date, MSI has demonstrated its versatility in clinical applications, such as biomarker diagnostics of different diseases, prognostics of disease severities and metabolic response to drug treatment, etc. These studies have provided significant insight in clinical studies over the years and current technical advances are further facilitating the improvement of this field. Although the underlying concept is simple, factors such as choice of ionization method, sample preparation, instrumentation and data analysis must be taken into account for successful applications of MSI. Herein, we briefly reviewed these key elements yet focused on the clinical applications of MSI that cannot be addressed by other means.nnnCONCLUSIONSnChallenges and future perspectives in this field are also discussed to conclude that the ever-growing applications with continuous development of this powerful analytical tool will lead to a better understanding of the biology of diseases and improvements in clinical diagnostics.